|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 276, Issue 41, 38121-38138, October 12, 2001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, June 12, 2001, and in revised form, July 13, 2001
Caveolin-1 is the principal structural protein of
caveolae membranes in fibroblasts and endothelia. Recently, we have
shown that the human CAV-1 gene is localized to a suspected
tumor suppressor locus, and mutations in Cav-1 have been implicated in
human cancer. Here, we created a caveolin-1 null (CAV-1 Caveolin was first identified in 1989 by Glenney and colleagues
(1, 2) as a major v-Src substrate in Rous sarcoma virus-transformed chicken embryo fibroblasts. Interestingly, this same protein was found
to be the primary structural component of caveolae microdomains, 50-100 nm vesicular invaginations of the plasma membrane (3).
Caveolae were morphologically described as early as the 1950s by Yamada
(4) and Palade (5). They are curious structures that can be found
individually or in clusters at the surfaces of numerous cell types, the
best examples of which are adipocytes, endothelial cells, muscle cells,
and fibroblasts. Research in the past decade has shown that caveolae
are specialized membrane microdomains formed as a result of
localized accumulation of cholesterol, glycosphingolipids, and caveolin
(6-8). Caveolin, an integral membrane protein that can directly bind
cholesterol, most likely plays a major role in the invagination of
caveolae from the plasma membrane proper, although our understanding of
the mechanisms behind this process remains rudimentary.
Two other members of the caveolin gene family have recently been
identified and cloned, caveolin-2 and caveolin-3 (9, 10); as a
consequence, caveolin has been re-termed caveolin-1
(Cav-1).1 Caveolin-2 has the
same tissue distribution as and co-localizes with caveolin-1, whereas
caveolin-3 is expressed only in cardiac, skeletal, and smooth muscle
cells (11, 12).
Although caveolae function in vesicular and cholesterol trafficking
(13, 14), they have also been implicated in signal transduction at the
plasma membrane (15, 16). Biochemical and morphological experiments
have shown that a variety of lipid-modified signaling molecules are
concentrated within these plasma membrane microdomains, such as Src
family tyrosine kinases, Ha-Ras, eNOS, and heterotrimeric G-proteins
(17-22).
In many ways, caveolin-1 is intricately involved in caveolar
functioning. In the years after the discovery that caveolae might serve
to compartmentalize signaling molecules and facilitate cross-talk among
signaling cascades (the so-called "caveolae signaling hypothesis" (16)), Cav-1 has been found to be a key regulator of some of these
proteins. Both in vitro and cell culture experiments
indicate that Cav-1 can directly interact with and maintain some of
these signaling molecules in an inactive conformation (reviewed in Ref. 23). In effect, Cav-1 seems to act as a scaffolding protein, able to
negatively regulate the activity of other molecules by binding to and
releasing them in a timely fashion.
Research in the past few years has established a recurring theme in
this regulation. Many of the proteins that either interact with,
transcriptionally repress, or are inhibited by Cav-1 fall under the
pro-proliferative, oncogenic, and anti-apoptotic category of molecules.
Cav-1 interacts with and negatively regulates the EGF-R,
platelet-derived growth factor receptor, and Neu tyrosine kinases
(24-26), Ha-Ras (17, 18), c-Src (17), and phosphatidylinositol 3-kinase (27). Conversely, caveolin-1 levels are transcriptionally reduced upon activation of the oncogenes Ha-ras,
v-abl, myc, neu, the HPV oncogene
E6, among others (26, 28-30). Therefore, it is not
surprising that we and others (26, 29, 31-37) observed undetectable or
very low expression levels of Cav-1 in numerous tumor-derived cell lines.
For some time, it has been known that a certain locus (D7S522; 7q31.1)
is an aphidicolin-induced fragile site in the human genome (38, 39) and
a hot spot for deletions in a variety of human tumors including
breast, prostate, colorectal, ovarian, pancreatic, and renal cell
carcinomas (38, 40-46). Interestingly, determination of the genomic
organization of the human CAV-1 locus revealed that
it maps to 7q31.1, adjacent to the LOH marker D7S522, and as of yet it
still remains the closest known gene to this putative tumor suppressor
locus (35, 47).
Taken together, the results described above have led many investigators
to propose the possibility that Cav-1 is indeed a "tumor
suppressor" whose reduction/deletion in cells would provide growth
advantages and expedite tumorigenesis. In support of this idea, the
only two methods thus far used to abolish Cav-1 expression have arrived
at similar conclusions. Antisense-mediated down-regulation of Cav-1 in
NIH-3T3 fibroblasts leads to a hyperactivation of the p42/44 MAP kinase
pathway and anchorage-independent growth (48). An RNA
interference-based ablation of Cav-1 in Caenorhabditis elegans leads to progression of the meiotic cell cycle, a
phenotype that mirrors that of Ras activation (49).
Furthermore, a recent report indicates that the caveolin-1 gene is
mutated in up to 16% of human breast cancer samples examined (50).
Recombinant expression of the caveolin-1 cDNA harboring this
mutation (P132L) was sufficient to transform NIH 3T3 cells (50). As
similar results have been obtained previously using an antisense
approach to ablate caveolin-1 expression (48), these results indicate
that the caveolin-1 (P132L) mutation may behave in a dominant-negative
fashion. Interestingly, an analogous mutation occurs within the
caveolin-3 gene (P104L) in patients with a novel form of autosomal
dominant limb-girdle muscular dystrophy (LGMD-1C) (51).
In order to gain a better understanding of caveolae and caveolin-1
functioning in a mammalian organism, we used a gene targeting strategy
to disrupt the Cav-1 locus in the mouse. In this way, we could
observe the role Cav-1 plays in animal physiology
(i.e. during development and into adult life) as well as
molecularly (i.e. caveolar biogenesis, its interaction with
caveolin-2, and its functional roles in endocytosis, cellular
proliferation, and signal transduction). In this study, we describe the
generation of mice lacking the cav-1 gene and determine some
of the molecular side effects that result from a deficiency of Cav-1 expression.
Undoubtedly, the generation of viable/fertile Cav-1-deficient mice (and
cells derived from these animals) will allow us and others to
critically evaluate the many proposed functions of caveolae organelles
and the caveolin-1 protein in vivo.
Materials--
Antibodies and their sources were as follows:
anti-caveolin-1 mAb 2297, anti-caveolin-2 mAb 65, and
anti-caveolin-3 mAb 26 (10, 11, 52) (gifts of Dr. Roberto
Campos-Gonzalez, BD Transduction Laboratories, Inc.); anti-caveolin-1
pAb N-20 (Santa Cruz Biotechnology); anti-p42/44 pAb and
phospho-specific anti-p42/44 pAb (New England Biolabs);
anti- Construction of the Targeting Vector--
Genomic clones
containing the murine Cav-1 locus were isolated from a
129/Sv(J1) Screening of Homologously Recombined ES Cells and Generation of
Germ Line Chimeras--
WW6 ES cells (gift of Dr. Pamela Stanley (56))
were electroporated with the linearized targeting construct (40 µg)
and selected with G418 (150 µg/ml of active component, Life
Technologies, Inc.) as described previously (57). Homologous
recombination in 360 selected ES clones was assessed via Southern blot
analysis. Briefly, genomic DNA was digested with PstI or
XbaI and hybridized with a 1.1-kb
XbaI-SacI probe; Cav-1+/
Animals were analyzed at 2-4.5 months of age. Experiments were
conducted under the direct supervision of the trained veterinarians of
the Einstein Animal Institute, and animal protocols were approved by
the Animal Use Committee.
Mouse Embryonic Fibroblast (MEF) Culture and Immortalization
Protocol--
Primary MEFs were obtained from Day 13.5 embryos
essentially as described (58). Briefly, embryos were decapitated,
thoroughly minced, and trypsinized in 1 ml of 0.05% trypsin, 0.53 mM EDTA (Life Technologies, Inc.) for 20 min at 37 °C.
Ten ml of complete medium (Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life
Technologies, Inc.)) was used to inactivate the trypsin and resuspend
the dissociated cells. Cells were plated on a 10-cm plate and cultured
in a 37 °C, 5% CO2 incubator. These "passage 1"
cells were further propagated using a defined 3T3 passaging protocol
(i.e. 3 × 105 cells were plated per 60-mm
dish every 3 days). For all experiments early passage primary MEFs
(<5) were used. To immortalize MEFs, cells were passaged according to
the 3T3 protocol continuously until growth rates in culture resumed the
rapid rates seen in early passage MEFs (i.e. Passage 25 cells and beyond).
Transmission Electron Microscopy--
MEFs were fixed with
2.5% glutaraldehyde in 0.1 M cacodylate buffer
post-fixed with OsO4, and stained with uranyl acetate and lead citrate. A cryotome was used to yield sections, and the samples were examined under a JEOL 1200EX transmission electron microscope and photographed at a magnification of × 16,000 (59-61). Caveolae were identified by their characteristic flask shape, size (50-100 nm), and location at or near the plasma membrane (28).
Expression Vectors--
The cDNA encoding full-length
caveolin-1 was subcloned into pCB7, a mammalian expression vector
driven by the cytomegalovirus promoter (62). The cDNAs encoding GFP
and GFP-Cav-1 (containing the full-length Cav-1 cDNA C-terminal to
GFP) were as described previously (63).
Immunoblot Analysis--
Cells were cultured in their respective
media and allowed to reach 80-90% confluency. Subsequently, they were
washed with PBS and incubated with lysis buffer (10 mM
Tris, pH 7.5; 50 mM NaCl; 1% Triton X-100; 60 mM octyl glucoside) containing protease inhibitors (Roche
Molecular Biochemicals). Protein concentrations were quantified using
the BCA reagent (Pierce), and the volume required for 10 µg of
protein was determined. Samples were separated by SDS-PAGE (12.5%
acrylamide) and transferred to nitrocellulose. The nitrocellulose
membranes were stained with Ponceau S (to visualize protein bands)
followed by immunoblot analysis. All subsequent wash buffers contained
10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween
20, which was supplemented with 1% bovine serum albumin (BSA) and 2%
nonfat dry milk (Carnation) for the blocking solution and 1% BSA for
the antibody diluent. Primary antibodies were used at a 1:500 dilution.
Horseradish peroxidase-conjugated secondary antibodies (1:5000
dilution, Pierce) were used to visualize bound primary antibodies with
the Supersignal chemiluminescence substrate (Pierce).
Purification of Caveolae-enriched Membrane
Fractions--
Caveolae-enriched membrane fractions were purified
essentially as we described previously (59). 200 mg of lung tissue was placed in 2 ml of MBS (25 mM Mes, pH 6.5, 150 mM NaCl) containing 1% Triton X-100 and solubilized by
using quick 10-s bursts of a rotor homogenizer and passing 10 times
through a loose fitting Dounce homogenizer. The sample was mixed with
an equal volume of 80% sucrose (prepared in MBS lacking Triton X-100),
transferred to a 12-ml ultracentrifuge tube, and overlaid with a
discontinuous sucrose gradient (4 ml of 30% sucrose, 4 ml of 5%
sucrose, both prepared in MBS, lacking detergent). The samples were
subjected to centrifugation at 200,000 × g (39,000 rpm
in a Sorval rotor TH-641) for 16 h. A light scattering band was
observed at the 5-30% sucrose interface. Twelve 1-ml fractions were
collected, and 50-µl aliquots of each fraction were subjected to
SDS-PAGE and immunoblotting.
Immunofluorescence Microscopy--
The procedure was performed
as we described previously (17). MEFs (either un-transfected or
transfected with the caveolin-1 cDNA) were fixed for 30 min in PBS
containing 2% paraformaldehyde, rinsed with PBS, and quenched with 50 mM NH4Cl for 10 min. The cells were then
incubated in permeabilization buffer (PBS; 0.2% BSA; 0.1% Triton
X-100) for 10 min, washed with PBS, and double-labeled with a 1:400
dilution of anti-caveolin-1 pAb N-20 and 1:200 dilution of
anti-caveolin-2 mAb for 60 min. After rinsing with PBS (3 times), secondary antibodies (7.5 µg/ml) ((lissamine-rhodamine-conjugated goat anti-rabbit and fluorescein (FITC)-conjugated goat anti-mouse) antibodies (Jackson ImmunoResearch) were added for a period of 60 min.
Cells were washed with PBS (3 times) and slides mounted with Slow-Fade
anti-fade reagent (Molecular Probes). A Bio-Rad MR600 confocal
fluorescence microscope was used for visualization of bound secondary antibodies.
Rescue of Caveolin-2 Levels in Caveolin-1-deficient
MEFs--
The construction and characterization of the Cav-1 and GFP
Adenoviruses were as we described previously (34). MEF infections were
conducted as follows: 105 cells were seeded in a series of
35-mm dishes. The desired viral titer (quantified as plaque-forming
units (PFUs)) was aliquoted and preincubated with 104
molecules of poly-L-lysine per viral particle in PBS for 30 min. Serum-free medium up to 1 ml was added to this solution and placed on the cells for 2 h. Cells were then cultured in regular growth medium (DMEM, 10% FBS) for 48 h and subjected to immunoblot
analysis. The lysosomal/proteasomal inhibitor experiments were
performed essentially as described (64). Briefly, 1.5 × 105 cells were seeded in a series of 35-mm dishes and
treated for the indicated times with vehicle alone
(Me2SO) or with either the proteasomal inhibitors
MG-132 (1 µM, Sigma) or MG-115 (1 µM, Sigma) or the lysosomal inhibitors chloroquine (50 µM,
Sigma) or NH4Cl (10 mM). Cells were then lysed
and subjected to immunoblot analysis.
Endocytosis Assays--
Wild-type and Cav-1 null mouse embryo
fibroblasts (MEFs) were plated on 18-mm glass coverslips (Fisher) in
12-well plates. Cells were grown in complete medium (DMEM supplemented
with 10% FBS, 2 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin (Life Technologies, Inc.)). When cells reached
~75-85% confluency, the media was replaced with 1 ml of complete
media containing FITC-conjugated albumin (Sigma) at a final
concentration of 10 µg/ml. Cells were incubated at 37 °C for 5, 15, and 30 min, washed in PBS, and fixed in 2% paraformaldehyde for 20 min. The cells were washed for 20 min in PBS and mounted on slides with
the Prolong anti-fade reagent (Molecular Probes) and imaged with an
Olympus IX 70 inverted microscope. Virtually identical experiments were carried out with FITC-conjugated transferrin (10 µg/ml; Sigma).
Caveolin-1 knockout MEFs were grown on 60-mm tissue culture dishes and
transiently transfected with the caveolin-1 cDNA in the pCB7 vector
using LiopfectAMINE Plus (Life Technologies, Inc.), according to the
manufacturer's instructions. The transfected cells were plated on
18-mm coverslips in 12-well plates after 24 h. FITC-albumin uptake
was then examined in the transfected cells 36 h after the initial
transfection. Cells transfected with caveolin-1 were detected by
immunostaining with anti-Cav-1 IgG (N-20; Santa Cruz Biotechnology) and
a rhodamine-conjugated secondary antibody.
MEF Proliferation Curves and Cell Cycle
Analysis--
Proliferation curves were conducted essentially as
described previously (65). Briefly, 15 × 103 cells
were seeded in a series of twenty 35-mm dishes and cultured under
regular growth conditions (DMEM, 10% FBS). Each day, two plates were
counted using a hemocytometer, and the medium was changed for the
remaining plates. Growth curves were continued for a 10-day time course.
Cell cycle analysis was conducted by Flow Cytometry essentially as
described (66). Briefly, 2 × 105 cells were seeded in
60-mm dishes and cultured under regular growth conditions (DMEM, 10%
FBS) for 24 h. Cells were trypsinized, washed in PBS, and fixed in
70% ethanol at 4 °C for at least 30 min. Fixed cells were
resuspended in PBS containing 0.25 mg/ml RNase A (Sigma) and 10 µg/ml
propidium iodide (Sigma) and subjected to univariate cell cycle
analysis using a Becton-Dickinson FACScan flow cytometer. The
G0/G1, S, and G2/M phases of the
cell cycle were quantified with CELLQUEST software.
DNA synthesis in MEFs was directly analyzed by
[3H]thymidine incorporation, essentially as described
(66). Briefly, 2 × 105 cells were seeded in five
60-mm dishes, allowed to adhere, and cultured overnight under regular
growth conditions (DMEM, 10% FBS) supplemented with 1 µCi of
[3H]thymidine. Cells were washed with PBS, lysed in 0.3 M NaOH, and fixed with 10% trichloroacetic acid at 4 °C
for 30 min. The precipitated material was centrifuged, washed, and
resuspended in 200 µl of 0.3 M NaOH, 100 µl of which
was quantified using a scintillation counter.
Light and Electron Microscopic Analysis of the Lung--
Lung
tissue samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium
tetroxide followed by 1% uranyl acetate, dehydrated through a graded
series of ethanol, and embedded in LX112 resin (LADD Research
Industries, Burlington VT). Ultrathin sections were cut on a Reichert
Ultracut E, stained with uranyl acetate followed by lead citrate, and
viewed using a JEOL 1200EX transmission electron microscope at 80 kV. One-µm thick sections were stained with toluidine blue and
imaged on a Zeiss Axiophot microscope.
Preparation of Lung Paraffin Sections--
Wild-type and Cav-1
null mice were sacrificed and the lungs removed and placed in 10%
formalin. The lungs were then inflated by injecting fixative with a
syringe directly into the lungs to preserve the alveolar structure. The
tissue was fixed for 2 h, washed in PBS for 20 min, and dehydrated
through a graded series of ethanol washes. The tissue was then treated
with xylene for 40 min all at room temperature and then paraffin for
1 h at 55 °C. The tissue was then embedded, and 5-µm thick
sections were prepared using a Microm (Baxter Scientific) microtome and
placed on super-frost plus slides (Fisher). Slides were then
hematoxylin and eosin-stained according to standard
laboratory protocols.
Quantitation of Nuclei--
Wild-type and Cav-1 null mouse lung
tissue sections stained with hematoxylin and eosin were examined
using a Zeis Axiophot. By using a 20× objective, six random
0.5-mm2 fields were photographed for each genotype, and all
the nuclei within those regions were manually tabulated using a
hand-held counter.
Immunostaining of Lung Paraffin Sections--
Sections of
wild-type and Cav-1 null mouse lung were de-paraffinized in xylene for
4 min and rehydrated through a graded series of ethanol and placed in
PBS. Sections were pre-blocked with 2% horse serum for 20 min and then
washed with PBS for 10 min. The sections were incubated with a given
primary antibody at room temperature for 1 h. An FITC-conjugated
or lissamine-rhodamine-conjugated secondary antibody was added
to the sections after a 10-min wash in PBS. After a 30-min incubation
with the secondary antibody, the sections were washed in PBS for 20 min. Prolong anti-fade reagent was then added to prevent bleaching of
the fluorochrome. Samples were imaged with an Olympus IX 70 inverted
microscope. Anti-VEGF-R IgG (Flk-1; rabbit pAb C-20) was purchased from
Santa Cruz Biotechnology, Inc. Anti-Ki67 IgG (rabbit pAb; NCL-Ki67p) was purchased from Novocastra, Ltd, UK, and used at a dilution of
1:500. For Ki67 immunostaining, sections were subjected to antigen
retrieval by microwave irradiation in 0.01 M, pH 6, trisodium citrate buffer.
Assessment of Exercise Tolerance--
A 4-liter beaker filled
with water (25 °C) was used as a "swimming pool" to assess the
exercise tolerance of male mice. Briefly, a very light weight (a paper
clip; 0.4 g; ~1.25% of their body weight) was attached to the
tail of a mouse with a body weight of ~32 g. The mouse was gently
placed in the water and carefully observed. The time at which the mouse
was initially unable to maintain complete buoyancy was recorded and the
mouse was immediately removed from the pool. No mice were injured in
these experiments; 5 animals were tested for each genotype.
Aortic Ring Studies of Vasoconstriction and
Vasorelaxation--
Wild-type and Cav-1 null male mice (4.5 months
old) were sacrificed by CO2 asphyxiation. The thoracic
aorta was dissected and cut into cylindrical segments of ~3 mm in
length. Five to six rings from 3 mice were obtained from each genotype
of mouse strain. Rings were immediately placed in ice-cold
Krebs-Henseleit buffer containing the following composition (in
mM): NaCl, 110, KCl, 4.8, CaCl2, 2.5, MgSO4, 1.2, KH2PO4, 1.2, NaHCO3, 25, glucose and dextrose, 11, in glass-distilled
water. Briefly, the rings were suspended by two hooks (25-µm
thickness) inserted into the lumen and mounted in a vessel myograph
system (7-ml organ bath, Danish Myo Technology, Aarhus, Denmark)
containing Krebs-Henseleit buffer. The organ chambers were maintained
at 32 ± 0.05 °C and continuously bubbled with 95%
O2 and 5% CO2 to maintain pH 7.4 ± 0.1. The mouse aortas were submitted to a resting tension of 1.2 g, and
isometric tension was recorded using a transducer coupled to a MacLab
data acquisition system. Following a 60-min equilibration period, with
frequents washings (every
Data are expressed as means ± S.E. Statistical differences were
measured by one-way analysis of variance followed by Newman-Keul post-hoc test.
Generation of Caveolin-1-deficient Mice via Targeted Disruption of
the Cav-1 Locus--
We previously determined the genomic organization
of the caveolin-1 (Cav-1) locus and found that exons 1 and 2 are
spaced within 2 kb of each other, whereas exon 3 is ~10 kb downstream (47). Therefore, we generated a targeting vector designed to replace
the first two exons and a small portion of the 5' promoter sequence
with the neomycin resistance cassette (neo) (Fig.
1A).
WW6 embryonic stem (ES) cells (56) were electroporated with the
targeting vector, and 360 clones were selected with G418. Homologous
recombination at this locus is predicted to create two new restriction
sites, PstI and XbaI, both of which can be used
to identify positive ES cell clones by Southern blot analysis (Fig.
1A); four clones were determined to be positive in this manner. Germ line chimeras were derived from only two of these clones,
as shown in the positive Southern blots in Fig. 1B. We subsequently mated these chimeras with C57Bl/6 mice to yield F1 heterozygous offspring, a cohort of which was interbred to produce the
Cav-1 null progeny. Southern blot and PCR-based methods of assessing
the targeted locus were performed on the first series of live
offspring, confirming the predicted loss of a wild-type 8.0-kb band on
Southern blot and the 500-bp band on PCR analysis (Fig. 1C).
Genotyping of offspring from such heterozygous inter-crosses revealed
that there is no reduced viability of the Cav-1-null mice and that mice
of all three genotypes are present at the expected Mendelian frequency
(Cav-1+/+ 25.2%, Cav-1 +/
Although Cav-1 is expressed in numerous tissues at varying levels, it
is found in abundance in certain terminally differentiated cells
(i.e. adipocytes, endothelial cells, type I pneumocytes, and
fibroblasts) (9, 11). Furthermore, Cav-1 expression is completely
absent in skeletal and cardiac muscle cells, and in contrast,
caveolin-3 (Cav-3), another member of the caveolin gene family, is
selectively expressed (10, 12). In order to verify whether the targeted
disruption of the Cav-1 locus led to a truly null mutation, we
determined the expression of the Cav-1 protein in adipose, lung, and
heart tissues from mice of all three genotypes (wild-type,
heterozygote, and knockout; Fig. 1C). In all tissues examined, ablation of the Cav-1 locus leads to a concomitant
loss of Cav-1 protein expression;
In addition, several points are worth noting. 1) These mice are
deficient in both Cav-1 isoforms (the full-length 178-amino acid
We also assessed Cav-1 expression in cultured mouse embryonic
fibroblasts (MEFs), another cell type with abundant Cav-1 expression (67). Two different clones of MEFs for each possible genotype were
extracted and cultured from day 13.5 embryos. Immunoblot analysis of
Cav-1 levels indicated similar findings to those above, namely a
complete ablation of Cav-1 expression in the knockout and a significant
reduction in Cav-1 expression in heterozygous MEFs (Fig.
1E).
Phenotype and Histopathological Examination of Cav-1 The Absence of Caveolin-1 Is Sufficient to Abrogate Caveolae
Formation--
The molecular components required for caveolar
biogenesis have been studied by numerous investigators. From the
following observations, the general consensus remains that caveolin-1
plays an essential role in caveolae formation. 1) Cholesterol is
required for caveolar invagination, because treatment with
cholesterol-depleting agents (e.g. filipin and
methyl-
Thus, the generation of caveolin-1 null mice provided an opportunity to
test this assertion under physiological conditions. In this manner,
nonspecific effects due to chemical treatments, cellular
transformation, and overexpression would not confound such a study.
MEFs derived from Cav-1+/+ and The Absence of Caveolin-1 Leads to Degradation and Redistribution
of Caveolin-2--
Currently, the caveolin gene family is composed of
caveolin-1, -2, and -3. All known terminally differentiated tissues
that express caveolin-1 also express the closely related family member caveolin-2 (Cav-2) (9, 11). In contrast, caveolin-3 (Cav-3), the
protein with the highest homology to Cav-1, is expressed
specifically in muscle cells (including cardiac, skeletal, and smooth
muscle). Therefore, we were interested to determine any possible
counter-regulatory or compensatory behavior by Cav-2 and Cav-3 in Cav-1
null tissues. We immunoblotted the same tissues samples used to compare
Cav-1 expression in mice of different genotypes (Fig. 1D)
for Cav-2 and Cav-3.
To our surprise, Cav-2 expression was greatly reduced in all the
Cav-1(
The in vivo relationship between these two proteins (Cav-1
and Cav-2) goes beyond simple co-expression however. Although
Cav-1 is able to form homo-oligomers consisting of 14-16
individual molecules (70, 71), it also is capable of forming similar size hetero-oligomers with Cav-2 (11, 72) and co-localizes with Cav-2
in caveolae microdomains (11). Additionally, it appears that Cav-2
requires the presence of Cav-1 for oligomerization and plasma membrane
localization; when Cav-2 is overexpressed alone, it behaves as a
mixture of monomers and dimers and is found in the Golgi complex
(73-75). However, down-regulation of caveolin-1 either by antisense
strategies or by oncogenic transformation (conditions where Cav-1 is
reduced to undetectable levels) has no effect on Cav-2 levels or their
localization (48).2 The
generation of Cav-1-deficient mice provided us the opportunity to
definitively resolve the relationship between Cav-1 and Cav-2 in
vivo.
Due to the abundance of Type I pneumocytes and endothelial cells, lung
tissue is a great source for the purification and molecular analysis of
caveolae (60). In order to determine the localization of Cav-2 in Cav-1
null mice, we subjected mouse lung tissue to extraction and sucrose
gradient ultracentrifugation, a procedure with which we have previously
separated caveolar microdomains from other cellular constituents. Via
this method, it is possible to dramatically concentrate Cav-1, the
caveolae marker protein, with respect to total cellular protein (60,
62). The outputs of this centrifugation consist of 12 equal fractions
(of which fractions 4-5 and 8-12 are considered of caveolar and
non-caveolar origin, respectively). As shown in Fig. 3C,
Cav-1 and -2 are enriched heavily in the caveolar fractions of
wild-type lungs. Interestingly, however, in Cav-1-deficient lungs,
Cav-2 is almost entirely excluded from such fractions. We also obtained
similar results in cultured MEFs (data not shown), indicating that a
lack of Cav-1 alters the fractionation of Cav-2.
We next attempted to uncover more definitively the subcellular
localization of Cav-2. Fig. 3D shows a series of micrographs of wild-type and knockout MEFs co-immunostained with anti-Cav-1 polyclonal and anti-Cav-2 monoclonal antibodies. There is a distinct and overlapping membrane localization for Cav-1 and -2 in
wild-type cells; in contrast, we found Cav-2 only in the perinuclear
Golgi compartments of Cav-1-deficient MEFs (Fig. 3D). We
reasoned that transient transfection of these Cav-1 knockout cells with
the Cav-1 cDNA should rescue and redistribute Cav-2 away from the Golgi, restoring its plasma membrane localization. Fig.
4A confirms this assumption,
showing co-localization of both proteins at the cell surface in the
Cav-1 transfected cell. It should be noted that the Golgi staining for
Cav-2 in neighboring untransfected cells is not viewable at this
exposure simply due to the immensely reduced Cav-2 expression.
We have described previously a high efficiency method of Cav-1
overexpression using adenovirus-mediated gene transfer (34). With this
strategy, we would be able to demonstrate biochemically a
Cav-1-mediated rescue of Cav-2 expression. Since the Cav-1 adenovirus contains a tet-responsive promoter ("tet-off" system), Cav-1
expression is possible only by co-infection of cells with a
tet-transactivator (tTA)-producing virus. Fig. 4B shows that
a steadily increasing dose of adenoviral titers achieves a concomitant
increase in Cav-1 expression. In turn, we observe a robust elevation of
Cav-2 expression;
To gain insight into possible mechanisms for the reduction in Cav-2
levels, we focused on the cellular degradative machinery. The obvious
requirement of Cav-1 for both the expression and membrane trafficking
of Cav-2 indicates that protein misfolding, a hang-up in the Golgi, and
subsequent degradation (proteasomal pathway) or an increase in
retrograde trafficking from the membrane (lysosomal pathway) are
possible areas of investigation. Therefore, we treated Cav-1 knockout
MEFs with MG-132 and MG-115 (two classically used proteasomal
inhibitors (76, 77)) and chloroquine and NH4Cl (two
lysosomal inhibitors) for a period of up to 24 h (Fig. 4, C and D). We discovered that only the proteasomal
inhibitors have a positive effect on Cav-2 expression. Over a time
course of 8, 16, and 24 h, Cav-2 levels increase substantially
from base line. It is interesting to note, however, that the increase
in Cav-2 expression is not complete. This could be due to the
following: 1) sub-optimal dosages of proteasomal inhibitors, a
condition not rectifiable in such experiments as higher dosages have
toxic effects on the MEF cells2 or 2) the presence of other
degradative processes not fully abrogated by our repertoire of chemical inhibitors.
Caveolin-1 Null MEFs Show Defects in the Endocytosis of Albumin but
Not Transferrin--
Albumin has been used extensively to monitor
caveolae-mediated endocytosis (78). Thus, we next examined the
ability of Cav-1-deficient MEFs to endocytose the fluorescent tracer,
FITC-albumin. Fig. 5, A and
B, shows the Cav-1-deficient MEFs fail to accumulate FITC-albumin even after 30-60 min of continuous incubation. In contrast, wild-type MEFs show cell surface labeling with FITC-albumin after only 5 min, with significant intracellular accumulation by 15 min
of incubation. These results support the idea that caveolae clearly
participate in endocytosis of specific ligands, such as albumin.
Importantly, transient expression of the caveolin-1 cDNA in
Cav-1-deficient MEFs was sufficient to restore the uptake of FITC-albumin (Fig. 5B).
To ensure that a lack of caveolin-1 expression did not globally affect
endocytosis, we also examined the fate of a second fluorescent tracer,
FITC-transferrin, which is endocytosed via clathrin-coated pits. Fig.
5C demonstrates that FITC-transferrin was rapidly
endocytosed in both wild-type and Cav-1-deficient MEFs, with no
apparent differences. After 30 min, FITC-transferrin accumulated in a
perinuclear compartment in both wild-type and Cav-1-deficient MEFs.
Thus, Cav-1-deficient MEFs show a selective defect in the uptake of a
known caveolar ligand, i.e. albumin. This is consistent with
our observation that Cav-1-deficient MEFs lack morphological caveolae,
as seen by transmission electron microscopy (Fig. 2).
The Growth Properties and Cell Cycle Analysis of Cav-1
However, all of this work has been conducted in immortalized cell lines
(i.e. cells that have perturbations in one or more genes
important for controlling cell cycle progression), a situation that may
confound the physiological behavior of Cav-1. Therefore, we attempted
to study Cav-1 function in cellular proliferation in cultured primary
MEFs. We first determined the growth potential of six independent MEF
cultures (two from each possible genotype) (also shown in Figs.
1E and 3B) over a 10-day period. Cells were plated sparsely in a series of 35-mm dishes, two of which were counted
each day. In each case, the Cav-1-deficient cells proliferated approximately 2-fold faster and to higher saturation densities than
their wild-type counterparts (Fig.
6A). Remarkably, cells heterozygous for Cav-1 show intermediate proliferation rates that seem
to inversely correlate with their levels of Cav-1 expression (see Fig.
1E for a comparison of Cav-1 protein levels). These experiments were also performed with MEFs derived from 2 other knockout
and wild-type embryos with similar results (data not shown).
We next conducted a more quantitative growth comparison of the Cav-1+/+
and
If increases in cellular proliferation and S phase are due to a Cav-1
deficiency, we reasoned that recombinant overexpression of Cav-1 in
knockout cells should act to revert their cell cycle profiles to
wild-type levels. In light of the fact that MEFs are relatively
resistant to high transfection efficiencies, the following strategy was
devised. We have described previously (63) the characterization of a
GFP-Cav-1 chimera that behaves indistinguishably from wild-type Cav-1.
Transient transfection of this GFP-Cav-1 chimera or GFP alone, followed
by flow cytometric analysis of GFP-positive cells, allowed us to
compare the cell cycle responses of transfected and untransfected
cells. Fig. 6D summarizes the results from such an
experiment, whereas Fig. 6E shows the relative changes in S
phase between each group of cells. Note that expression of GFP alone
was insufficient to complement the cell cycle defect in Cav-1
The relative inability of Cav-1-deficient cells to control cellular
proliferation could be a result of lost functional interactions with
any of the signaling molecules previously implicated to interact with
caveolin-1. Based on several important observations, one of the most
attractive candidates in this process is the Ras/p42/44 MAP kinase
signaling cascade. Cholesterol depletion of cellular membranes, a
process that abolishes caveolae formation and Cav-1 membrane
localization (3), leads to a hyperactivation of the p42/44 MAP kinase
cascade (81). Antisense-mediated reductions of Cav-1 in NIH 3T3
fibroblasts leads to a similar hyperactivation of p42/44 MAP kinase
cascade (48). Finally, the ablation of Cav-1 levels in C. elegans by RNA interference produces a meiotic phenotype that
mirrors that of Ras activation (49). We attempted to recapitulate the
above results in MEFs derived from wild-type and knockout embryos. Fig.
7A shows that under conditions
of normal serum, serum starvation, and serum re-introduction
(situations that activate, depress, and reactivate the p42/44 kinase
cascade, respectively), Cav-1 deficiency does not affect the activation state of this cascade. We independently corroborated these results by
observing a lack of altered kinetics in Cav-1-deficient cells under
conditions of EGF-stimulated p42/44 phosphorylation (Fig. 7B). Therefore, we conclude that the hyperproliferative
effect of Cav-1 deficiency on primary cultured cells is independent of the Ras/p42/44 MAP kinase cascade.
Caveolin-1 levels have previously been shown to increase at the onset
of senescence in primary human fibroblasts (82). Therefore, we were
interested in assessing the levels of Cav-1 in wild-type MEFs and the
relative proliferation capacity of Cav-1 null MEFs at higher passages
of culture. In this way, we could observe both the senescence and
subsequent immortalization responses of Cav-1 knockout MEFs. By using a
standard 3T3 passaging protocol, two wild-type and two Cav-1 knockout
MEF cultures were serially propagated until immortalization. Fig.
8A shows the growth of these
cells at each passage. There was no observable differences in
senescence and subsequent immortalization in the Cav-1 null cells. We
additionally performed immunoblot analysis on lysates taken from
wild-type cells at passages 4 and 14 of culture. Fig. 8B
shows a dramatic increase in Cav-1 and -2 expression at the higher
passage, even though this phenomenon apparently does not appear to be
important for age-dependent cellular senescence and growth
arrest.
Caveolin-1-deficient Mice Show Lung Abnormalities, with Thickened
Alveolar Septa and Hypercellularity, and Exercise Intolerance--
As
a consequence of the hyperproliferative phenotype we observed with
Cav-1 null MEFs, we extensively examined our pathology specimens for
evidence of hypercellularity. Interestingly, the lung appeared
abnormal, and thin sections (1 µm) were cut to better evaluate this
possible phenotype.
Fig. 9A shows toluidine
blue-stained thin sections of lung parenchyma from wild-type and
caveolin-1-deficient animals. Note that in caveolin-1-deficient mice,
the alveolar spaces appeared significantly smaller or appeared
constricted, with thickened alveolar septa and hypercellularity. In
caveolin-1-deficient mice, reticulin staining showed increased basement
membranes in the thickened alveolar walls (Fig. 9B). There
was increased density and thickness of the basement membranes and loose
arrays of reticulin fibers. However, lung fibrosis was not detected
with trichrome staining (not shown). The presence of thickened alveolar
septa and hypercellularity was indeed confirmed by transmission
electron microscopy at low magnification. Montages of images taken of
wild-type and Cav-1 null alveoli are shown in Fig. 9C.
As these lung tissue sections appeared hypercellular, we next
quantitated the number of nuclei per high power field using hematoxylin and eosin-stained paraffin sections. Our results
indicate that Cav-1 null mice lung sections show an ~2-fold increase
in cellularity. This is consistent with our observation that
Cav-1-deficient MEFs proliferate ~2-fold faster and to higher
saturation densities than their wild-type counterparts (Fig.
9D).
As endothelial cells are one of the major cell types in lung tissue,
and they are known to normally express high levels of caveolin-1, we
examined the status of lung endothelial cells in both wild-type and
caveolin-1-deficient animals. Transmission electron microscopy
revealed that endothelial cells from Cav-1 null mice lack caveolae,
whereas their normal counterparts in wild-type mice showed abundant
caveolae (Fig. 10A). We also
used antibodies to VEGF-R (Flk-1) as a marker for endothelial cells. Immunostaining of paraffin sections with anti-VEGF-R revealed that the
number of lung endothelial cells were increased in Cav-1 null animals.
For example, in wild-type animals, we routinely observed ~1-2
VEGF-R-positive endothelial cells per 60× field, whereas in
Cav-1-deficient animals there were ~6-10 VEGF-R-positive endothelial
cells per 60× field. Also, in Cav-1-deficient animals, the
VEGF-R-positive endothelial cells were sometimes present in discrete
clusters, i.e. reminiscent of a focus of cellular growth. Two representative images for each genotype are shown in Fig. 10,
panels B and C.
The Ki67 "proliferation" antigen is a nuclear protein that is
highly expressed in proliferating cells (late G1, S,
G2, and M phases of the cell cycle) and is undetectable in
cells in the G0 phase of the cell cycle (83, 84). Fig.
11 shows that Ki67 immunoreactivity is
dramatically increased in lung tissue sections from Cav-1 null mice, as
compared with wild-type controls. This is consistent with the idea that
a lack of caveolin-1 can lead to hyperproliferation, as we have shown
with Cav-1 null MEFs in culture (Fig. 6A).
To grossly assess the possible physical consequences of these lung
abnormalities, we examined the exercise tolerance of wild-type and
Cav-1-deficient mice. For this purpose, we subjected these animals to a
"swimming test" (see "Experimental Procedures"). Fig.
12 shows that wild-type animals were
able to swim for up to 40 min, whereas Cav-1-deficient animals only
swam for ~10 min. Thus, Cav-1-deficient mice clearly show exercise
intolerance when compared with their wild-type littermates.
Caveolin-1-deficient Mice Show Abnormal Vasoconstriction and
Vasorelaxation Responses--
Caveolin-1 is highly expressed in
endothelial cells where caveolae are abundant. In addition, in
vitro studies have shown that caveolin-1 functions as a tonic
inhibitor of eNOS (85-87). Thus, we next assessed the vascular tone of
isolated mouse aortic rings by using an established physiological
method that measures tension in response to vasoconstriction or vasorelaxation.
For this purpose, we employed phenylephrine (PE; an
As shown in Fig. 13, aortic rings
isolated from the Cav-1 null mice were significantly different from
their wild-type counterparts in all parameters examined. The results of
a representative experiment on an aortic ring from each genotype (WT
versus KO) are shown in Fig. 13A. As illustrated,
PE was first used to elicit a contractile response. Upon achieving
steady-state, relaxation was induced by adding acetylcholine (Ach) in
gradually increasing doses (from 10
There are several important observations to note. 1) The steady-state
maximal tension response to PE in the wild-type aortic rings was nearly
2-fold greater than that observed for the Cav-1 null aortic rings (Fig.
13B, p < 0.05). It should be noted that over the same experimental time course, there was a less than 10%
variation in tension development in wild-type and Cav-1 null aortic
rings. 2) Although Ach induced a concentration-dependent relaxation response in aortic rings from both wild-type and Cav-1 null mice, significantly greater relaxation was observed in Cav-1 null
aortic rings at all Ach concentrations examined (Fig. 13C). 3) After addition of L-NAME, the steady-state contractile
response in the continuing presence of PE was significantly greater in aortic rings from both the wild-type and Cav-1 null mice; however, the
percent increase was significantly greater for the Cav-1 null mice (see
Fig. 13, A and B). Moreover, the steady-state
PE-induced contractile response after addition of L- NAME
in the Cav-1 null mice was indistinguishable from that observed in the
wild-type mice (Fig. 13, A and B).
In summary, we observed that Cav-1 null mice showed an impaired
vasoconstrictor response to PE. This impaired response was due to
increased eNOS activity as the proper vasoconstrictor response could be
restored by treatment with L-NAME, a well characterized NOS
inhibitor. Conversely, Ach-induced relaxation (a
NO-dependent phenomenon) of the aortic rings was clearly
potentiated by loss of caveolin-1 expression.
The discovery of caveolae by pioneering cell biologists in the
1950s added yet another major organelle to the cellular repertoire. Although the field remained relatively dormant for several decades, the
advent of caveolar biology occurred in 1992 with the discovery of Cav-1
as the marker protein for such microdomains. It has become clear over
the ensuing years that caveolar function is intimately liked to this
marker protein.
In this study, we describe the generation of a new mouse model
with an ablation of the gene encoding the Cav-1 protein. We show that
the cells derived from these mice are deficient in caveolae, as
determined ultrastructurally, thereby conclusively demonstrating that
Cav-1 is required for caveolae formation in primary cells. Surprisingly, despite a lack of such prevalent and conspicuous organelles, these mice are both viable and fertile.
Cav-1 null MEFs are perturbed in several other ways, however. First, we
show that Cav-2, a protein that is co-expressed, co-localizes, and
hetero-oligomerizes with Cav-1 is severely affected in Cav-1 null
cells. In the absence of caveolin-1, Cav-2 levels are reduced by
~95%. In addition, the remaining Cav-2 no longer targets to the
plasma membrane but instead is sequestered within the Golgi complex. We
further show that re-introduction of Cav-1 in these deficient cells can
rescue this effect by elevating Cav-2 levels and recruiting it to the
plasma membrane. Thus, the reduction of Cav-2 protein seems to be
independent of transcriptional repression and is rather mediated by
proteasomal degradation, as two inhibitors of the 26 S proteasome are
able to partially reverse this effect. Second, we demonstrate the Cav-1
null MEFs fail to endocytose a known caveolar ligand, i.e.
FITC-albumin, but show no defects in the uptake of FITC-transferrin, a
marker for clathrin-mediated endocytosis. Importantly, transient
expression of the caveolin-1 cDNA in Cav-1-deficient MEFs was
sufficient to restore the uptake of FITC-albumin. Third, we show that
Cav-1 null MEFs reveal a hyperproliferative phenotype. Cav-1 null MEFs
are able to grow approximately 2-fold faster during the exponential
phase and reach higher densities at confluence. These effects are due
to an increase of ~25-30% in the S phase fraction. Furthermore, we
demonstrate a reversion of this excess proliferation to wild-type
levels by re-expressing Cav-1 in knockout cells. However, we do not
find any evidence that the observed growth augmentation is due to a hyperactivation of the p42/44 MAP kinase cascade, a signaling pathway
reported by many investigators to be intimately linked to
caveolae/caveolin functioning. Furthermore, although we show that Cav-1
levels increase in higher passage cells (i.e. cells at or
near senescence), a deficiency in Cav-1 is not sufficient to expedite
immortalization in primary fibroblasts.
Caveolae are thought to form as a result of a local accumulation of
cholesterol, glycosphingolipids, and caveolin-1 (8, 88, 89). Caveolin-1
can bind cholesterol in vitro (8); also, Cav-1 is a major
protein bound to photoactivable forms of both cholesterol and
glycolipids in vivo (88, 90). Although in this study we have
demonstrated that physiological levels of Cav-1 protein are required
for caveolae formation (in corroboration of previous overexpression
studies), the mechanisms underlying this process remain entirely
unknown. Primarily, this is due to the fact that the Cav-1 protein is
not readily amenable to mutational analysis. Due to its ability to form
a large oligomeric complex with itself and with Cav-2 (11, 70, 72, 91),
its ability to coalesce into even larger macrostructures (62), its
binding to cholesterol and glycosphingolipids (8, 88-90), and its
membrane-spanning properties, any deletion/mutation of the protein can
confound an analysis of caveolae formation in numerous ways. For
example, baculovirus-mediated expression of Cav-1 proteins lacking
their oligomerization domain or C-terminal domains (i.e.
Cav-1 Based on the current study, the intricate dependence of Cav-2 on the
presence of Cav-1 is obvious. Cav-2 is present at astonishingly lower
levels (~5% of wild-type) in knockout tissues, is localized in the
Golgi compartment, and is degraded by the proteasomal pathway. However,
the degradation of Cav-2 is perhaps not entirely surprising. Rather
elaborate mechanisms of quality surveillance have developed at various
levels of the secretory pathway. Incompletely folded or assembled
proteins are often sequestered at the endoplasmic reticulum where they
are eventually degraded by the 26 S proteasome (reviewed in Refs. 92
and 93)). The few molecules that "escape" detection, traffic to the
Golgi where they can again be detected and re-routed to the endoplasmic
reticulum for degradation. Since Cav-2 cannot homo-oligomerize but
rather hetero-oligomerizes with Cav-1, it is possible that in the
absence of Cav-1, several critical hydrophobic regions remain exposed,
thereby affecting not only folding of Cav-2 but increasing the
probability of recognition by the proteasomal apparatus. To date,
however, much less is known about Cav-2 function than is for Cav-1
function. Cav-2 does not contain a scaffolding domain (the primary
proposed region of interaction between Cav-1 and signaling molecules)
and has rarely been implicated in signal transduction processes.
Although there is no overt reason to believe that its severely reduced
levels in Cav-1-deficient cells can compound any phenotypic analyses,
the fact remains that Cav-1 knockout mice are in effect deficient in
two caveolins. The generation of Cav-2 knockout mice will ultimately
resolve this issue.
Based on the growth curves of MEFs and the corresponding cell
cycle analyses, we have shown in this study that a deficiency in Cav-1
leads to higher proliferation rates. This is the first direct
demonstration of a relationship between Cav-1 and the cell cycle under
physiological circumstances. Our results corroborate previous data
showing that Cav-1 overexpression can reduce cell proliferation and/or
abrogate anchorage-independent growth in several cancer cell lines (29,
31, 61). Surprisingly, we found that this proliferation is not due to a
hyperactivation of the p42/44 MAP kinase cascade, a signaling pathway
that had been shown in numerous ways to be reciprocally regulated by
Cav-1 (28, 48, 49, 61, 81, 94).
The main difference in our study lies in the use of embryonic
fibroblasts, instead of immortalized and transformed cell lines. This
type of discrepancy is not unusual and necessitates the analysis of
proteins in primary culture systems, such as this one. For example, the
ras oncogene, a potent transforming agent when used in
immortalized cells (95, 96), actually induces cell cycle arrest and
premature senescence in MEFs (97, 98). In the same way, the mechanistic
explanation of the excessive proliferation of Cav-1-deficient primary
cells may depend on other signal transduction processes. Although
several other pro-proliferative signaling molecules have been
shown to be regulated by Cav-1, further work is required to determine
more closely their physiological relevance. Analysis of these pathways
in knockout versus wild-type MEFs will eventually shed light
on the detailed mechanism for the observed hyperproliferation. In
addition, although we have not noticed any spontaneous tumors in Cav-1
null mice at 9 months of age, they may have a higher susceptibility
than wild-type mice to tumors induced either chemically or by breeding
with other tumor-prone mice (e.g. the p53- or
INK4a-deficient mice (79, 99)).
It should be noted that the lack of spontaneous tumor formation and the
modest proliferation defect observed in the Cav-1 null setting is
reminiscent of several previously described mice lacking inhibitory
cell cycle proteins. For example, mice deficient in the
cyclin-dependent kinase inhibitor, p21 (which functions in
G1 phase progression (100, 101) and is a major target of p53 (102)), do not develop tumors, and their MEFs display only a modest
proliferative advantage over the wild-type counterparts (65). The
ablation of the p19INK4d, a member of the INK4 (inhibitor
of cyclin-dependent kinase 4/6) family of proteins, also
does not predispose mice to tumors or cell cycle defects (103). A
deficiency of p15INK4b predisposes only a small percentage
of mice to tumors (104). Although mice lacking p27, another important
cyclin-dependent kinase inhibitor, can develop pituitary
tumors, cells derived from these mice only show subtle cell cycle
defects (105, 106). In many instances, the lack of an overt phenotype
can be due to compensatory proteins (i.e. compensation
derived from parallel-acting cell cycle and checkpoint control
pathways). We assessed a possible up-regulation of caveolin-3, the
highly homologous muscle-specific caveolin family member, in several
Cav-1 null tissues, and we found it to remain unperturbed. Therefore,
if there are any counter-regulatory mechanisms involved, they are
independent of the caveolin gene family.
In accordance with the hyperproliferative phenotype we observed with
Cav-1 null MEFs, the lung parenchyma of Cav-1 null animals appeared
hypercellular with thickened alveolar septa. Quantitation of the number
of nuclei per high power field using hematoxylin-eosin-stained paraffin
sections revealed an ~2-fold increase in cellularity. The Ki67
"proliferation" antigen is a nuclear protein that is highly
expressed in proliferating cells and is undetectable in cells in the
G0 phase of the cell cycle (83). Interestingly, Ki67
immunoreactivity was also dramatically increased in lung tissue
sections from Cav-1 null mice. This is consistent with our observation
that Cav-1-deficient MEFs proliferate faster and to higher saturation
densities. We also found that the number of VEGF-R-positive lung
endothelial cells were increased in Cav-1 null animals. These
VEGF-R-positive endothelial cells were sometimes present in discrete
clusters, i.e. reminiscent of a focus of cellular growth.
Transmission electron microscopy revealed that lung endothelial cells
from Cav-1 null mice lack caveolae, whereas their normal counterparts
in wild-type mice showed abundant caveolae. Taken together, these
findings are consistent with the idea that a lack of caveolin-1
expression and caveolae organelles can lead to hyperproliferation in
certain cell types. These lung abnormalities appeared to have physical
consequences, as the Cav-1-deficient mice clearly showed exercise intolerance.
Several in vitro studies employing recombinant expression
and peptide-based analyses have strongly suggested that caveolin-1 can
function as an endogenous negative regulator of eNOS, by providing tonic inhibition of eNOS enzymatic activity (85-87). Here, by using isolated mouse aortic rings, we evaluated the effect of loss of caveolin-1 expression on the vasoconstrictor actions of PE, an Our current studies directly support the findings of Sessa and
colleagues (107) who used a chimeric peptide containing a cellular
internalization signal and the caveolin-1 scaffolding domain (CSD,
residues 82-101). By using mouse aortic rings from wild-type mice,
they showed that the CSD could potentiate the vasoconstrictor response
to PE and that the CSD could inhibit Ach-induced relaxation of the
blood vessel. The actions of the CSD could be mimicked by using the NOS
inhibitor, L-NAME. In further support of the specificity of
the CSD, a scrambled peptide version of the CSD had no activity in this
assay system.
In summary, by using targeted disruption of the caveolin-1 gene in
mice, we have shown that Cav-1 expression is required to stabilize the
Cav-2 protein product, to mediate the caveolar endocytosis of specific
ligands, to negatively regulate the proliferation of certain cell
types, and to provide tonic inhibition of eNOS activity in endothelial
cells. The availability of a viable Cav-1-deficient mouse model will
allow investigators to critically evaluate the many proposed functions
of the Cav-1 protein and caveolae organelles in vivo.
We thank Drs. David Baltimore and Anthony J. Koleske for participating in the early stages of this study. We also
thank Dr. Roberto Campos-Gonzalez for donating mAbs directed against
caveolin-1, caveolin-2, and caveolin-3; Dr. Tony Karnezis for help with
cell cycle analysis; and Dr. Michael Cammer for help with photography.
After this paper was published online
(July 16, 2001), another report appeared online describing the
generation of caveolin-1 knockout mice (August 9, 2001; Ref. 108). In
accordance with our results, these authors demonstrate that loss of
caveolin-1 expression prevents caveolae formation, induces a loss of
caveolin-2 expression, and causes lung hypercellularity and vascular
defects, as well as exercise intolerance. However, these authors did
not examine the phenotypic behavior of MEFs in culture or further study
the hyperproliferative phenotype. In addition, they did not examine the
endocytic behavior of caveolin-1 deficient cells. Both reports (our
paper and Ref. 108) conclude that the observed caveolin-1 null
phenotype is consistent with the idea that caveolin-1 is a negative
regulator of signal transduction and that caveolin-1 may indeed
function as a tumor suppressor gene.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.This
work was supported in part by grants from the
National Institutes of Health, the Muscular Dystrophy Association, the
American Heart Association, and the Komen Breast Cancer Foundation (to M. P. L.).
§
Supported by a National Institutes of Health (NIH) Medical
Scientist Training Grant T32-GM07288.
***
Supported by NIH Grants R01-CA70897, R01-CA86072, and R01-CA75503,
the Komen Breast Cancer Foundation, and the Department of Defense.
Recipient of a Hirschl/Weil-Caulier Career Scientist Award.
Published, JBC Papers in Press, July 16, 2001, DOI 10.1074/jbc.M105408200
2
B. Razani, J. A. Engelman, X. B. Wang, W. Schubert, X. L. Zhang, C. B. Marks, F. Macaluso,
R. G. Russell, M. Li, R. G. Pestell, D. Di Vizio, H. Hou,
Jr., B. Knietz, G. Lagaud, G. J. Christ, W. Edelmann, and M. P. Lisanti, unpublished observations.
The abbreviations used are:
Cav-1, caveolin-1;
Cav-2, caveolin-2;
Cav-3, caveolin-3;
eNOS, endothelial nitric-oxide
synthase;
FITC, fluorescein isothiocyanate;
L-NAME, nitro-L-arginine methyl ester;
MAP, mitogen-activated
protein;
EGF, epidermal growth factor;
EGF-R, EGF receptor;
VEGF-R, vascular endothelial growth factor receptor;
mAb, monoclonal antibody;
kb, kilobase pair;
bp, base pair;
PCR, polymerase chain reaction;
MEF, mouse embryonic fibroblast;
PAGE, polyacrylamide gel electrophoresis;
GFP, green fluorescent protein;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
PFUs, plaque-forming units;
KO, knockout;
NO, nitric oxide;
NOS, nitric-oxide synthase;
PE, phenylephrine;
Ach, acetylcholine;
pAb, polyclonal antibody;
Mes, 4-morpholine-ethanesulfonic acid;
ES, embryonic stem;
CSD, caveolin-1
scaffolding domain.
Caveolin-1 Null Mice Are Viable but Show Evidence of
Hyperproliferative and Vascular Abnormalities*
§,,,,,
,***,,
Department of Molecular Pharmacology and The
Albert Einstein Cancer Center, the ¶ Analytical Imaging Facility,
the Department of Pathology and Institute for Animal Studies,
the ** Department of Pathology,
Departments of Developmental and Molecular
Biology and Medicine, and The Albert Einstein Cancer Center, the
§§ Department of Cell Biology and The Albert
Einstein Cancer Center, the ¶¶ Departments of Urology,
Physiology, and Biophysics, Institute for Smooth Muscle Biology, The
Albert Einstein College of Medicine, Bronx, New York 10461
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) mouse
model, using standard homologous recombination techniques, to assess
the role of caveolin-1 in caveolae biogenesis, endocytosis, cell
proliferation, and endothelial nitric-oxide synthase (eNOS) signaling.
Surprisingly, Cav-1 null mice are viable. We show that these mice lack
caveolin-1 protein expression and plasmalemmal caveolae. In addition,
analysis of cultured fibroblasts from Cav-1 null embryos reveals the
following: (i) a loss of caveolin-2 protein expression; (ii) defects in
the endocytosis of a known caveolar ligand, i.e.
fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative
phenotype. Importantly, these phenotypic changes are reversed by
recombinant expression of the caveolin-1 cDNA.
Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an
increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted,
endothelial cells from Cav-1 null mice lack caveolae membranes.
Finally, we examined eNOS signaling by measuring the physiological
response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity
can be blunted by using a specific NOS inhibitor,
nitro-L-arginine methyl ester. These findings are in
accordance with previous in vitro studies showing that
caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1
expression is required to stabilize the caveolin-2 protein product, to
mediate the caveolar endocytosis of specific ligands, to negatively
regulate the proliferation of certain cell types, and to provide tonic
inhibition of eNOS activity in endothelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin mAb TUB-2.1 and anti--actin mAb AC-15 (Sigma). A
variety of other reagents were purchased commercially as follows: cell
culture reagents and the LipofectAMINE liposomal transfection reagent
were from Life Technologies, Inc.
-phage genomic library (53, 54) by using probes
corresponding to the murine Cav-1 cDNA. The genomic organization of
the locus was determined by subcloning portions of these genomic inserts into the vector pBS-SK+ (Stratagene) and using
Southern blotting to determine a detailed restriction map of the region
(55). One of the genomic clones (containing the first and second exons
of Cav-1) was used to construct the targeting vector. Briefly, a 2.7-kb
NotI-EcoRI fragment that is immediately 5' to the
first exon and a 2.1-kb BamHI-BamHI fragment that
is immediately 3' to the second exon of the cav-1 gene were used to flank the NEO cassette in the targeting vector pGT-N29 (New
England Biolabs) (as shown in Fig. 1).
clones produced an
8.0-kb wild-type and a 5.5-kb knockout band (PstI digest) or
a 10.0-kb wild-type and a 4.0-kb knockout band (XbaI digest)
(as shown in Fig. 1). Four Cav-1+/ ES clones were microinjected into
C57BL/6 blastocysts, and three gave rise to male chimeras with a
significant ES cell contribution (as determined by an Agouti coat
color). By mating with C57BL/6 females and genotyping of offspring tail
DNA via Southern and PCR analysis, germ line transmission was confirmed for two separate clones (Fig. 1). F1 male and female heterozygous animals were interbred to obtain Cav-1-deficient animals. To facilitate the genotyping of all future mice, we also devised a 3-primer PCR-based
screening strategy. The wild-type specific forward primer was derived
from Cav-1 exon 2 (5'-GTGTATGACGCGCACACCAAG-3'); the knockout-specific
forward primer was derived from the neomycin cassette
(5'-CTAGTGAGACGTGCTACTTCC-3'), and the common reverse primer was
derived from Cav-1 intron 2 (5'-CTTGAGTTCTGTTAGCCCAG-3'). PCR
conditions were 95 °C/5 min, 35 cycles of (95 °C/1 min,
56 °C/1 min, 72 °C/1 min 20 s) and then 72 °C/7 min,
which resulted in a ~650-bp wild-type band and a ~330-bp knockout band.
15 min), the rings were preconstricted
with a submaximal concentration of PE (10 µM), and
concentrations of Ach (10
8 to 104
M) or L-NAME (100 µM) were
injected when the PE-contractile response achieved steady state (5 min).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (44K):
[in a new window]
Fig. 1.
Targeted disruption of the cav-1
gene produces a null mutation. A, the
Cav-1 locus (containing the first two exons) and the targeting
construct (containing the neomycin (NEO) cassette with
flanking segments homologous to the locus) are shown in schematic
format. The transcriptional orientation of neomycin cassette and the
Cav-1 locus are delineated by arrows. Note that
homologous recombination would eliminate a 2.2-kb genomic segment
containing Cav-1 exons 1 and 2 and introduce two new restriction sites
(PstI and XbaI), both of which can be used to
screen for positive ES clones. The 1.1-kb
SacI-XbaI probe used for Southern blot analysis
is located 3' of the targeting vector, as shown. B, Southern
blot analysis of two positive and wild-type ES cell clones. The probe
used is a 1.1-kb SacI-XbaI fragment located 3' of
the targeting vector shown in A. The 4-kb (XbaI
digest) or 5.5-kb (PstI digest) bands signify appropriate
targeted disruption of the Cav-1 locus. C, Southern
blot analysis of PstI-digested tail DNA from the offspring
of Cav-1 heterozygote inter-crosses. The absence of a wild-type 8.0 kb
signifies the generation of the Cav-1 knockout animal. An alternative
PCR-based strategy used to determine the genotype of animals is also
shown. The absence of a 500-bp wild-type band signifies the generation
of a Cav-1 knockout animal. D, lysates from three tissues
with varying levels of Cav-1 expression (fat, lung, and heart) were
prepared from mice of all three genotypes. 30 µg of protein was
loaded in each lane, subjected to SDS-PAGE, and immunoblotted with
anti-Cav-1 mAb (clone 2297). A longer exposure of the same blot fails
to detect any Cav-1 expression in knockout tissues. Equal protein
loading was assessed using the anti- -tubulin mAb (clone 2.1).
E, MEFs were derived from 13.5-day-old embryos. Two
independent embryos of each genotype were selected for subsequent
analysis. 20 µg of cell lysate was loaded in each lane, subjected to
SDS-PAGE, and immunoblotted with anti-Cav-1 mAb (clone 2297).
49.2%, Cav-1/ 25.6%;
n = 305 animals).
-tubulin is shown as an equal
protein loading control.
-isoform and the shorter 146-amino acid -isoform (52)). 2) The
ablation of the Cav-1 locus in only one chromosome, as in the
heterozygous animals, is sufficient to reduce protein levels by
approximately half. 3) Although Cav-1 is expressed in the cardiac tissue of wild-type and heterozygous mice (Fig. 1D), Cav-1
expression is derived from endothelial and fibroblastic cells within
the heart and not the cardiac myocytes themselves (10, 12).
/
Mice--
Caveolin-1 null mice are both viable and fertile. We
initially established a large cohort consisting of mice from all
genotypes, the eldest of which are now 9 months of age. Although no
overt phenotypic abnormalities (including tumors) have been detected, only two mice have thus far died of unknown causes, both of which were
Cav-1-deficient (autolysis prevented a pathological work-up). Follow-up
of this cohort in the coming months will establish whether Cav-1
deficiency can precipitate tumorigenesis and/or a reduction in life
span. A routine histopathological examination of Cav-1 null mice at
4-5 months of age (n = 4 male, n = 4 female) failed to show any evidence of abnormalities, with the
exception of lung tissue (see below). We have noticed, however, that
although in the first few months of life there is no overt difference
between wild-type and knockouts, older Cav-1-deficient mice are more
likely to be smaller in size than their wild-type littermates.
-cyclodextrin) ablates caveolar structures (68). 2)
Caveolin-1 is a cholesterol-binding protein, possibly facilitating the
concentration of the critical mass of cholesterol required for
invagination (8, 69). 3) Down-regulation of caveolin-1 in Ha-Ras and
v-Abl-transformed NIH 3T3 fibroblasts or by antisense strategies
results in a concomitant reduction of caveolae at the membrane (28,
48). 4) Overexpression of caveolin-1 in a lymphocytic cell line, cells
that do not endogenously express the protein, is sufficient to allow
the formation of caveolae (6).
/ embryos were cultured to near
100% confluency, conditions that have been shown to result in optimal
caveolin-1 expression and caveolae formation. Standard transmission
electron microscopy was used to visualize the plasma membrane (Fig.
2). While wild-type MEFs have numerous uniformly sized caveolae, the Cav-1-deficient cells are conspicuously devoid of caveolae. An exhaustive search of the plasma membrane from
Cav-1 knockout MEFs failed to show any invaginations resembling caveolae. However, we did observe the occasional clathrin-coated pit
(invaginations typically 5 times larger than caveolae) in MEFs of both
genotypes (data not shown), indicating that their number or presence is
not affected by a Cav-1 deficiency.
View larger version (41K):
[in a new window]
Fig. 2.
A deficiency in Cav-1 is sufficient to
disrupt caveolae formation. Cav-1 wild-type and knockout MEFs were
grown to near-confluence on 60-mm dishes and prepared for transmission
electron microscopy as described under "Experimental Procedures."
All analyses were performed at × 16,000 magnification (for ease
of view, images shown are further magnified to × 43,500). The
plasma membranes of numerous cells were exhaustively scanned for
caveolae, defined as uniform 50-100-nm flask-shaped membrane
invaginations. The scale bar is shown at the lower left
corner (bar, 150 nm).
/) tissues examined (Fig.
3A). A longer exposure of the
same blots shows that caveolin-2 is in fact expressed, albeit at ~5%
of wild-type levels. Cav-3 levels remained unperturbed, however,
and showed the expected expression pattern (i.e.
muscle-specific expression) (Fig. 3A). A -tubulin
immunoblot indicates equal protein loading in all lanes (Fig.
3A). More importantly, in heterozygous animals, Cav-2 levels
remain unperturbed despite the reduction in caveolin-1 (Fig. 1,
D and E). Similar results were observed in
Cav-1-null MEFs (Fig. 3B). Therefore, it seems that the
absence, but not the reduction of Cav-1, is sufficient to cause a
near-total deficiency in Cav-2.
View larger version (47K):
[in a new window]
Fig. 3.
Phenotypic behavior of the caveolin-2 protein
in Cav-1 null cells. A and B, the absence of
Cav-1 leads to severely reduced caveolin-2 levels. A, 30 µg of lysates from tissues used in Fig. 1D were loaded in
each lane, subjected to SDS-PAGE, and immunoblotted with anti-Cav-2 mAb
(clone 26) and anti-Cav-3 mAb (clone 65). A longer exposure of the same
blot shows that Cav-2 is expressed in Cav-1 knockout mice, albeit at
extremely reduced levels (~5% of wild-type). Equal protein loading
was assessed using the anti- -tubulin mAb (clone 2.1). Note that the
expression of caveolin-3 remains muscle-specific. B, 20 µg
of lysates from MEFs used in Fig. 1E were subjected to
SDS-PAGE and immunoblotting with anti-Cav-2 mAb (clone 26).
C and D, caveolin-2 is displaced from caveolae
microdomains and localizes to a perinuclear compartment in Cav-1 null
cells. C, lung tissue from wild-type and knockout mice was
homogenized thoroughly in lysis buffer containing 1% Triton X-100 and
subjected to sucrose gradient centrifugation, a procedure that
separates caveolar microdomains from other cellular constituents (60).
Twelve fractions, of which fractions 4-5 and 8-12 are considered of
caveolar and non-caveolar origin, respectively, were collected and
subjected to SDS-PAGE. Immunoblotting with anti-Cav-1 mAb (clone 2297)
and anti-Cav-2 mAb (clone 26) was used to determine the localization of
Cav-1 and Cav-2 in these gradient fractions. Note that the Western blot
showing the distribution Cav-2 in Cav-1 null lung tissue is overexposed
to illustrate the distribution of residual Cav-2, as Cav-2 protein
levels are down-regulated to ~5% of normal levels in Cav-1 null
animals. D, formaldehyde-fixed wild-type and knockout MEFs
were doubly immunostained with anti-Cav-1 pAb (N20) and anti-Cav-2 mAb
(clone 26). Bound 1° antibodies were visualized with distinctly
tagged 2° antibodies (see "Experimental Procedures"). Note the
perinuclear/Golgi localization of caveolin-2 in Cav-1 knockout
MEFs.
View larger version (16K):
[in a new window]
Fig. 4.
Rescue of caveolin-2 expression in Cav-1 null
cells. A and B, caveolin-2 localization and
expression can be rescued by recombinantly expressing Cav-1 in Cav-1
knockout cells. A, Cav-1 / MEFs were transiently
transfected with the full-length cDNA encoding Cav-1. Thirty six
hours post-transfection, cells were formaldehyde-fixed and doubly
immunostained with anti-Cav-1 pAb (N20) and anti-Cav-2 mAb (clone 26)
as in Fig. 3D. The image shown is that of a
Cav-1-transfected cell. B, in order to obtain a higher
efficiency of Cav-1 expression in Cav-1/ MEFs, an adenoviral
strategy was used (34). The Cav-1 and GFP adenoviruses (Ad-Cav-1 and
Ad-GFP) contain a tet-responsive promoter that can only be induced by
co-infection of cells with the tet-transactivator (Ad-tTA) "tet off" system. MEFs were co- infected with Ad-Cav-1 and Ad-tTA at varying titers (100,400, and
1000 PFUs/cell). Controls included infection with Ad-Cav-1 alone (1000 PFUs/cell) or co-infection with Ad-GFP and Ad-tTA (1000 PFUs/cell).
C and D, in the absence of Cav-1, caveolin-2 is
partially degraded through the proteasomal pathway. C,
Cav-1/ MEFs grown to near-confluence were treated with the
proteasomal inhibitors MG-132 (1 µM) and MG-115 (1 µM) for a series of time points (8, 16, and 24 h) or
with vehicle (Me2SO). Whole cell lysis and subsequent
SDS-PAGE allowed the comparison of Cav-2 levels with that of wild-type
MEFs. D, Cav-1(/) MEFs, grown to near-confluence, were
treated with the lysosomal inhibitors chloroquine (50 µM)
and NH4Cl (10 mM) or vehicle
(Me2SO) for 24 h. Whole cell lysis and subsequent
SDS-PAGE allowed comparison of Cav-2 levels with that of wild-type
MEFs. Equal protein loading was assessed in A and
B with anti--actin mAb (clone AC-15).
-actin is used as an equal protein loading
control. Note that expression of equal titers of the irrelevant protein
GFP have no effect on Cav-2 expression (Fig. 4B).
View larger version (30K):
[in a new window]
Fig. 5.
Caveolin-1-deficient MEFs show defects in the
endocytosis of albumin but not transferrin. A,
wild-type and Cav-1 null MEFs were incubated in normal medium
supplemented with FITC-albumin (10 µg/ml). After 5, 15, and 30 min at
37 °C, cells were formaldehyde-fixed and visualized by fluorescence
microscopy. Note that Cav-1 null MEFs fail to internalize FITC-albumin.
Left panels, wild-type MEFs (WT); right
panels, Cav-1 null MEFs (KO). B, Cav-1 null
MEFs were transiently transfected with the full-length cDNA
encoding caveolin-1. Thirty six hours post-transfection, cells were
allowed to continuously endocytose FITC-albumin for 30 min, as in
A. Cells were then formaldehyde-fixed and immunostained with
anti-Cav-1 IgG (rabbit pAb N-20). Note that in cells recombinantly
expressing the caveolin-1 cDNA that uptake of FITC-albumin is
clearly restored (left panels). However, untransfected cells
in the same cell population failed to internalize FITC-albumin
(right panels). Upper panels, FITC-albumin
uptake; middle panels, Cav-1 immunostaining; lower
panels, phase images. C, wild-type and Cav-1 null MEFs
were incubated in normal medium supplemented with FITC-transferrin (10 µg/ml). After 5, 15, and 30 min at 37 °C, cell were
formaldehyde-fixed and visualized by fluorescence microscopy. Note that
both wild-type and Cav-1 null MEFs internalize FITC-transferrin,
without any apparent differences. Left panels, wild-type
MEFs (WT); right panels, Cav-1 null MEFs
(KO).
/
MEFs--
In the past decade, several important observations have
implicated Cav-1 as a negative regulator of signaling pathways involved in pro-proliferative responses; as a result, Cav-1 has been suggested to function as a putative tumor suppressor. Caveolin-1 levels are
drastically reduced upon oncogenic transformation of several cell lines
(28-30, 33-37, 61). More importantly, overexpression of Cav-1 is
sufficient to abrogate anchorage-independent growth in these
transformed cells (29-32, 34, 61). Cav-1 also interacts with
and negatively regulates several pro-proliferative signaling molecules,
such as certain receptor-tyrosine kinases (including EGF-R,
platelet-derived growth factor receptor, and Neu), Ha-Ras, c-Src, and
phosphatidylinositol 3-kinase, among others (17, 18, 24-27). Finally,
down-regulation of Cav-1 by an antisense strategy in NIH-3T3
fibroblasts leads to a tumorigenic phenotype, enabling the these cells
to grow in soft agar and nude mice (48).
View larger version (27K):
[in a new window]
Fig. 6.
Growth properties and cell cycle
analysis of Cav-1 /
MEFs. A-C, Cav-1-deficient MEFs proliferate
faster and have increased S phase fractions. A, six
independent MEF cultures, consisting of two Cav-1+/+, two Cav-1+/,
and two Cav-1/ genotypes, were plated at a density of 15 × 103 cells/dish on a series of 35-mm dishes. Duplicate
plates from each MEF culture were then counted each day for a period of
10 days. Cell numbers at the indicated time points (Days
1-10) are the average of duplicate plates. B,
wild-type and knockout MEFs were plated at a density of 2 × 105 in 60-mm dishes. At the exponential phase of growth
they were ethanol-fixed, stained with propidium iodide, and analyzed by
flow cytometry for the G0/G1, S, and
G2/M phases of the cell cycle. Parameters indicated are the
percentage of cells in each phase of the cell cycle out of a total of
10,000 cells analyzed. C, wild-type and knockout MEFs were
plated at a density of 2 × 105 in 60-mm dishes. Upon
adherence to the plates they were supplemented with 1 µCi/ml
[3H]thymidine and cultured overnight. Incorporated
tritium was determined by scintillation counts of alkaline lysed
cells/trichloroacetic acid-precipitated DNA (see "Experimental
Procedures"). Data shown are the average and standard deviation of
counts from five plates. D and E, the increased S
phase in Cav-1 null cells can be rescued by re-introduction of Cav-1.
D, Cav-1 knockout MEFs were transfected with either GFP
alone or GFP-Cav-1. Untransfected plates of wild-type and knockout
cells were similarly cultured. Thirty six hours
post-transfection, all cells were trypsinized, live-stained with
Hoechst 33342, and subjected to flow cytometry. Transfected
(i.e. GFP-positive) cells were distinguished from
non-transfected cells (GFP-negative) by using appropriate fluorescence
channels. Cell cycle parameters for all populations were analyzed as shown. The
indicated numbers represent the percentages of cells in each phase out
of a total of 10,000 cells. E, the percentage of increase in
S phase fraction as compared with wild-type MEFs for all transfected
and untransfected cell populations analyzed in D.
/ cells. Fig. 6B shows a representative flow cytometric analysis of the cell cycle of wild-type and knockout MEFs in
their exponential phase of growth. Note that there is a reproducible
increase of ~25-30% in the S phase population of the
Cav-1-deficient cells, with concomitant decreases in
G0/G1 and to a lesser extent G2/M.
We quantitatively assessed this S phase increase in another way,
i.e. by calculating the incorporation of
[3H]thymidine in randomly cycling wild-type and knockout
MEFs. Fig. 6C shows the rates of S phase increase in line
with the cell cycle analysis above (with increases in thymidine
incorporation of ~25%). Interestingly, an increase in S phase of
similar magnitude is observed in MEFs harboring a deletion of several
classically known cell cycle inhibitors, such as
p16INK4A/p19ARF and Rb (79, 80).
/
MEFs, whereas expression of GFP-Cav-1 successfully complemented this
defect. Thus, we conclude that the observed increase in cell
proliferation in Cav-1 null MEFs reflects a decrease in the number of
cells in the G0/G1 phase of the cell cycle,
with a corresponding proportional increase in the number of cells in the S phase.
View larger version (44K):
[in a new window]
Fig. 7.
Hyperproliferation of Cav-1-deficient MEFs is
not related to hyperactivation of the p42/44 MAP kinase cascade.
A, MEFs derived from embryos of all three genotypes were
cultured in triplicate at 70-80% confluence. One set of cultures was
grown continuously under normal serum conditions (DMEM, 10% FBS), and
the other two were serum-starved for 12 h. Serum (10% FBS) was
reintroduced into only one set of the serum-starved cells for 40 min.
Cells were lysed, subjected to SDS-PAGE, and immunoblotted with
phospho-specific anti-p42/44 pAb, anti-p42/44 antibody, and anti-Cav-1
mAb (2297). B, wild-type and knockout MEFs were plated in a
series of 60-mm dishes, serum-starved for 12 h, and stimulated
with EGF (50 ng/ml) for either 0, 5, 10, 20, or 40 min. Cells were
lysed, subjected to SDS-PAGE, and immunoblotted for the same antibodies
used in A.
View larger version (19K):
[in a new window]
Fig. 8.
Although Cav-1 levels increase at higher
passages of culture, the absence of Cav-1 does not impart an advantage
in senescence and subsequent immortalization. A, four
independent MEF cultures (two wild-type and two knockout) were
propagated according to the 3T3 protocol (i.e. 3 × 105 cells were plated in 60-mm dishes every 3 days). At
each passage (2-16), the combined cell counts from three 60-mm dishes
were determined for each MEF culture by hemocytometer. B,
wild-type and Cav-1 knockout MEFs were propagated according to the 3T3
protocol (i.e. 3 × 105 cells were plated
in 60-mm dishes every 3 days). Cell lysates were prepared from Passage
4 and Passage 14 MEFs, subjected to SDS-PAGE, and immunoblotted with
anti-Cav-1 mAb (clone 2297), anti-Cav-2 mAb (clone 65), or
anti- -actin mAb (clone AC-15).
View larger version (59K):
[in a new window]
Fig. 9.
Caveolin-1-deficient mice show lung
abnormalities, with thickened alveolar septa and
hypercellularity. A, light microscopy.
One-µm sections of lung parenchyma were cut and stained
with toluidine blue. Note that in caveolin-1-deficient mice the
alveolar spaces appeared significantly smaller or appeared constricted,
with thickened alveolar septa and hypercellularity. These images were
acquired with a 60× objective. Upper panel, wild-type mice
(WT); lower panel, Cav-1 null mice
(KO). B, reticulin staining. Wild-type and Cav-1
null mouse lung tissue sections were subjected to reticulin staining
and examined using a Zeis Axiophot with a 20× objective. Note that in
caveolin-1-deficient mice, reticulin staining showed increased basement
membranes in the thickened alveolar walls. There was increased density
and thickness of the basement membranes and loose arrays of reticulin
fibers. Upper panel, wild-type mice (WT);
lower panel, Cav-1 null mice (KO). C,
transmission electron microscopy. Lung tissue samples were processed
for electron microscopy, as detailed under "Experimental
Procedures." Images were acquired at low magnification (× 2,000), and montages were assembled to illustrate the detailed
morphology of the alveolar architecture. Note the presence of thickened
alveolar septa and hypercellularity in Cav-1-deficient mice.
Upper panel, wild-type mice (WT);
lower panel, Cav-1 null mice (KO). D,
quantitation of nuclei. Wild-type and Cav-1 null mouse lung tissue
sections stained with hematoxylin and eosin were examined using
a Zeis Axiophot. Using a 20× objective, six random 0.5-mm2
fields were photographed for each genotype, and all the nuclei within
those regions were manually tabulated using a hand-held counter. Note
that lung sections from Cav-1 null mice show an ~2-fold increase in
overall cellularity.
View larger version (60K):
[in a new window]
Fig. 10.
Lung endothelial cells in Cav-1-deficient
mice lack caveolae and are more numerous. A,
transmission electron microscopy. Lung tissue samples were processed
for electron microscopy as detailed under "Experimental
Procedures." Images were acquired at high magnification (× 33,000).
Note that endothelial cells from Cav-1 null mice lack caveolae, whereas
their normal counterparts in wild-type mice showed abundant caveolae
(see arrows). Endothelial cells were identified by their
proximity to red blood cells (RBC) that are electron dense
(due to their high iron content) and appear black.
Similarly, type I pneumocytes also lacked caveolae in the Cav-1 null
mice (not shown). Upper panel, wild-type mice
(WT); lower panel, Cav-1 null mice
(KO). B and C, immunostaining. Lung
paraffin sections from wild-type and caveolin-1-deficient mice were
immunostained with an endothelial marker, anti-VEGF-R (Flk-1) IgG.
Bound primary antibodies were detected with a fluorescently labeled
secondary antibody. Arrows point at VEGF-R-positive
endothelial cells, which appear more numerous in lung sections from
Cav-1-deficient animals. Two representative fields are shown for each
genotype. B, wild-type mice (WT); C,
Cav-1 null mice (KO). Long overexposures are shown to
illustrate better the overall architecture of the adjacent
VEGF-R-negative lung parenchyma.
View larger version (6K):
[in a new window]
Fig. 11.
Immunostaining of the lung parenchyma with
the Ki67 "proliferation" antigen. Lung paraffin sections from
wild-type and caveolin-1-deficient mice were immunostained with a
widely used proliferation marker, Ki67. Bound primary antibodies were
detected with a fluorescently labeled secondary antibody. Note that
Ki67 immunoreactivity is dramatically increased in lung tissue sections
from Cav-1 null mice, as compared with wild-type controls. Upper
panel, wild-type mice (WT); lower panel,
Cav-1 null mice (KO).
View larger version (49K):
[in a new window]
Fig. 12.
Exercise tolerance in wild-type and
Cav-1-deficient mice. Weight-matched, age-matched (4.5 months),
same-sex littermates for each genotype were subjected to a "swimming
test" (see "Experimental Procedures"). Note that wild-type
animals were able to swim for up to 40 min, whereas Cav-1-deficient
animals only swam for ~10 min (an ~4-fold change). Thus,
Cav-1-deficient mice clearly show exercise intolerance, as would be
predicted based on the morphology of the lung. Similar exercise
intolerance was also observed in 1-month-old mice (not shown).
1-adrenergic receptor agonist) as a vasoconstrictor and
acetylcholine (Ach) to induce NO-dependent relaxation. To
demonstrate a role for eNOS in these physiological responses, we took
advantage of the availability of a well characterized arginine-based
NOS inhibitor, known as L-NAME
(nitro-L-arginine methyl ester).
8 to 104
M), thereby creating a dose-response curve. Finally, in
order to dissociate the PE-induced contractility from NO-mediated
relaxation, the NOS inhibitor L-NAME was added to all
rings.
View larger version (11K):
[in a new window]
Fig. 13.
Caveolin-1-deficient mice show abnormal
endothelium/NO-dependent modulation of mouse aortic
contraction. A, representative trace of
concentration-dependent acetylcholine
(10 8-104 M)-induced
relaxation, followed by addition of L-NAME (100 µM), which induced further contraction of the mouse
aorta. Note the appearance of spontaneous oscillatory contractions
present in the Ach concentration response curve in aortic rings from
the wild-type mouse but largely absent from tracings obtained on aortic
rings from the Cav-1 null mouse. As such, for comparative purposes, the
% relaxation (see C) was calculated from the
"steady-state" trough of relaxation observed for each Ach
concentration. The asterisks indicate the times of addition
of increasing amounts of Ach (108, 3 × 108,
107, 3 × 107, 106, 3 × 106, 105, 3 × 105,
and 104 M (molar)). B,
L-NAME (100 µM) modulation of PE-induced
contraction in mouse aorta from wild-type (WT) and Cav-1
null (KO) mice. Points represent the mean ± S.E. of 5 (KO) and 6 (WT) rings from 3 mice each. *,
p < 0.05 versus control WT; ***,
p < 0.0001; ###, p < 0.0001 versus KO; two-way analysis of variance for repeated
measures. Note that Cav-1 null mice showed (i) an impaired
vasoconstrictor response to PE, and (ii) this impaired response could
be rescued by treatment with L-NAME, a well characterized
NOS inhibitor. C, concentration-dependent
relaxation induced by acetylcholine (expressed as the log of molar
concentration) in aortas pre-constricted with 10 µM
phenylephrine from wild-type (WT; open squares)
and Cav-1 null (KO; black squares) mice. Points
represent mean ± S.E. of 5 (KO) or 6 (WT)
rings from 3 mice each. ***, p < 0.0001 versus WT. Note that Ach-induced relaxation (a
NO-dependent phenomenon) of the aortic rings was clearly
potentiated by the loss of caveolin-1 expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
61-100 and 140-178) in Sf21 insect cells can
induce vesicle formation albeit with sizes 10× normal caveolae. Our
establishment of Cav-1-deficient cells can aid future studies in
several ways. First, determinations of the composition of the plasma
membrane in Cav-1-deficient cells could possibly establish whether the
absence of caveolae is due to relative reductions in
cholesterol/glycosphingolipid content or to simply the Cav-1 protein
itself. Second, overexpression of Cav-1 mutants in these cells will
establish an elegant screening strategy for de novo caveolae formation.
1-adrenergic receptor agonist. We observed that Cav-1
null mice showed an impaired vasoconstrictor response to PE. This
impaired response was due to increased eNOS activity as the proper
vasoconstrictor response could be restored by treatment with
L-NAME, a well characterized NOS inhibitor. Conversely,
acetylcholine (Ach)-induced relaxation (a NO-dependent
phenomenon) of the aortic rings was clearly potentiated by loss of
caveolin-1 expression. These physiological observations provide strong
in vivo evidence that caveolin-1 indeed functions as a tonic
inhibitor of eNOS-mediated signal transduction.
ACKNOWLEDGEMENTS
Note Added in Proof
FOOTNOTES
Recipient of a Hirschl/Weil-Caulier Career Scientist
Award. To whom correspondence should be addressed: Dept. of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti@aecom.yu.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Glenney, J. R., Jr.
(1989)
J. Biol. Chem.
264,
20163-20166
2.
Glenney, J. R., Jr.,
and Zokas, L.
(1989)
J. Cell Biol.
108,
2401-2408
3.
Rothberg, K. G.,
Heuser, J. E.,
Donzell, W. C.,
Ying, Y. S.,
Glenney, J. R.,
and Anderson, R. G.
(1992)
Cell
68,
673-682
4.
Yamada, E.
(1955)
J. Biophys. Biochem. Cytol.
1,
445-458
5.
Farquhar, M.,
and Palade, G.
(1963)
J. Cell Biol.
17,
375-412
6.
Fra, A. M.,
Williamson, E.,
Simons, K.,
and Parton, R. G.
(1995)
Proc. Natl. Acad. Sci., U. S. A.
92,
8655-8659
7.
Li, S.,
Song, K. S.,
Koh, S. S.,
Kikuchi, A.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
28647-28654
8.
Murata, M.,
Peranen, J.,
Schreiner, R.,
Weiland, F.,
Kurzchalia, T.,
and Simons, K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10339-10343
9.
Scherer, P. E.,
Okamoto, T.,
Chun, M.,
Nishimoto, I.,
Lodish, H. F.,
and Lisanti, M. P.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
131-135
10.
Song, K. S.,
Scherer, P. E.,
Tang, Z.-L.,
Okamoto, T.,
Li, S.,
Chafel, M.,
Chu, C.,
Kohtz, D. S.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
15160-15165
11.
Scherer, P. E.,
Lewis, R. Y.,
Volonte, D.,
Engelman, J. A.,
Galbiati, F.,
Couet, J.,
Kohtz, D. S.,
van Donselaar, E.,
Peters, P.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
29337-29346
12.
Tang, Z.-L.,
Scherer, P. E.,
Okamoto, T.,
Song, K.,
Chu, C.,
Kohtz, D. S.,
Nishimoto, I.,
Lodish, H. F.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
2255-2261
13.
Galbiati, F.,
Razani, B.,
and Lisanti, M. P.
(2001)
Cell
106,
403-411
14.
Razani, B.,
Schlegel, A.,
and Lisanti, M. P.
(2000)
J. Cell Sci.
113,
2103-2109
15.
Couet, J.,
Li, S.,
Okamoto, T.,
Scherer, P. S.,
and Lisanti, M. P.
(1997)
Trends Cardiovasc. Med.
7,
103-110
16.
Lisanti, M. P.,
Scherer, P.,
Tang, Z.-L.,
and Sargiacomo, M.
(1994)
Trends Cell Biol.
4,
231-235
17.
Li, S.,
Couet, J.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
29182-29190
18.
Song, K. S.,
Li, S.,
Okamoto, T.,
Quilliam, L.,
Sargiacomo, M.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
9690-9697
19.
Song, K. S.,
Sargiacomo, M.,
Galbiati, F.,
Parenti, M.,
and Lisanti, M. P.
(1997)
Cell. Mol. Biol.
43,
293-303
20.
Garcia-Cardena, G.,
Oh, P.,
Liu, J.,
Schnitzer, J. E.,
and Sessa, W. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6448-6453
21.
Shaul, P. W.,
Smart, E. J.,
Robinson, L. J.,
German, Z.,
Yuhanna, I. S.,
Ying, Y.,
Anderson, R. G. W.,
and Michel, T.
(1996)
J. Biol. Chem.
271,
6518-6522
22.
Li, S.,
Okamoto, T.,
Chun, M.,
Sargiacomo, M.,
Casanova, J. E.,
Hansen, S. H.,
Nishimoto, I.,
and Lisanti, M. P.
(1995)
J. Biol. Chem.
270,
15693-15701
23.
Okamoto, T.,
Schlegel, A.,
Scherer, P. E.,
and Lisanti, M. P.
(1998)
J. Biol. Chem.
273,
5419-5422
24.
Couet, J.,
Sargiacomo, M.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
30429-30438
25.
Yamamoto, M.,
Toya, Y.,
Jensen, R. A.,
and Ishikawa, Y.
(1999)
Exp. Cell Res.
247,
380-388
26.
Engelman, J. A.,
Lee, R. J.,
Karnezis, A.,
Bearss, D. J.,
Webster, M.,
Siegel, P.,
Muller, W. J.,
Windle, J. J.,
Pestell, R. G.,
and Lisanti, M. P.
(1998)
J. Biol. Chem.
273,
20448-20455
27.
Zundel, W.,
Swiersz, L. M.,
and Giaccia, A.
(2000)
Mol. Cell. Biol.
20,
1507-1514
28.
Koleske, A. J.,
Baltimore, D.,
and Lisanti, M. P.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1381-1385
29.
Razani, B.,
Altschuler, Y.,
Zhu, L.,
Pestell, R. G.,
Mostov, K. E.,
and Lisanti, M. P.
(2000)
Biochemistry
39,
13916-13924
30.
Park, D. S.,
Razani, B.,
Lasorella, A.,
Schreiber-Agus, N.,
Pestell, R. G.,
Iavarone, A.,
and Lisanti, M. P.
(2001)
Biochemistry
40,
3354-3362
31.
Lee, S. W.,
Reimer, C. L.,
Oh, P.,
Campbel, L. D. B.,
and Schnitzer, J. E.
(1998)
Oncogene
16,
1391-1397
32.
Bender, F. C.,
Reymond, M. A.,
Bron, C.,
and Quest, A.
(2000)
Cancer Res.
60,
5870-5878
33.
Bagnoli, M.,
Tomassetti, A.,
Figini, M.,
Flati, S.,
Dolo, V.,
Canevari, S.,
and Miotti, S.
(2000)
Oncogene
19,
4754-4763
34.
Zhang, W.,
Razani, B.,
Altschuler, Y.,
Bouzahzah, B.,
Mostov, K. E.,
Pestell, R. G.,
and Lisanti, M. P.
(2000)
J. Biol. Chem.
275,
20717-20725
35.
Engelman, J. A.,
Zhang, X. L.,
and Lisanti, M. P.
(1999)
FEBS Lett.
448,
221-230
36.
Racine, C.,
Belanger, M.,
Hirabayashi, H.,
Boucher, M.,
Chakir, J.,
and Couet, J.
(1999)
Biochem. Biophys. Res. Commun.
255,
580-586
37.
Suzuki, T.,
Suzuki, Y.,
Hanada, K.,
Hashimoto, A.,
Redpath, J. L.,
Stanbridge, E. J.,
Nishijima, M.,
and Kitagawa, T.
(1998)
J. Biochem. (Tokyo)
124,
383-388
38.
Shridhar, V.,
Sun, Q. C.,
Miller, O. J.,
Kalemkerian, G. P.,
Petros, J.,
and Smith, D. I.
(1997)
Oncogene
15,
2727-2733
39.
Jenkins, R. B.,
Qian, J.,
Lee, H. K.,
Huang, H.,
Hirasawa, K.,
Bostwick, D. G.,
Proffitt, J.,
Wilber, K.,
Lieber, M. M.,
Liu, W.,
and Smith, D. I.
(1998)
Cancer Res.
58,
759-766
40.
Zenklusen, J. C.,
Thompson, J. C.,
Troncoso, P.,
Kagan, J.,
and Conti, C. J.
(1994)
Cancer Res.
54,
6370-6373
41.
Zenklusen, J. C.,
Bieche, I.,
Lidereau, R.,
and Conti, C. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12155-12158
42.
Huang, H.,
Qian, C.,
Jenkins, R. B.,
and Smith, D. I.
(1998)
Genes Chromosomes Cancer
21,
152-159
43.
Zenklusen, J. C.,
Thompson, J. C.,
Klein-Szanto, A. J.,
and Conti, C. J.
(1995)
Cancer Res.
55,
1347-1350
44.
Zenklusen, J. C.,
Weitzel, J. N.,
Ball, H. G.,
and Conti, C. J.
(1995)
Oncogene
11,
359-363
45.
Koike, M.,
Takeuchi, S.,
Yokota, J.,
Park, S.,
Hatta, Y.,
Miller, C. W.,
Tsuruoka, N.,
and Koeffler, H. P.
(1997)
Genes Chromosomes Cancer
19,
1-5
46.
Achille, A.,
Biasi, M. O.,
Zamboni, G.,
Bogina, G.,
Magalini, A. R.,
Pederzoli, P.,
Perucho, M.,
and Scarpa, A.
(1996)
Cancer Res.
56,
3808-3813
47.
Engelman, J. A.,
Zhang, X. L.,
Galbiati, F.,
Volonte, D.,
Sotgia, F.,
Pestell, R. G.,
Minetti, C.,
Scherer, P. E.,
Okamoto, T.,
and Lisanti, M. P.
(1998)
Am. J. Hum. Genet.
63,
1578-1587
48.
Galbiati, F.,
Volonté, D.,
Engelman, J. A.,
Watanabe, G.,
Burk, R.,
Pestell, R.,
and Lisanti, M. P.
(1998)
EMBO J.
17,
6633-6648
49.
Scheel, J.,
Srinivasan, J.,
Honnert, U.,
Henske, A.,
and Kurzchalia, T. V.
(1999)
Nat. Cell Biol.
1,
127-129
50.
Hayashi, K.,
Matsuda, S.,
Machida, K.,
Yamamoto, T.,
Fukuda, Y.,
Nimura, Y.,
Hayakawa, T.,
and Hamaguchi, M.
(2001)
Cancer Res.
61,
2361-2364
51.
Minetti, C.,
Sotgia, F.,
Bruno, C.,
Scartezzini, P.,
Broda, P.,
Bado, M.,
Masetti, E.,
Mazzocco, P.,
Egeo, A.,
Donati, M. A.,
Volonte', D.,
Galbiati, F.,
Cordone, G.,
Bricarelli, F. D.,
Lisanti, M. P.,
and Zara, F.
(1998)
Nat. Genet.
18,
365-368
52.
Scherer, P. E.,
Tang, Z.-L.,
Chun, M. C.,
Sargiacomo, M.,
Lodish, H. F.,
and Lisanti, M. P.
(1995)
J. Biol. Chem.
270,
16395-16401
53.
Wu, H.,
Liu, X.,
and Jaenisch, R.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
2819-2823
54.
Wu, H.,
Liu, X.,
Jaenisch, R.,
and Lodish, H. F.
(1995)
Cell
83,
59-67
55.
Engelman, J. A.,
Zhang, X. L.,
Galbiati, F.,
and Lisanti, M. P.
(1998)
FEBS Lett.
429,
330-336
56.
Ioffe, E.,
Liu, Y.,
Bhaumik, M.,
Poirier, F.,
Factor, S. M.,
and Stanley, P.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7357-7361
57.
Edelmann, W.,
Yang, K.,
Umar, A.,
Heyer, J.,
Lau, K.,
Fan, K.,
Liedtke, W.,
Cohen, P. E.,
Kane, M. F.,
Lipford, J. R., Yu, N.,
Crouse, G. F.,
Pollard, J. W.,
Kunkel, T.,
Lipkin, M.,
Kolodner, R.,
and Kucherlapati, R.
(1997)
Cell
91,
467-477
58.
Kamijo, T.,
Zindy, F.,
Roussel, M. F.,
Quelle, D. E.,
Downing, J. R.,
Ashmun, R. A.,
Grosveld, G.,
and Sherr, C. J.
(1997)
Cell
91,
649-659
59.
Sargiacomo, M.,
Sudol, M.,
Tang, Z.-L.,
and Lisanti, M. P.
(1993)
J. Cell Biol.
122,
789-807
60.
Lisanti, M. P.,
Scherer, P. E.,
Vidugiriene, J.,
Tang, Z.-L.,
Hermanoski-Vosatka, A.,
Tu, Y.-H.,
Cook, R. F.,
and Sargiacomo, M.
(1994)
J. Cell Biol.
126,
111-126
61.
Engelman, J. A.,
Wycoff, C. C.,
Yasuhara, S.,
Song, K. S.,
Okamoto, T.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
16374-16381
62.
Song, K. S.,
Tang, Z.-L.,
Li, S.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
4398-4403
63.
Volonte, D.,
Galbiati, F.,
and Lisanti, M. P.
(1999)
FEBS Lett.
445,
431-439
64.
Galbiati, F.,
Volonte, D.,
Minetti, C.,
Chu, J. B.,
and Lisanti, M. P.
(1999)
J. Biol. Chem.
274,
25632-25641
65.
Deng, C.,
Zhang, P.,
Harper, J. W.,
Elledge, S. J.,
and Leder, P.
(1995)
Cell
82,
675-684
66.
LeCouter, J. E.,
Kablar, B.,
Hardy, W. R.,
Ying, C.,
Megeney, L. A.,
May, L. L.,
and Rudnicki, M. A.
(1998)
Mol. Cell. Biol.
18,
7455-7465
67.
Glenney, J. R.,
and Soppet, D.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
10517-10521
68.
Hailstones, D.,
Sleer, L. S.,
Parton, R. G.,
and Stanley, K. K.
(1998)
J. Lipid Res.
39,
369-379
69.
Smart, E. J.,
Ying, Y.-S.,
Donzell, W. C.,
and Anderson, R. G. W.
(1996)
J. Biol. Chem.
271,
29427-29435
70.
Monier, S.,
Parton, R. G.,
Vogel, F.,
Behlke, J.,
Henske, A.,
and Kurzchalia, T.
(1995)
Mol. Biol. Cell
6,
911-927
71.
Monier, S.,
Dietzen, D. J.,
Hastings, W. R.,
Lublin, D. M.,
and Kurzchalia, T. V.
(1996)
FEBS Lett.
388,
143-149
72.
Das, K.,
Lewis, R. Y.,
Scherer, P. E.,
and Lisanti, M. P.
(1999)
J. Biol. Chem.
274,
18721-18726
73.
Li, S.,
Galbiati, F.,
Volonte, D.,
Sargiacomo, M.,
Engelman, J. A.,
Das, K.,
Scherer, P. E.,
and Lisanti, M. P.
(1998)
FEBS Lett.
434,
127-134
74.
Mora, R.,
Bonilha, V. L.,
Marmorstein, A.,
Scherer, P. E.,
Brown, D.,
Lisanti, M. P.,
and Rodriguez-Boulan, E.
(1999)
J. Biol. Chem.
274,
25708-25717
75.
Parolini, I.,
Sargiacomo, M.,
Galbiati, F.,
Rizzo, G.,
Grignani, F.,
Engelman, J. A.,
Okamoto, T.,
Ikezu, T.,
Scherer, P. E.,
Mora, R.,
Rodriguez-Boulan, E.,
Peschle, C.,
and Lisanti, M. P.
(1999)
J. Biol. Chem.
274,
25718-25725
76.
Rock, K. L.,
Gramm, C.,
Rothstein, L.,
Clark, K.,
Stein, R.,
Dick, L.,
Hwang, D.,
and Goldberg, A. L.
(1994)
Cell
78,
761-771
77.
Lowe, J.,
Stock, D.,
Jap, B.,
Zwickl, P.,
Baumeister, W.,
and Huber, R.
(1995)
Science
268,
533-539
78.
Ghitescu, L.,
Fixman, A.,
Simionescu, M.,
and Simionescu, N.
(1986)
J. Cell Biol.
102,
1304-1311
79.
Serrano, M.,
Lee, H.,
Chin, L.,
Cordon-Cardo, C.,
Beach, D.,
and DePinho, R. A.
(1996)
Cell
85,
27-37
80.
Herrera, R. E.,
Sah, V. P.,
Williams, B. O.,
Makela, T. P.,
Weinberg, R. A.,
and Jacks, T.
(1996)
Mol. Cell. Biol.
16,
2402-2407
81.
Furuchi, T.,
and Anderson, R. G. W.
(1998)
J. Biol. Chem.
273,
21099-21104
82.
Park, W. Y.,
Park, J. S.,
Cho, K. A.,
Kim, D. I.,
Ko, Y. G.,
Seo, J. S.,
and Park, S. C.
(2000)
J. Biol. Chem.
275,
20847-20852
83.
Schluter, C.,
Duchrow, M.,
Wohlenberg, C.,
Becker, M. H.,
Key, G.,
Flad, H. D.,
and Gerdes, J.
(1993)
J. Cell Biol.
123,
513-522
84.
Gerdes, J.,
Li, L.,
Schlueter, C.,
Duchrow, M.,
Wohlenberg, C.,
Gerlach, C.,
Stahmer, I.,
Kloth, S.,
Brandt, E.,
and Flad, H. D.
(1991)
Am. J. Pathol.
138,
867-873
85.
Garcia-Cardena, G.,
Martasek, P.,
Siler-Masters, B. S.,
Skidd, P. M.,
Couet, J. C.,
Li, S.,
Lisanti, M. P.,
and Sessa, W. C.
(1997)
J. Biol. Chem.
272,
25437-25440
86.
Ju, H.,
Zou, R.,
Venema, V. J.,
and Venema, R. C.
(1997)
J. Biol. Chem.
272,
18522-18525
87.
Michel, J. B.,
Feron, O.,
Sacks, D.,
and Michel, T.
(1997)
J. Biol. Chem.
272,
15583-15586
88.
Fra, A. M.,
Masserini, M.,
Palestini, P.,
Sonnino, S.,
and Simons, K.
(1995)
FEBS Lett.
375,
11-14
89.
Li, S.,
Song, K. S.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
568-573
90.
Thiele, C.,
Hannah, M. J.,
Fahrenholz, F.,
and Huttner, W. B.
(2000)
Nat. Cell Biol.
2,
42-49
91.
Sargiacomo, M.,
Scherer, P. E.,
Tang, Z.-L.,
Kubler, E.,
Song, K. S.,
Sanders, M. C.,
and Lisanti, M. P.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
9407-9411
92.
Wickner, S.,
Maurizi, M. R.,
and Gottesman, S.
(1999)
Science
286,
1888-1893
93.
Ellgaard, L.,
Molinari, M.,
and Helenius, A.
(1999)
Science
286,
1882-1888
94.
Engelman, J. A.,
Chu, C.,
Lin, A.,
Jo, H.,
Ikezu, T.,
Okamoto, T.,
Kohtz, D. S.,
and Lisanti, M. P.
(1998)
FEBS Lett.
428,
205-211
95.
Shimizu, K.,
Goldfarb, M.,
Suard, Y.,
Perucho, M.,
Li, Y.,
Kamata, T.,
Feramisco, J.,
Stavnezer, E.,
Fogh, J.,
and Wigler, M. H.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
2112-2116
96.
Newbold, R. F.,
and Overell, R. W.
(1983)
Nature
304,
648-651
97.
Serrano, M.,
Lin, A. W.,
McCurrach, M. E.,
Beach, D.,
and Lowe, S. W.
(1997)
Cell
88,
593-602
98.
Lin, A. W.,
Barradas, M.,
Stone, J. C.,
van Aelst, L.,
Serrano, M.,
and Lowe, S. W.
(1998)
Genes Dev.
12,
3008-3019
99.
Sah, V. P.,
Attardi, L. D.,
Mulligan, G. J.,
Williams, B. O.,
Bronson, R. T.,
and Jacks, T.
(1995)
Nat. Genet.
10,
175-180
100.
Harper, J.,
Adam, G.,
Wei, N.,
Keyomarsi, K.,
and Elledge, S.
(1993)
Cell
75,
805-816
101.
Harper, J. W.,
Elledge, S. J.,
Keyomarsi, K.,
Dynlacht, B.,
Tsai, L. H.,
Zhang, P.,
Dobrowolski, S.,
Bai, C.,
Connell-Crowley, L.,
and Swindell, E.
(1995)
Mol. Biol. Cell
6,
387-400
102.
el-Deiry, W. S.,
Tokino, T.,
Velculescu, V. E.,
Levy, D. B.,
Parsons, R.,
Trent, J. M.,
Lin, D.,
Mercer, W. E.,
Kinzler, K. W.,
and Vogelstein, B.
(1993)
Cell
75,
817-825
103.
Zindy, F.,
van Deursen, J.,
Grosveld, G.,
Sherr, C. J.,
and Roussel, M. F.
(2000)
Mol. Cell. Biol.
20,
372-378
104.
Latres, E.,
Malumbres, M.,
Sotillo, R.,
Martin, J.,
Ortega, S.,
Martin-Caballero, J.,
Flores, J. M.,
Cordon-Cardo, C.,
and Barbacid, M.
(2000)
EMBO J.
19,
3496-3506
105.
Fero, M. L.,
Rivkin, M.,
Tasch, M.,
Porter, P.,
Carow, C. E.,
Firpo, E.,
Polyak, K.,
Tsai, L. H.,
Broudy, V.,
Perlmutter, R. M.,
Kaushansky, K.,
and Roberts, J. M.
(1996)
Cell
85,
733-744
106.
Coats, S.,
Whyte, P.,
Fero, M. L.,
Lacy, S.,
Chung, G.,
Randel, E.,
Firpo, E.,
and Roberts, J. M.
(1999)
Curr. Biol.
9,
163-173
107.
Bucci, M.,
Gratton, J. P.,
Rudic, R. D.,
Acevedo, L.,
Roviezzo, F.,
Cirino, G.,
and Sessa, W. C.
(2000)
Nat. Med.
6,
1362-1367
108.
Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B.,
Menne, J., Lindschau, C., Mende, F., Luft, F. C., Schedl, A., Haller,
H., and Kurzchalia, T. V. (2001) Science, in press,
Published Online August 9, 2001, 10.1126/science.1062688
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Xu, G. Yang, and G. Hu Binding of IFITM1 enhances the inhibiting effect of caveolin-1 on ERK activation Acta Biochim Biophys Sin, June 1, 2009; 41(6): 488 - 494. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. G. Hansen and B. J. Nichols Molecular mechanisms of clathrin-independent endocytosis J. Cell Sci., June 1, 2009; 122(11): 1713 - 1721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Witkiewicz, A. Dasgupta, F. Sotgia, I. Mercier, R. G. Pestell, M. Sabel, C. G. Kleer, J. R. Brody, and M. P. Lisanti An Absence of Stromal Caveolin-1 Expression Predicts Early Tumor Recurrence and Poor Clinical Outcome in Human Breast Cancers Am. J. Pathol., June 1, 2009; 174(6): 2023 - 2034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Guo, I. Hernandez, B. Isermann, T.-b. Kang, L. Medved, R. Sood, E. J. Kerschen, T. Holyst, M. W. Mosesson, and H. Weiler Caveolin-1-dependent apoptosis induced by fibrin degradation products Blood, April 30, 2009; 113(18): 4431 - 4439. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Russo, U. J. K. Soh, M. M. Paing, P. Arora, and J. Trejo Caveolae are required for protease-selective signaling by protease-activated receptor-1 PNAS, April 14, 2009; 106(15): 6393 - 6397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Zaas, Z. D. Swan, B. J. Brown, G. Li, S. H. Randell, S. Degan, M. E. Sunday, J. R. Wright, and S. N. Abraham Counteracting Signaling Activities in Lipid Rafts Associated with the Invasion of Lung Epithelial Cells by Pseudomonas aeruginosa J. Biol. Chem., April 10, 2009; 284(15): 9955 - 9964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Russo, U. J. K. Soh, and J. Trejo Proteases Display Biased Agonism at Protease-Activated Receptors: Location matters! Mol. Interv., April 1, 2009; 9(2): 87 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Balligand, O. Feron, and C. Dessy eNOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues Physiol Rev, April 1, 2009; 89(2): 481 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Mercier, M. C. Casimiro, J. Zhou, C. Wang, C. Plymire, K. G. Bryant, K. M. Daumer, F. Sotgia, G. Bonuccelli, A. K. Witkiewicz, et al. Genetic Ablation of Caveolin-1 Drives Estrogen-Hypersensitivity and the Development of DCIS-Like Mammary Lesions Am. J. Pathol., April 1, 2009; 174(4): 1172 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. G. Romanenko, K. S. Roser, J. E. Melvin, and T. Begenisich The role of cell cholesterol and the cytoskeleton in the interaction between IK1 and maxi-K channels Am J Physiol Cell Physiol, April 1, 2009; 296(4): C878 - C888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Haram, O. J. Kemi, S. J. Lee, M. O. Bendheim, Q. Y. Al-Share, H. L. Waldum, L. J. Gilligan, L. G. Koch, S. L. Britton, S. M. Najjar, et al. Aerobic interval training vs. continuous moderate exercise in the metabolic syndrome of rats artificially selected for low aerobic capacity Cardiovasc Res, March 1, 2009; 81(4): 723 - 732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Marmon, J. Hinchey, P. Oh, M. Cammer, C. J. de Almeida, L. Gunther, C. S. Raine, and M. P. Lisanti Caveolin-1 Expression Determines the Route of Neutrophil Extravasation through Skin Microvasculature Am. J. Pathol., February 1, 2009; 174(2): 684 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Peterson, L. V. d'Uscio, S. Cao, X.-L. Wang, and Z. S. Katusic Guanosine Triphosphate Cyclohydrolase I Expression and Enzymatic Activity Are Present in Caveolae of Endothelial Cells Hypertension, February 1, 2009; 53(2): 189 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Le Saux, K. Teeters, S. Miyasato, J. Choi, G. Nakamatsu, J. A. Richardson, B. Starcher, E. C. Davis, E. K. Tam, and C. Jourdan-Le Saux The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties Am J Physiol Lung Cell Mol Physiol, December 1, 2008; 295(6): L1007 - L1017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. C. Matthews, M. J. Taggart, and M. Westwood Modulation of Caveolin-1 Expression Can Affect Signalling through the Phosphatidylinositol 3-Kinase/Akt Pathway and Cellular Proliferation in Response to Insulin-Like Growth Factor I Endocrinology, October 1, 2008; 149(10): 5199 - 5208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Lizarbe, C. Garcia-Rama, C. Tarin, M. Saura, E. Calvo, J. A. Lopez, C. Lopez-Otin, A. R. Folgueras, S. Lamas, and C. Zaragoza Nitric oxide elicits functional MMP-13 protein-tyrosine nitration during wound repair FASEB J, September 1, 2008; 22(9): 3207 - 3215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhong, E. J. Smart, B. Weksler, P.-O. Couraud, B. Hennig, and M. Toborek Caveolin-1 Regulates Human Immunodeficiency Virus-1 Tat-Induced Alterations of Tight Junction Protein Expression via Modulation of the Ras Signaling J. Neurosci., July 30, 2008; 28(31): 7788 - 7796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Shi and J. Sottile Caveolin-1-dependent {beta}1 integrin endocytosis is a critical regulator of fibronectin turnover J. Cell Sci., July 15, 2008; 121(14): 2360 - 2371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-L. He, A. B. Deora, H. Xiong, Q. Ling, B. B. Weksler, R. Niesvizky, and K. A. Hajjar Endothelial Cell Annexin A2 Regulates Polyubiquitination and Degradation of Its Binding Partner S100A10/p11 J. Biol. Chem., July 11, 2008; 283(28): 19192 - 19200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Frank, S. Pavlides, M. W.-C. Cheung, K. Daumer, and M. P. Lisanti Role of caveolin-1 in the regulation of lipoprotein metabolism Am J Physiol Cell Physiol, July 1, 2008; 295(1): C242 - C248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Peng, B. Zhang, D. Wu, A. J. Ingram, B. Gao, and J. C. Krepinsky TGF{beta}-induced RhoA activation and fibronectin production in mesangial cells require caveolae Am J Physiol Renal Physiol, July 1, 2008; 295(1): F153 - F164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Hu, S. M. Vogel, D. E. Schwartz, A. B. Malik, and R. D. Minshall Intercellular Adhesion Molecule-1-Dependent Neutrophil Adhesion to Endothelial Cells Induces Caveolae-Mediated Pulmonary Vascular Hyperpermeability Circ. Res., June 20, 2008; 102(12): e120 - e131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hiroi, Z. Guo, Y. Li, A. H. Beggs, and J. K. Liao Dynamic regulation of endothelial NOS mediated by competitive interaction with {alpha}-actinin-4 and calmodulin FASEB J, May 1, 2008; 22(5): 1450 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Maniatis, V. Shinin, D. E. Schraufnagel, S. Okada, S. M. Vogel, A. B. Malik, and R. D. Minshall Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L865 - L873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Tourkina, M. Richard, P. Gooz, M. Bonner, J. Pannu, R. Harley, P. N. Bernatchez, W. C. Sessa, R. M. Silver, and S. Hoffman Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L843 - L861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. W. Ryter and A. M. K. Choi Caveolin-1: a critical regulator of pulmonary vascular architecture and nitric oxide bioavailability in pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L862 - L864. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Garg and A. K. Agarwal Caveolin-1: A New Locus for Human Lipodystrophy J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1183 - 1185. [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hernandez-Deviez, M. T. Howes, S. H. Laval, K. Bushby, J. F. Hancock, and R. G. Parton Caveolin Regulates Endocytosis of the Muscle Repair Protein, Dysferlin J. Biol. Chem., March 7, 2008; 283(10): 6476 - 6488. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Langlois, K. N. Cowan, Q. Shao, B. J. Cowan, and D. W. Laird Caveolin-1 and -2 Interact with Connexin43 and Regulate Gap Junctional Intercellular Communication in Keratinocytes Mol. Biol. Cell, March 1, 2008; 19(3): 912 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Pojoga, T. M. Yao, S. Sinha, R. L. Ross, J. C. Lin, J. D. Raffetto, G. K. Adler, G. H. Williams, and R. A. Khalil Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1258 - H1265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Santibanez, F. J. Blanco, E. M. Garrido-Martin, F. Sanz-Rodriguez, M. A. del Pozo, and C. Bernabeu Caveolin-1 interacts and cooperates with the transforming growth factor-{beta} type I receptor ALK1 in endothelial caveolae Cardiovasc Res, March 1, 2008; 77(4): 791 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Le Saux, K. Teeters, S. K. Miyasato, P. R. Hoffmann, O. Bollt, V. Douet, R. V. Shohet, D. H. Broide, and E. K. Tam Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-{beta} Signaling, in Acute Allergen-induced Airway Remodeling J. Biol. Chem., February 29, 2008; 283(9): 5760 - 5768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu and P. F. Pilch A Critical Role of Cavin (Polymerase I and Transcript Release Factor) in Caveolae Formation and Organization J. Biol. Chem., February 15, 2008; 283(7): 4314 - 4322. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Tahir, G. Yang, A. A. Goltsov, M. Watanabe, K.-i. Tabata, J. Addai, E. M. A. Fattah, D. Kadmon, and T. C. Thompson Tumor Cell-Secreted Caveolin-1 Has Proangiogenic Activities in Prostate Cancer Cancer Res., February 1, 2008; 68(3): 731 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Salani, L. Briatore, S. Garibaldi, R. Cordera, and D. Maggi Caveolin-1 Down-Regulation Inhibits Insulin-Like Growth Factor-I Receptor Signal Transduction in H9C2 Rat Cardiomyoblasts Endocrinology, February 1, 2008; 149(2): 461 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Ramos, M. W. Lame, H. J. Segall, and D. W. Wilson Smad Signaling in the Rat Model of Monocrotaline Pulmonary Hypertension Toxicol Pathol, February 1, 2008; 36(2): 311 - 320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zemans and G. P. Downey Role of caveolin-1 in regulation of inflammation: different strokes for different folks Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L175 - L177. [Full Text] [PDF]
|
|
|
|
|
|
D. J. Kennedy, J. Elkareh, A. Shidyak, A. P. Shapiro, S. Smaili, K. Mutgi, S. Gupta, J. Tian, E. Morgan, S. Khouri, et al. Partial nephrectomy as a model for uremic cardiomyopathy in the mouse Am J Physiol Renal Physiol, February 1, 2008; 294(2): F450 - F454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bianco, L. Strizzi, M. Mancino, K. Watanabe, M. Gonzales, S. Hamada, A. Raafat, L. Sahlah, C. Chang, F. Sotgia, et al. Regulation of Cripto-1 Signaling and Biological Activity by Caveolin-1 in Mammary Epithelial Cells Am. J. Pathol., February 1, 2008; 172(2): 345 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tang, M. H. Gao, N. C. Lai, A. L. Firth, T. Takahashi, T. Guo, J. X.-J. Yuan, D. M. Roth, and H. K. Hammond Adenylyl Cyclase Type 6 Deletion Decreases Left Ventricular Function via Impaired Calcium Handling Circulation, January 1, 2008; 117(1): 61 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gosens, G. L. Stelmack, G. Dueck, M. M. Mutawe, M. Hinton, K. D. McNeill, A. Paulson, S. Dakshinamurti, W. T. Gerthoffer, J. A. Thliveris, et al. Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1406 - L1418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. F. Pilch, R. P. Souto, L. Liu, M. P. Jedrychowski, E. A. Berg, C. E. Costello, and S. P. Gygi Cellular spelunking: exploring adipocyte caveolae J. Lipid Res., October 1, 2007; 48(10): 2103 - 2111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Murata, M. I. Lin, Y. Huang, J. Yu, P. M. Bauer, F. J. Giordano, and W. C. Sessa Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice J. Exp. Med., October 1, 2007; 204(10): 2373 - 2382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Predescu, D. N. Predescu, and A. B. Malik Molecular determinants of endothelial transcytosis and their role in endothelial permeability Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L823 - L842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yang, C. Ying, M. Xu, X. Zuo, X. Ye, L. Liu, Y. Nara, and X. Sun High-fat diet up-regulates caveolin-1 expression in aorta of diet-induced obese but not in diet-resistant rats Cardiovasc Res, October 1, 2007; 76(1): 167 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. D. Hardie, T. R. Korfhagen, M. A. Sartor, A. Prestridge, M. Medvedovic, T. D. Le Cras, M. Ikegami, S. C. Wesselkamper, C. Davidson, M. Dietsch, et al. Genomic Profile of Matrix and Vasculature Remodeling in TGF-{alpha} Induced Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 309 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. H. Patel, S. Zhang, F. Murray, R. Y. S. Suda, B. P. Head, U. Yokoyama, J. S. Swaney, I. R. Niesman, R. T. Schermuly, S. S. Pullamsetti, et al. Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension FASEB J, September 1, 2007; 21(11): 2970 - 2979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Albinsson, Y. Shakirova, A. Rippe, M. Baumgarten, B.-I. Rosengren, C. Rippe, R. Hallmann, P. Hellstrand, B. Rippe, and K. Sward Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1222 - R1231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Nixon, A. Carter, J. Wegner, C. Ferguson, M. Floetenmeyer, J. Riches, B. Key, M. Westerfield, and R. G. Parton Caveolin-1 is required for lateral line neuromast and notochord development J. Cell Sci., July 1, 2007; 120(13): 2151 - 2161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kamishima, T. Burdyga, J. A. Gallagher, and J. M. Quayle Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H204 - H214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. B. Atkins and M. K. Jain Role of Kruppel-Like Transcription Factors in Endothelial Biology Circ. Res., June 22, 2007; 100(12): 1686 - 1695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Predescu, D. N. Predescu, I. Knezevic, I. K. Klein, and A. B. Malik Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells J. Biol. Chem., June 8, 2007; 282(23): 17166 - 17178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Gilad and B. Schwartz Association of estrogen receptor {beta} with plasma-membrane caveola components: implication in control of vitamin D receptor J. Mol. Endocrinol., June 1, 2007; 38(6): 603 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Grande-Garcia, A. Echarri, J. de Rooij, N. B. Alderson, C. M. Waterman-Storer, J. M. Valdivielso, and M. A. del Pozo Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases J. Cell Biol., May 21, 2007; 177(4): 683 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Clarke, V. Ohanian, and J. Ohanian Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2248 - H2256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Costa, M. Senou, F. Van Rode, J. Ruf, M. Capello, D. Dequanter, P. Lothaire, C. Dessy, J. E. Dumont, M.-C. Many, et al. Reciprocal Negative Regulation between Thyrotropin/3',5'-Cyclic Adenosine Monophosphate-Mediated Proliferation and Caveolin-1 Expression in Human and Murine Thyrocytes Mol. Endocrinol., April 1, 2007; 21(4): 921 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Jasmin, S. Malhotra, M. Singh Dhallu, I. Mercier, D. M. Rosenbaum, and M. P. Lisanti Caveolin-1 Deficiency Increases Cerebral Ischemic Injury Circ. Res., March 16, 2007; 100(5): 721 - 729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Lin, J. Yu, T. Murata, and W. C. Sessa Caveolin-1-Deficient Mice Have Increased Tumor Microvascular Permeability, Angiogenesis, and Growth Cancer Res., March 15, 2007; 67(6): 2849 - 2856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-H. Sun, D. C. Flynn, V. Castranova, L. L. Millecchia, A. R. Beardsley, and J. Liu Identification of a Novel Domain at the N Terminus of Caveolin-1 That Controls Rear Polarization of the Protein and Caveolae Formation J. Biol. Chem., March 9, 2007; 282(10): 7232 - 7241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Adebiyi, G. Zhao, S. Y. Cheranov, A. Ahmed, and J. H. Jaggar Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1584 - H1592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Schubert, F. Sotgia, A. W. Cohen, F. Capozza, G. Bonuccelli, C. Bruno, C. Minetti, E. Bonilla, S. DiMauro, and M. P. Lisanti Caveolin-1(-/-)- and Caveolin-2(-/-)-Deficient Mice Both Display Numerous Skeletal Muscle Abnormalities, with Tubular Aggregate Formation Am. J. Pathol., January 1, 2007; 170(1): 316 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Peng, D. Wu, A. J. Ingram, B. Zhang, B. Gao, and J. C. Krepinsky RhoA Activation in Mesangial Cells by Mechanical Strain Depends on Caveolae and Caveolin-1 Interaction J. Am. Soc. Nephrol., January 1, 2007; 18(1): 189 - 198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Trushina, R. D. Singh, R. B. Dyer, S. Cao, V. H. Shah, R. G. Parton, R. E. Pagano, and C. T. McMurray Mutant huntingtin inhibits clathrin-independent endocytosis and causes accumulation of cholesterol in vitro and in vivo Hum. Mol. Genet., December 15, 2006; 15(24): 3578 - 3591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Medina, C. J. de Almeida, E. Dew, J. Li, G. Bonuccelli, T. M. Williams, A. W. Cohen, R. G. Pestell, P. G. Frank, H. B. Tanowitz, et al. Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection Infect. Immun., December 1, 2006; 74(12): 6665 - 6674. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shakirova, J. Bonnevier, S. Albinsson, M. Adner, B. Rippe, J. Broman, A. Arner, and K. Sward Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1326 - C1335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Frank and M. P. Lisanti Zebrafish as a Novel Model System to Study the Function of Caveolae and Caveolin-1 in Organismal Biology Am. J. Pathol., December 1, 2006; 169(6): 1910 - 1912. [Full Text] [PDF]
|
|
|
|
|
|
P.-K. Fang, K. R. Solomon, L. Zhuang, M. Qi, M. McKee, M. R. Freeman, and P. C. Yelick Caveolin-1{alpha} and -1{beta} Perform Nonredundant Roles in Early Vertebrate Development Am. J. Pathol., December 1, 2006; 169(6): 2209 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ushio-Fukai and R. W. Alexander Caveolin-Dependent Angiotensin II Type 1 Receptor Signaling in Vascular Smooth Muscle Hypertension, November 1, 2006; 48(5): 797 - 803. [Full Text] [PDF]
|
|
|
|
|
|
O. M. Tirado, S. Mateo-Lozano, J. Villar, L. E. Dettin, A. Llort, S. Gallego, J. Ban, H. Kovar, and V. Notario Caveolin-1 (CAV1) Is a Target of EWS/FLI-1 and a Key Determinant of the Oncogenic Phenotype and Tumorigenicity of Ewing's Sarcoma Cells. Cancer Res., October 15, 2006; 66(20): 9937 - 9947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, W. R. Thelin, B. Yang, S. L. Milgram, and K. Jacobson Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides J. Cell Biol., October 9, 2006; 175(1): 169 - 178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Cabrita, F. Jaggi, S. P. Widjaja, and G. Christofori A Functional Interaction between Sprouty Proteins and Caveolin-1 J. Biol. Chem., September 29, 2006; 281(39): 29201 - 2912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Okazaki, F. Tazoe, S. Okazaki, N. Isoo, K. Tsukamoto, M. Sekiya, N. Yahagi, Y. Iizuka, K. Ohashi, T. Kitamine, et al. Increased cholesterol biosynthesis and hypercholesterolemia in mice overexpressing squalene synthase in the liver J. Lipid Res., September 1, 2006; 47(9): 1950 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gosens, G. L. Stelmack, G. Dueck, K. D. McNeill, A. Yamasaki, W. T. Gerthoffer, H. Unruh, A. S. Gounni, J. Zaagsma, and A. J Halayko Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L523 - L534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Jasmin, I. Mercier, J. Dupuis, H. B. Tanowitz, and M. P. Lisanti Short-Term Administration of a Cell-Permeable Caveolin-1 Peptide Prevents the Development of Monocrotaline-Induced Pulmonary Hypertension and Right Ventricular Hypertrophy Circulation, August 29, 2006; 114(9): 912 - 920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-W. Lin, Y.-C. Lin, T.-Y. Chang, S.-H. Tsai, H.-C. Ho, Y.-T. Chen, and V. C. Yang Caveolin-1 Expression Is Associated with Plaque Formation in Hypercholesterolemic Rabbits J. Histochem. Cytochem., August 1, 2006; 54(8): 897 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Frank, M. W.-C. Cheung, S. Pavlides, G. Llaverias, D. S. Park, and M. P. Lisanti Caveolin-1 and regulation of cellular cholesterol homeostasis Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H677 - H686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Nevins and D. C. Thurmond Caveolin-1 Functions as a Novel Cdc42 Guanine Nucleotide Dissociation Inhibitor in Pancreatic beta-Cells J. Biol. Chem., July 14, 2006; 281(28): 18961 - 18972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Prisby, M. K. Wilkerson, E. M. Sokoya, R. M. Bryan Jr., E. Wilson, and M. D. Delp Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms J Appl Physiol, July 1, 2006; 101(1): 348 - 353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhang, K. Furukawa, H.-H. Chen, T. Sakakibara, T. Urano, and K. Furukawa Metastatic Potential of Mouse Lewis Lung Cancer Cells Is Regulated via Ganglioside GM1 by Modulating the Matrix Metalloprotease-9 Localization in Lipid Rafts J. Biol. Chem., June 30, 2006; 281(26): 18145 - 18155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Vihanto, C. Vindis, V. Djonov, D. P. Cerretti, and U. Huynh-Do Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase J. Cell Sci., June 1, 2006; 119(11): 2299 - 2309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Lu, F. Kambe, X. Cao, T. Yoshida, S. Ohmori, K. Murakami, T. Kaji, T. Ishii, D. Zadworny, and H. Seo DHCR24-Knockout Embryonic Fibroblasts Are Susceptible to Serum Withdrawal-Induced Apoptosis Because of Dysfunction of Caveolae and Insulin-Akt-Bad Signaling Endocrinology, June 1, 2006; 147(6): 3123 - 3132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Li, F. Sotgia, M. A. Vuolo, M. Li, W. C. Yang, R. G. Pestell, J. A. Sparano, and M. P. Lisanti Caveolin-1 Mutations in Human Breast Cancer: Functional Association with Estrogen Receptor {alpha}-Positive Status Am. J. Pathol., June 1, 2006; 168(6): 1998 - 2013. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Hassan, T. M. Williams, P. G. Frank, and M. P. Lisanti Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2393 - H2401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Cheng and J. H. Jaggar Genetic ablation of caveolin-1 modifies Ca2+ spark coupling in murine arterial smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2309 - H2319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Torres, J. C. Tapia, D. A. Rodriguez, M. Parraga, P. Lisboa, M. Montoya, L. Leyton, and A. F. G. Quest Caveolin-1 controls cell proliferation and cell death by suppressing expression of the inhibitor of apoptosis protein survivin J. Cell Sci., May 1, 2006; 119(9): 1812 - 1823. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-b. Chen, S. Jia, A. G. King, A. Barker, S.-m. Li, E. Mata-Greenwood, J. Zheng, and R. R. Magness Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy Biol Reprod, May 1, 2006; 74(5): 832 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Schwencke, R. C. Braun-Dullaeus, C. Wunderlich, and R. H. Strasser Caveolae and caveolin in transmembrane signaling: Implications for human disease Cardiovasc Res, April 1, 2006; 70(1): 42 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. O. D. Achcar, Y. Demura, P. R. Rai, L. Taraseviciene-Stewart, M. Kasper, N. F. Voelkel, and C. D. Cool Loss of Caveolin and Heme Oxygenase Expression in Severe Pulmonary Hypertension Chest, March 1, 2006; 129(3): 696 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Yang and X.-F. Ming Recent advances in understanding endothelial dysfunction in atherosclerosis. Clin. Med. Res., March 1, 2006; 4(1): 53 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fleming Segregation and integration: Roles played by caveolae and caveolins in the cardiovascular system Cardiovasc Res, March 1, 2006; 69(4): 784 - 787. [Full Text] [PDF]
|
|
|
|
|
|
O. Feron and J.-L. Balligand Caveolins and the regulation of endothelial nitric oxide synthase in the heart Cardiovasc Res, March 1, 2006; 69(4): 788 - 797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Hardin and J. Vallejo Caveolins in vascular smooth muscle: Form organizing function Cardiovasc Res, March 1, 2006; 69(4): 808 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zulli, B. F. Buxton, M. J. Black, Z. Ming, A. Cameron, and D. L. Hare The Immunoquantification of Caveolin-1 and eNOS in Human and Rabbit Diseased Blood Vessels J. Histochem. Cytochem., February 1, 2006; 54(2): 151 - 159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Miyawaki-Shimizu, D. Predescu, J. Shimizu, M. Broman, S. Predescu, and A. B. Malik siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L405 - L413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hernandez-Deviez, S. Martin, S. H. Laval, H. P. Lo, S. T. Cooper, K. N. North, K. Bushby, and R. G. Parton Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3 Hum. Mol. Genet., January 1, 2006; 15(1): 129 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Sotgia, T. M. Williams, W. Schubert, F. Medina, C. Minetti, R. G. Pestell, and M. P. Lisanti Caveolin-1 Deficiency (-/-) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness Am. J. Pathol., January 1, 2006; 168(1): 292 - 309. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |