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J. Biol. Chem., Vol. 277, Issue 43, 41046-41059, October 25, 2002
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From the
Received for publication, October 1, 2001, and in revised form, August 22, 2002
Hyaluronan (HA) is a large nonsulfated
glycosaminoglycan and an important regulator of angiogenesis, in
particular, the growth and migration of vascular endothelial
cells. We have identified some of the key intermediates
responsible for induction of mitogenesis and wound recovery. Treatment
of bovine aortic endothelial cells with oligosaccharides of hyaluronan
(o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane
translocation of phospholipase C Angiogenesis, the formation of new blood vessels, is essential for
the growth and repair of tissues and is prevalent in a variety of
pathological conditions. Excessive vascularization occurs in rheumatoid
arthritis, diabetic retinopathy, psoriasis, and neoplasia (1, 2).
Conversely, in myocardial infarction and cerebrovascular diseases such
as stroke, there is considerable destruction of the vasculature (3).
Further knowledge of the mechanisms that regulate angiogenesis is
required for the development of strategies to control it.
Hyaluronan (HA)1 is a
nonsulfated, linear glycosaminoglycan, consisting of repeating units of
( The biological functions of HA/o-HA are thought to be initiated through
cell surface receptors or HA-binding proteins, resulting in signal
transduction activation and ultimately mitogenesis (26-28). Native HA
binds to a 34-kDa member of the hyaladherins, and increases general protein tyrosine phosphorylation and that of PLC Materials--
Primary monoclonal antibodies to H-Ras,
anti-phosphotyrosine (PY99), and phospho-ERK-1/ERK-2
(pERK1/2), polyclonal antibodies, and their specific
blocking peptides against G Preparation of o-HA--
The method of preparation is
described in full elsewhere (14). Briefly, native rooster comb HA (500 mg) was dissolved in sodium acetate buffer (50 ml, 0.1 M,
pH 5.4) containing 0.15 M NaCl and treated with 20,000 units of bovine testicular hyaluronidase at 37 °C. After 2, 4, 6, 8, and 24 h, aliquots (10 ml) were treated with 1 ml of
trichloroacetic acid. Mixtures were centrifuged, and supernatants were
dialyzed against distilled water for 24-48 h at 4 °C in Spectra-Por
tubing (Pierce-Warriner, Chester, UK) with at least 4 changes. They
were then re-centrifuged and lyophilized. The powder was dissolved in
20 ml of 0.1% acetic acid and applied to a G50 Sephadex column
(2.6 × 180 cm). Fractions (10 ml) were collected, assayed for
uronic acid, and combined to yield three pools (F1, F2, and F3). The
size range of oligosaccharides in each pool was determined after
incorporation of [3H]glucosamine-labeled HA, SDS-PAGE,
and fluorography, as described previously (14). Successive bands
differed on SDS gels by one disaccharide unit, and precise definition
of the size range was determined by comparison with labeled octa-,
hexa-, and tetrasaccharides of known molecular size. F1, F2, and F3
fractions consisted of disaccharide units of >16, 10-16, and 3-10,
i.e. of approximately >7200, 4500-7200, and 1350-4500 Da,
respectively. Angiogenic activity was determined by adding freeze-dried
samples of each fraction onto the chorioallantoic membrane of the chick
embryo. Only fraction F3 (o-HA) produced a consistent angiogenic
response (14, 36) and was used in this study. Angiogenic activity
resided only in the hyaluronate fragments, because the activity of
hyaluronan preparations digested with denatured hyaluronidase and a
24-h normal digest were found to lack biological activity, suggesting that there was no contamination with vascular permeability factor. Fraction F3 was further digested with Streptomyces
hyaluronidase, and lost its biological activity, as determined by the
chorioallantoic membrane of the chick embryo assay (14).
Isolation and Routine Culture of BAECs--
BAEC were obtained
and characterized as endothelial by the presence of von Willebrand
factor and the uptake of Dil-labeled acetylated low density
lipoprotein, as described previously (12). They were routinely cultured
in Dulbecco's modified Eagle's medium containing 15% fetal calf
serum, 1.5 mM glutamine, 100 IU/ml penicillin, and 50 ng/ml
streptomycin (complete medium). Culture flasks were maintained at
37 °C in a humidified atmosphere of 5% CO2 and 95% air.
Cell Proliferation Studies--
BAEC were seeded at a
concentration of 2 × 104/ml in 2 ml of complete
medium, in triplicate 6-well plates. After attachment, medium was
replaced with serum poor medium (SPM), containing 2.5% fetal calf
serum in which the cells grew at a significantly reduced rate (31). In
some cases, specific enzyme inhibitors, pertussis toxin (10-500 ng/ml,
6 h), Go 6976 (1-100 nM, 24 h),
Wound Recovery Assays (Cell Migration)--
Semiconfluent cells
cultured on Thermanox plastic coverslips in 24-well plates were
grown to confluence in SPM (24-48 h). Medium was replaced with
Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum
and cells were incubated for a further 48 h with or without
inhibitors/antisense reagents, in triplicate, as described above.
Coverslips were washed in PBS, the cellular layer wounded using a
mechanical wounder (37), rinsed again in PBS to remove loose and
dislodged cells, and placed into a fresh 24-well plate containing
inhibitors or the appropriate vehicles. Some coverslips were
immediately fixed in 100% ethanol (time zero controls). o-HA (0.5 µg/ml) was added to some of the wells and the plate was incubated at
37 °C for 18 h. Pilot studies demonstrated that BAEC wounded
under these conditions underwent negligible proliferation up to 24 h (even in the presence of o-HA), however, cell movement resulting in
wound closure was almost complete in cells treated with o-HA at this
concentration after 18 h (native HA had no effect, data not
shown). The coverslips were rinsed (×3) in PBS, fixed in 100% ethanol
(5 min), and allowed to air dry. The mechanical wounder produced 11 parallel lesions 400-µm wide across the cell monolayer.
Movement of cells into the denuded area was quantified using a Seescan
computerized image analysis system (Manchester, UK). Each field of view
covered ~2% of the total coverslip area. For each coverslip, 10 fields of view (each ~2 mm by 1.45 mm) were examined at random. The
lesion area in each field of view was measured and using the data from
time 0 (T0), the wound area was then converted
to give mean % recovery from 3 identically treated coverslips
(%r) using the equation: %r = [1 Intracellular Delivery of Inhibitory
Antibodies--
Semiconfluent BAEC were cultured in 6-well plates in
SPM for 48 h and the medium was replaced with Ca2+-
and Mg2+-free bicarbonate buffer (pH 7.3) containing
glycerol (1.2 M) at 37 °C (38, 39). Cells were placed
immediately on ice for 10 min and the plasma cell membrane made
transiently permeable by addition of chilled
L- Use of Antisense Oligonucleotides--
Phosphorothioate
oligonucleotides corresponding to bovine PKC Western Blotting--
Semiconfluent BAEC cultured in 6- or
24-well plates in SPM (48 h) were incubated with specific enzyme
inhibitors or oligonucleotides before addition of o-HA (1 µg/ml,
2-10 min) as described previously. Total cell lysates were collected
in RIPA buffer and processed as described earlier, whereas plasma cell
membrane and cytoplasmic fractions were separated using a
digitonin-based buffer system (31). Briefly, after washing in ice-cold
PBS, cells in 6-well plates were agitated at 4 °C for 5 min in 300 µl/well of ice-cold buffer containing 140 mM NaCl, 25 mM KCl, 5 mM MgCl2, 2 mM EDTA and EGTA, 10 µg/ml leupeptin and pepstatin, 1 mM phenylmethylsulfonyl fluoride, 20 mM
Tris-HCl (pH 7.5), and 0.5 µg/ml digitonin (44). The buffer, now
containing the cytoplasmic contents was removed and stored at 4 °C.
The remaining "membrane" fraction was again rinsed in ice-cold PBS
and solubilized in 300 µl of the same buffer containing 1% (w/v)
Triton X-100. Both fractions were centrifuged (10,000 × g, 15 min at 4 °C) to remove insoluble debris, and stored at Antibody Specificity Studies--
Equal concentrations of
protein (15 µg) from cell lysates were resolved in duplicate by 12%
SDS-PAGE as described above. Polyclonal antibodies (1 µg/ml) were
incubated with or without matching blocking peptides specific for a
particular epitope (Santa Cruz) (10 µg/ml) overnight at 4 °C on a
rotating mixer. Antibodies were then diluted in the appropriate
blocking buffer, and identical blots were stained with antibody, with
or without peptide treatment using the method described previously.
Specificity of Src antibodies was assessed by comparing staining in
total BAEC extracts separated by Western blotting (as described above)
with positive control cell lysates (WEHI-231). We have previously
characterized all remaining antibodies (31).
Immunoprecipitation Studies--
Equal concentrations of protein
from total cell lysates (100 µg in 0.5 ml of RIPA buffer) were
incubated with 2 µg of primary antibody overnight at 4 °C on a
rotating mixer. Antibodies were then attached to protein A/G-agarose
beads (20 µl, 30 min, 4 °C with continuous mixing). Alternatively,
cell lysates were mixed directly with antibodies already conjugated to
protein-agarose beads (Shc, PLC Determination of ERK1/2 Activity--
The assay kit
was supplied by Upstate Biotechnology, and the protocol was as per the
manufacturers instructions. Briefly, semiconfluent BAEC, cultured in
6-well plates in SPM (48 h), were treated with the appropriate enzyme
inhibitors or neutralizing antibodies as described earlier, prior to
addition of o-HA (1 µg/ml, 5 min). ERK1/2 was immunoprecipitated from
the cell lysates, attached to an agarose complex, and then incubated
with a substrate mixture containing myelin basic protein. After
SDS-PAGE, blots were stained with anti-myelin basic protein antibody
and developed using ECL plus.
Determination of Ras Activity--
Based on the method described
previously (30), semiconfluent BAEC cultured in 6-well plates in SPM
(48 h) were incubated with the appropriate enzyme
inhibitors/oligonucleotides described earlier, prior to addition of
o-HA (1 µg/ml, 2-10 min). Cells were lysed in 500 µl of a
Mg2+ buffer (pH 7.5) containing 25 mM HEPES,
150 mM NaCl, 1% Igepal Ca-630, 10 mM
MgCl2, 1 mM EDTA, 2% glycerol, 10 µg of
leupeptin and aprotinin, and 1 mM sodium orthovanadate on
ice. After centrifugation (10,000 × g/20 min, 4 °C)
to remove insoluble material, 500 µg of each sample was incubated
with 10 µl of Raf-1 RBD-agarose conjugate overnight at 4 °C on a
rotating mixer. Beads containing activated Ras were centrifuged
(13,000 × g/10 min at 4 °C), the supernatant was
removed, and the beads were washed 3× in 0.5 ml of the same buffer.
Protein complexes were solubilized by boiling (5 min) in 50 µl of 2×
Laemmli sample buffer and separated by 12% SDS-PAGE. Resulting blots
were blocked for 1.5 h in TBS-Tween containing 5% defatted milk
and stained overnight with monoclonal anti-H-Ras antibody (RAS10,
1:1000) diluted in the same buffer (4 °C with constant mixing).
Washing, secondary horseradish peroxidase antibody staining, and
protein visualization were carried out as described for the Western
blots above. 10 µg of the original cell lysate was stained with
anti-H-Ras monoclonal antibody and served as a control to show total
Ras content of each sample.
Determination of Src Activity--
Semiconfluent BAEC cultured
in SPM for 48 h in 6-well plates were treated with o-HA (1 µg/ml) for 2, 5, and 10 min or for 8 min, after preincubation for
24 h with PP2 (100 nM) or pertussis toxin (100 ng/ml).
Src was immunoprecipitated in RIPA lysates under nondenaturing
conditions with anti-Src monoclonal antibody (2 µg/sample, overnight
at 4 °C). Bound Src was attached to protein A/G-agarose beads (20 µl/sample, 30 min, 4 °C) and the beads subsequently washed (1×)
with RIPA buffer and then (3×) with Src kinase reaction buffer
containing 100 mM Tris-HCl (pH 7.2), 125 mM
MgCl2, 25 mM MnCl2, 2 mM EGTA, 0.25 mM sodium orthovanadate, and 2 mM dithiothreitol. Samples were incubated at 30 °C in 15 µl of kinase buffer containing 25 µM ATP, 1 µg/sample
of Src substrate peptide, and 10 µCi of [ Analysis of Relative Protein
Concentrations--
Semiquantitative analysis of protein concentration
from Western blots was carried out using a scanning densitometer
(Amersham Biosciences). Results accompanying the figures are given as
numerical increase or decrease compared with the control untreated
cells, which were assigned an arbitrary optical density of 1.0. The
intensity of staining between different gels could not be compared
because of variation in ECL development time.
As a continuation of our previous work (31), we studied in detail
the up-regulation (between 2 and 10 min) of key signaling proteins and
their role in mediating ERK1/2 activation, proliferation, and cell
migration (wound recovery).
Involvement of PLC Isoforms in o-HA Signaling--
Western
blot analysis of BAEC extracts showed expression of PLC Role of PKC Isoforms in o-HA-induced Mediation of ERK1/2
Activation, Mitogenesis, and Wound Recovery in BAEC--
We showed
previously that o-HA-induced ERK1/2 activation, and subsequent
mitogenesis, were partially dependent on activation and associated
plasma membrane translocation of PKC
To distinguish between the functional importance of PKC Activation of Ras in o-HA-treated BAEC: Role in Cell Mitogenesis
and Wound Healing--
Ras activity increased 5.5-fold within 2 min,
and remained higher than control levels 10 min after o-HA treatment (1 µg/ml) (Fig. 4A). Analysis
of total Ras expression indicated equality of protein loading. Pilot
studies using FTI-277, a potent and specific inhibitor of Ras
farnesyltransferase processing (50), showed an optimum inhibitory
concentration that avoided cellular cytotoxicity of 1 µM
over 24 h. BAEC pretreated with FTI-277 before addition of o-HA (1 µg/ml, 5 min) showed a 71% reduction in Ras activity (Fig.
4B), as well as the degree of pERK1/2 formation
(62 and 74%, respectively) and ERK activity (83%) (Fig.
4C). A significant reduction in both o-HA-induced cell
proliferation and wound recovery occurred in the presence of FTI-277
(51%, p < 0.05, Fig. 4D, and 63%,
p < 0.05, Fig. 4E, respectively). To confirm these findings, Ras protein was down-regulated using specific Ras antisense oligonucleotides. Pilot studies showed
exposure of BAEC cultured in SPM to 10 µM antisense
oligonucleotides with 10 µg/ml LipofectAMINE 2000 reduced Ras protein
expression by 70% after 72 h compared with sense controls (Fig.
5A). o-HA (1 µg/ml, 5 min)-induced activation of Ras, measured in Raf-1 kinase RBD-agarose
immunoprecipitates, was reduced by 72% (Fig. 5B), whereas
pERK1/2 formation decreased by 31 and 33%, respectively,
in antisense-treated BAEC (Fig. 5C). BAEC cultured with
antisense reagent in the presence of o-HA demonstrated a significant
reduction in cell proliferation after 72 h (55%,
p < 0.05, Fig. 5D), and wound recovery
after 18 h (62%, p < 0.05, Fig. 5E),
compared with sense controls. Neither Go 6976 nor PKC
antisense reagents notably reduced Ras activity (data not shown),
suggesting Ras activation was independent of PKC.
Involvement of G-proteins in o-HA-induced Signal
Transduction--
Little is known of the mechanisms through which o-HA
receptors initiate second messenger activation. Because we have
demonstrated activation of several isoforms of PLC, we examined the
involvement of heterotrimeric G-proteins in this process. BAEC
expressed the G o-HA Activates Ras via Src and Subsequent Mobilization of Shc in
BAEC--
Previous work has demonstrated that both recruitment to and
interaction of Src with CD44 following HA binding promoted cell migration in human ovarian tumor cells (SK-OV-3 ipc) (54). Activation of Src is one mechanism through which the adapter protein Shc can
stimulate Ras. Specificity of Src antibodies was determined by
comparison with staining in a control whole cell lysate (WEHI-231, Fig.
9A). Activation (within 5 min)
and tyrosine phosphorylation (within 2 min) of Src lasted up to 10 min
in o-HA (1 µg/ml)-treated BAEC (Fig. 9B). The Src family
kinase inhibitor PP2, used at the optimum inhibitory concentration of
100 nM/24 h (determined from pilot studies), reduced
tyrosine phosphorylation and activation of Src in o-HA (1 µg/ml, 5 min)-treated BAEC (68 and 88%, respectively, Fig. 9C).
Pretreatment of BAEC with pertussis toxin had no effect (Fig.
9D), suggesting that upstream activating components may not
include heterotrimeric G proteins.
Preincubation of BAEC with PP2 before addition of o-HA (1 and 0.5 µg/ml, respectively), resulted in a significant decrease in cell
proliferation after 72 h (65%, p < 0.05, Fig.
10A), and wound recovery
after 18 h treatment (82%, p < 0.05, Fig.
10B). PP2 also inhibited activation of Ras (53%, Fig.
10C), pERK1/2 formation (70 and 56%,
respectively), and ERK activity (82%, Fig. 10D), suggesting
that it could be an essential upstream component. We attempted to
identify the mechanism through which c-Src induced activation of Ras.
Ras activation commonly occurs following Shc·Sos·Grb2
complex formation, after autophosphorylation of tyrosine kinase
receptors and subsequent dimerization (55). Shc tyrosine
phosphorylation may also be dependent on Src (54). BAEC expressed both
the p60 and p66 isoforms of Shc, with specificity being determined by
peptide inhibition studies (Fig.
11A). Anti-Shc immunoprecipitates from o-HA (1 µg/ml)-treated cells showed an increase in tyrosine phosphorylation of both p60 and p66 isoforms within 2 min (Fig. 11B), which was reduced by 65% following
preincubation of cells with PP2 (Fig. 11C). Shc
co-precipitated with Src within 2 min of o-HA (1 µg/ml) treatment
(Fig. 11D), and this was reduced in the presence of PP2 (91 and 72% respectively, Fig. 11E). Shc did not co-precipitate
with heterotrimeric G proteins, G
Many receptors activate the Ras-MAP kinase pathway following
redistribution and phosphorylation of the adapter protein Shc and its
subsequent association with the Sos·Grb2 complex (55). BAEC expressed
Sos2 (p170), specificity of the antibody being confirmed using
inhibitory peptides (Fig. 11F). Co-precipitation studies
showed o-HA-induced association of Sos2 with Shc after 2 min of
treatment (Fig. 11G).
Knowledge of the mechanisms through which HA stimulates
angiogenesis will help understand its central role in tissue remodeling and the pathobiology of diseases such as rheumatoid arthritis, diabetic
retinopathy, psoriasis, neoplasia, and stroke. We have previously shown
that o-HA-stimulated second messenger activity in BAEC involved PKC and
MAP kinase (ERK1/2), and resulted in mitogenesis (31). Native, high
molecular weight HA that is anti-angiogenic in vivo had no
effect on signal transduction activity, proliferation, or wound
recovery in these cells, but inhibited activation by o-HA (3, 11, 35),
making BAEC an appropriate and relevant model for the study of the
action of o-HA on vascular EC. In this study we show rapid o-HA-induced
phosphorylation and membrane translocation of PLC Cytoplasmic introduction of inhibitory antibodies to either
G Inhibition of ERK1/2 activity by PP2 prompted us to investigate
possible downstream signaling intermediates. Ras is a key enzyme
necessary for activation of the Raf-1 kinase-Mek-MAP kinase pathway in
response to a variety of ligand-receptor interactions, resulting in
mitogenesis and oncogenesis (64). Activation of Ras by native HA in rat
embryonic fibroblasts (3Y1) (29), and by o-HA in a CD44- and
PKC Conventional growth factor receptor activation of Ras involves
phosphotyrosine-dependent association of Shc with an
autophosphorylated receptor (51). The subsequent interaction between
phosphorylated Shc and the adapter protein Grb2 causes membrane
translocation of the Grb2·Sos complex where Sos mediates guanine
nucleotide exchange on Ras (65). Release of G Elsewhere, we have shown that plasma membrane translocation and
activation of PKC Here we have described for the first time, the signaling mechanisms
through which o-HA stimulates angiogenesis in vascular EC (see Fig.
12). At least two distinct pathways
converge upstream of the MAP kinase resulting in cell activation.
Heterotrimeric G-proteins and in particular, release of G We thank Mick Hoult for help in preparation of
the figures.
*
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.
§
To whom correspondence should be addressed. Fax: 0161247-6365;
E-mail: m.a.slevin@mmu.ac.uk.
Published, JBC Papers in Press, August 22, 2002, DOI 10.1074/jbc.M109443200
2
M. Slevin, S. Kumar, and J. Gaffney,
unpublished data.
The abbreviations used are:
HA, hyaluronan;
EC, endothelial cells, o-HA, oligosaccharides of
hyaluronan;
MAP, mitogen-activated protein kinase;
RHAMM, receptor for
hyaluronan mediated motility;
ERK1/2, extracellular signal-regulated
kinase;
BAEC, bovine aortic endothelial cells;
PLC, phospholipase C;
FITC, fluorescein isothiocyanate;
SPM, serum poor medium;
PBS, phosphate-buffered saline;
FACS, fluorescence-activated cell sorter;
pERK, phospho-ERK-1/ERK-2.
Angiogenic Oligosaccharides of Hyaluronan Induce Multiple
Signaling Pathways Affecting Vascular Endothelial Cell Mitogenic
and Wound Healing Responses*
§,
Department of Biological Sciences,
Manchester Metropolitan University, Manchester M1 5GD, United
Kingdom and the ¶ Department of Pathological Sciences,
Stopford Building, Manchester Victoria University,
Manchester M1 5GD, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (PLC1). Cytoplasmic loading
with inhibitory antibodies to PLC1, G
, and
G
i/o/t/z inhibited activation of
extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the
Gi/o inhibitor, pertussis toxin, reduced o-HA-induced
PLC1 tyrosine phosphorylation, protein kinase C (PKC) and 1/2
membrane translocation, ERK1/2 activation, mitogenesis, and wound
recovery, suggesting a mechanism for o-HA-induced angiogenesis through
G-proteins, PLC1, and PKC. In particular, we demonstrated a possible
role for PKC in mitogenesis and PKC1/2 in wound recovery. Using
antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277,
we showed that o-HA-induced bovine aortic endothelial cell
proliferation, wound recovery, and ERK1/2 activation were also
partially dependent on Ras activation, and that o-HA-stimulated
tyrosine phosphorylation of the adapter protein Shc, as well as its
association with Sos1. Binding of Src to Shc was required for its
activation and for Ras-dependent activation of ERK1/2, cell
proliferation, and wound recovery. Neither Src nor Ras activation was
inhibited by pertussis toxin, suggesting that their activation was
independent of heterotrimeric G-proteins. However, the specific Src
kinase inhibitor PP2 inhibited G subunit co-precipitation with
PLC1, suggesting a possible role for Src in activation of PLC1
and interaction between two distinct o-HA-induced signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,1-4)-D-glucuronic
acid-(,1-3)-N-acetyl-D-glucosamine. HA is
found in its native state as a high molecular mass polymer (>106 kDa) in the extracellular matrix of almost all
animal tissues and in significant quantities in the skin (dermis and
epidermis), brain, and central nervous system (4). Apart from
its role as an inert viscoelastic lubricant, essential for healthy
joint function (5), HA has a crucial role in regulation of the
angiogenic process. In particular, HA is a potent regulator of vascular
endothelial cell (EC) function. Native high molecular weight HA is
anti-angiogenic, inhibiting EC proliferation and migration (6-8) as
well as capillary formation in a three-dimensional collagen gel model
(9), whereas degradation products of specific size (3-10 disaccharide
units; o-HA) stimulate EC proliferation (10-11), migration (12),
sprout formation (13), and result in angiogenesis in the chick
chorioallantoic membrane (14) and in myocardial infarction (15).
Generation of this "angiogenic" o-HA from the naturally occurring
HA polymer is mediated by the endoglycosidase hyaluronidase (16), in
association with tissue damage, inflammatory disease, and in certain
tumors (10, 16, 17). In addition, the degree of invasiveness and metastasis of some tumors has been specifically linked to elevated HA
expression (18-20). Hyaluronidase produced by tumor cells could induce
angiogenesis and be used by tumor cells as a "molecular saboteur"
to depolymerize HA to facilitate invasion (21). Hyaluronan was
intrinsically associated with metastasis in prostate and bladder cancer
(22-24) although high levels of o-HA were shown in children with a
bone-metastasizing variant of renal tumor (25).
1 in a
variety of cell lines, although the role of this protein in mediating
cell behavioral effects is unknown (27). o-HA-induced Ras-dependent activation of mitogen-activated protein (MAP)
kinase was shown in rat embryonic 3Y1 fibroblasts (29). In T24 bladder carcinoma cells, o-HA induced activation of NF-
B via CD44 in a
pathway involving Ras, protein kinase C (PKC) 
, and a complex containing IB kinase 1 and 2 (30). In vascular EC, both CD44 (31,
32) and RHAMM (receptor for HA mediated motility) (33) have been
identified as potential targets for transduction of o-HA-induced
mitogenesis. Inhibition of the CD44/o-HA interaction using anti-CD44
antibodies (J173) reduced proliferation and migration of calf pulmonary
artery EC and human microvessel EC (HMEC-1) (34). In three types of
primary human EC, o-HA bound to the RHAMM receptor and induced tyrosine
phosphorylation of p125FAK, paxillin, and p42/44
extracellular signal-regulated kinase (ERK1/2) resulting in cell
proliferation (33). We have previously demonstrated that o-HA but not
native HA induced up-regulation of the immediate early response genes
c-jun, junB, Krox 20,
Krox 24, and c-fos in bovine aortic EC (BAEC)
(11, 35). Similarly, o-HA induced a rapid CD44- dependent
activation of multiple isoforms of PKC (, , and 
), Raf-1
kinase, MEK-1, and ERK1/2 resulting in mitogenesis in these cells (31).
These limited studies have so far failed to identify all of the key
intermediates involved in transduction of the o-HA-induced mitogenic
and wound recovery responses in vascular EC. This information could be
important in the development of novel therapeutic strategies for
treatment of angiogenic diseases. In this study, an extension of our
findings from previous work (31), we have examined in detail rapid
up-regulation of associated signaling proteins and have characterized
the pathways responsible for o-HA-induced angiogenesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i/o/t/z,
Gs/olf, Gq/11, G, PLC1, PLC2,
PLC1-3, PLC
1, PKC, PKC1-2, PKC, Sos, Shc, and
-actin as well as mouse B cell lymphoma cell lysate (WEHI-231) were
obtained from Autogen Bioclear (Wiltshire, United Kingdom). PKC
isoform-specific inhibitors (Go 6976 and PKC translocation inhibitor
peptide), H-Ras inhibitor FTI-277, Gq protein inhibitor GP
antagonist-2A, Gi/o inhibitor pertussis toxin, and PP2
Src family inhibitor were from Calbiochem. Manufacture of
phosphorothioate antisense and matching sense oligonucleotides directed
to bovine PKC was by VHBio (Newcastle-upon-Tyne, UK). Antisense, matching scrambled, and FITC-labeled oligonucleotides directed against bovine PKC1-2 and H-Ras
were from Biognostik (Gottingen, Germany). Ras and ERK activity assay
kits, as well as the Src substrate peptide (KVEKIGEGTYGVVYK), mouse
monoclonal anti-Src family tyrosine kinase, and anti-phospho-Src
antibodies were all from Upstate Biotechnology (Buckingham, UK).
Thermanox plastic coverslips were from Nunc (Naperville, IL),
ECL and ECL plus kits were from Amersham Biosciences (Bucks,
UK) and the protein detection reagent was obtained from Bio-Rad. All
other materials and chemicals were from Sigma.
-translocation inhibitor (ti, 1-20 µM, 24 h), FTI 277 (100 nm to 5 µM, 24 h), GP Ant-2A (100 nM-25 µM, 4 h), PP2 (10 nM
TO 10 µM), or sense/antisense oligonucleotides
directed toward PKC/ and H-Ras (10 µM, 72 h) were added before incubation with o-HA (1 µg/ml) for a further 72 h. Control wells contained appropriate
concentrations of the vehicle (Me2SO or ethanol).
Concentration ranges and preincubation times of inhibitors were based
on previously published information and our own unpublished pilot
studies.2 Trypan blue
exclusion studies confirmed that the inhibitors were not cytotoxic to
cells at the concentrations tested (data not included) and wells
treated with inhibitors without o-HA were included as controls. Fresh
medium and inhibitors were added every 72 h where necessary. After
72 h, cells were washed in PBS, detached in 0.05% trypsin/PBS,
and counted on a Coulter counter (Coulter Electronics, Hialeah, FL) set
to a threshold of 30 µm. Statistical significance was determined by
one-way analysis of variance.
(wound area at Tt/wound area at
T0)] × 100. Where Tt is the
wounded area 18 h post-injury. Statistical significance was
determined by one-way analysis of variance.
-lysophosphatidylcholine (40 µg/ml) for a further 7 min. Pre-warmed SPM (1 ml, 37 °C), containing 5 µg of rabbit
anti-mouse control IgG, FITC-labeled IgG, or antibodies to PLC1-2,
PLC1-3, Gi/o/t/z, G, Gs/olf, or
Gq/11 was added and the cells were incubated at 37 °C
for a further 1 h. Cells regained their impermeability during this
phase as determined by the recovery of trypan blue exclusion (data not
shown). Uptake and cytoplasmic expression of antibodies after 1 h
was confirmed by fluorescent microscopy of FITC-labeled cells, using a
Leitz microscope (Leica, Bensheim, Germany) and the
appropriate filter, and by FACS analysis (FACScan, BD
Biosciences) following washing (PBS), trypsinization, and
fixation of cells in 4% formaldehyde, PBS. After a 1-h
recovery, o-HA (1 µg/ml, 5 min) was added to some of the wells that
were then washed in PBS and cell lysates were collected in 0.5 ml of
ice-cold radioimmunoprecipitation (RIPA) buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5% sodium
deoxycholate, 0.5% (w/v) Nonidet P-40, 0.1% SDS, 1 mM
Na3VO4, and 5 µg/ml aprotinin (40). After
sonication, lysates were centrifuged (10,000 × g, 15 min at 4 °C) and the supernatants were collected and stored at
70 °C.
12 and
H-Ras were synthesized by Biognostik (GmbH, Gottingen, Germany) using R.A.D.A.R sequence design. Sequences of antisense nucleotides for PKC12 were 5'-TCAGCTGGAATCTAAATG and
matched scrambled sense/FITC-labeled nucleotides were
5'-ACTACTACACTAGACTAC (41), whereas H-Ras antisense
nucleotides were 5'-GCTTATACTCCGTCATTG and matched sense nucleotides
were 5'-GTTACTGCCTCATATTCG (42). Antisense and control sense
oligonucleotides directed toward bovine PKC were
synthesized by VHBio (Newcastle upon Tyne, UK). Antisense sequences
were 5'-GTCCCTCGCCGCCTCCTG-3' and sense, 5'-GTCCTCCGCCGCTCCCTG-3' as
described elsewhere (43). Semiconfluent BAEC were cultured at 37 °C
in 24-well plates in SPM with or without oligonucleotides (10 µM/72 h) together with LipofectAMINE 2000 (Invitrogen, 10 µg/ml). Cell lysates were collected at 4 °C
in RIPA buffer (300 µl) and processed as described previously (31).
Delivery of oligonucleotides into the cell cytoplasm was monitored by
addition of FITC-labeled PKC oligonucleotides (10 µM,
4-72 h) to semiconfluent BAEC cultured on glass coverslips in SPM.
Pilot studies were carried out to optimize the effects of
oligonucleotides, and showed a notable reduction in specific protein
expression determined by Western blotting, after 72 h exposure to
10 µM antisense oligonucleotide.
70 °C. Complete separation of cytoplasmic and membrane
fractions was demonstrated using the lactate dehydrogenase assay.
Protein concentration of cell lysates was determined using a
modification of the Bradford assay (Bio-Rad) and equal quantities of
protein (15 µg) were mixed with 2× Laemmli sample buffer, vortex
mixed, and boiled in a water bath for 15 min. Samples were separated along with prestained molecular weight markers (32,000-200,000) by 12% SDS-PAGE. Proteins were electroblotted (Hoefer, Bucks, UK) onto
nitrocellulose filters (1 h) and the filters were blocked for 1 h
at room temperature in TBS-Tween (pH 7.4) containing 5% (w/v)
de-fatted milk (PKC antibodies) or containing 1% (w/v) bovine serum
albumin (all other antibodies). Filters were stained with the following
primary antibodies diluted in the appropriate blocking buffer,
overnight at 4 °C on a rotating mixer: rabbit polyclonal anti-PLC1-2, PLC1-3, PLC1 (1:500); Gi/o/t/z,
G, Gs/olf, Gq/11 (1:750); PKC,
PKC1-2, PKC (1:100); H-Ras (1:400), Shc and Sos (1:1000); mouse
monoclonal antibodies to pERK1/2, Src, and phospho-Src
(1:1000); phosphotyrosine PY99 (1:1500), and phospho-myelin basic
protein (1:1500). After washing (5× 10 min in TBS-Tween at room
temperature), filters were stained with either goat anti-rabbit or
rabbit anti-mouse horseradish peroxidase-conjugated secondary
antibodies diluted in TBS-Tween containing 5% (w/v) de-fatted milk
(1:1000, 1 h, room temperature) with continuous mixing. After a
further 5 washes in TBS-Tween, proteins were visualized using ECL
chemiluminescent detection.
1, 20 µl, overnight, 4 °C). The
beads were pelleted by centrifugation (13,000 × g/10
min, 4 °C), the supernatant was removed, and the pellet washed 3×
in ice-cold RIPA buffer (0.5 ml). Excess buffer was removed from the
beads and protein-antibody complexes were solubilized in 50 µl of 2×
Laemmli sample buffer and subjected, in duplicate, to 12% SDS-PAGE
followed by blotting as described previously. One of the blots was
stained with the original immunoprecipitating primary antibody to
confirm equality of protein loading.
-32P]ATP as
described previously (45). The reaction was terminated by addition of
20 µl of 2× Laemmli sample buffer, the samples were boiled for 5 min, and proteins were separated by SDS-PAGE. Incorporated
radioactivity was visualized by autoradiography of the dried gel.
Active Src kinase enzyme (Upstate Biotech) was used as a positive
control in pilot experiments used to optimize the protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
1, 2, and 1 (Fig.
1A). Disappearance of these
proteins in antibody mixtures pretreated with specific
peptides that blocked the antibody epitope confirmed their
identity. Cytoplasm to plasma membrane translocation of PLC
isoforms is usually associated with their activation (46, 47).
o-HA (1 µg/ml)-treated BAEC showed a rapid increase (within
2 min and maintained after 5 and 10 min) in plasma membrane
expression of PLC1, and more transiently (maximal after 5 min), in
PLC1 and 2 (Fig. 1B). An increase in phosphotyrosine staining (Tyr(P)-99) of anti-PLC1 immunoprecipitates also
occurred within 2 min of treatment (Fig. 1C). We next
examined the involvement of PLC isoforms in ERK1/2 activation.
L--Lysophospatidylcholine-permeabilized BAEC
incorporated higher levels of FITC-labeled IgG than nonpermeabilized cells, as shown by fluorescent microscopy and FACS analysis (Fig. 1D, i and ii, respectively).
Degradation of loaded antibodies occurred after about 4 h making
this technique unsuitable for long term studies (data not shown). Only
anti-PLC1 (5 µg/ml) reduced the o-HA (1 µg/ml, 5 min)-induced
pERK1/2 formation (74 and 80%) and ERK activity (70%),
respectively, compared with IgG control antibodies. (Fig.
1E). These results suggest that o-HA-induced signaling
through PLC1 may be primarily involved in stimulating
mitogenesis.
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Fig. 1.
Involvement of PLC in o-HA-induced
signaling. A, Western blot detected expression
of the 1, 1, 2, and 1 isoforms of PLC in BAEC with (+,
lane 2) and without (, lane 1) neutralizing
peptide treatment. B, membrane translocation of PLC1 was
observed in Western blots within 2 min (lanes 2-4,
left panel). Translocation of 1 and 2 isoforms was more
transient, reaching a maximum after 5 min (lane 3,
middle and right panels). C, Western blots
showed an increase in antiphosphotyrosine staining of PLC1 (in
PLC1 immunoprecipitates) increasing after 2, 5, and 10 min treatment
with o-HA (1 µg/ml) (lanes 2-4). Total PLC1 expression
demonstrates equality of loading and a comparison with
pERK1/2 expression together with -actin loading control
is shown in the same samples. D, BAEC were made transiently
permeable with L--lysophosphatidylcholine and loaded
with FITC-labeled IgG antibodies. Delivery was monitored by (i)
fluorescent microscopy where + represents permeabilized cells, and (ii)
FACS analysis where M1 represents the fluorescent intensity
beyond which 95% of the control cells were negative. E,
BAEC, preloaded with anti-PLC1, antibodies (5 µg/ml) reduced o-HA
(1 µg/ml, 5 min), and induced pERK1/2 expression and
activity (lane 3). All experiments were carried out at least
three times and a representative example is shown. WB,
Western blot. MBP, myosin basic protein.
, -1, -2, and - (31).
Pilot studies based on previous published data (48) showed that Go 6976 (100 nM/24 h preincubation) reduced plasma membrane
translocation of PKC, -1, and -2 (84, 73, and 90%,
respectively) but not after addition of o-HA (1 µg/ml, 5 min).
Similarly, the -translocation inhibitor (TI, 20 µM/24 h preincubation) attenuated o-HA-induced plasma
membrane translocation of PKC (71%), but had no effect on PKC,
-1, or -2 (Fig. 2, A and
B) (49). The inhibitors were not cytotoxic as shown by cell
growth assays in SPM and trypan blue exclusion. Only Go 6976 (100 nM/24 h) notably reduced o-HA-induced formation of
pERK1/2 (75 and 73%, respectively) and ERK activity (78%)
compared with untreated cells (Fig. 2C). Pretreatment with
Go 6976 significantly inhibited o-HA-induced cell proliferation (70%)
compared with untreated cells (p < 0.05) although
TI had no inhibitory effect (Fig. 2D). Similarly, the
presence of Go 6976 almost completely inhibited o-HA-induced wound
healing (p < 0.001), although TI had a much smaller
(18%) but still significant effect (p < 0.05, Fig.
2E).
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Fig. 2.
Specific isoform inhibitors of PKC affect
o-HA-induced signal transduction, mitogenesis, and wound recovery.
A, Go 6976 (100 nM), and B, TI (20 µM) inhibited plasma membrane translocation of PKC,
1, 2, and , respectively, in o-HA (1 µg/ml, 5 min)-treated
cells (lane 3). C, preincubation with
Go 6976 (100 nM/24 h) reduced o-HA (1 µg/ml, 5 min)-induced pERK1/2 expression and activity (lane
3). D, Go 6976 (100 nM) significantly
inhibited o-HA (1 µg/ml)-induced cell proliferation over 72 h.
E, Go 6976 strongly inhibited o-HA (0.5 µg/ml/18
h)-induced wound healing (lane 6, *, p < 0.01), whereas TI had a much weaker, but still significant effect
(lane 5, *, p < 0.05). In the bar
chart, the first unfilled bar denotes cells with 100%
re-growth and the second bar (T0)
shows totally denuded, freshly wounded cells. All experiments were
carried out at least three times and a representative example is shown.
WB, Western blot. MBP, myosin basic
protein.
and -
isoforms, BAEC were exposed to specific antisense or scrambled oligonucleotides. Fluorescent monitoring of FITC-labeled scrambled PKC showed appreciable cytoplasmic incorporation over
24-72 h (Fig. 3A). Addition
of PKC antisense reagent (10 µM) with
LipofectAMINE 2000 (10 µg/ml), to semiconfluent BAEC, resulted in
down-regulation in PKC protein expression after 72 h (55%)
without affecting 1, 2, or isoforms, compared with the
controls. PKC1/2 antisense reagent (10 µM)
produced 70 and 75% reduction in expression of those isoforms,
respectively, without affecting PKC or - (Fig. 3B).
Antisense inhibition of PKC reduced pERK1/2
expression in o-HA (1 µg/ml, 5 min)-treated cells (73 and 72%,
respectively), whereas PKC inhibition resulted in a
smaller reduction (41 and 37%, respectively) (Fig. 3C).
Scrambled oligonucleotides had no effect on pERK1/2
expression. Incorporation of PKC antisense
oligonucleotide also significantly reduced o-HA-induced BAEC
proliferation by 49% (p < 0.05, analysis of variance)
but only weakly inhibited wound recovery (15% p < 0.05) (Fig. 3, D and E, respectively). Treatment
with PKC antisense oligonucleotide had no effect on proliferation, but strongly inhibited o-HA-induced wound recovery (85%, p < 0.001). In conclusion, the isoform may
be more important in regulating mitogenesis through ERK1/2, whereas the
isoforms, and to a lesser extent , are primarily involved in
cell migration/wound recovery.
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Fig. 3.
Effect of antisense reagents on PKC-induced
signaling and cell growth. A, shows oligonucleotide
(PKC control sequence-FITC) incorporation into BAEC over
0-72 h. Uptake was observed after 24 h (panel 3).
B, BAEC exposed to specific PKC and 1/2
antisense oligonucleotides (lane 3) (10 µM,
72 h), showed protein down-regulation compared with cells treated
with scrambled control oligonucleotides (lane 2) and
untreated cells (lane 1). C, after antisense
treatment, BAEC were exposed to o-HA (1 µg/ml, 5 min). A reduction in
pERK1/2 expression was seen in the presence of
PKC (left panel, lane 3), and to a
lesser extent PKC1/2 (left panel, lane
4) antisense oligonucleotides. D, antisense
down-regulation of PKC protein expression significantly reduced
o-HA-induced cell proliferation (*, p < 0.05).
E, PKC1/2 antisense treatment significantly
inhibited wound recovery (lane 7, *, p < 0.05). In the bar chart, the first unfilled bar
denotes cells with 100% re-growth, whereas the second bar
(T0), shows freshly wounded, maximally denuded
cells. All experiments were repeated at least three times and the
figure shows a representative example. WB, Western
blot.
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Fig. 4.
Inhibition of o-HA-induced Ras activation by
FTI-277 reduces BAEC mitogenesis and wound recovery. A,
BAEC showed an increase in active Ras expression within 2 min of o-HA
(1 µg/ml) treatment (lanes 2-4). Total Ras expression is
shown as a loading control. B, FTI-277 (1 µM,
24 h) inhibited o-HA (1 µg/ml, 5 min)-induced Ras activation
(lane 3). C, o-HA (1 µg/ml, 5 min)-induced
pERK1/2 expression and activity were markedly reduced in
FTI-277 (1 µM, 24 h)-treated cells (lane
3). -Actin loading controls are also shown. D, o-HA
(1 µg/ml)-induced BAEC proliferation was significantly reduced after
72 h (*, p < 0.05) in the presence of FTI-277 (1 µM). E, FTI-277 (1 µM, 24 h) significantly inhibited o-HA (0.5 µg/ml, 18 h)-induced wound
recovery (lane 5, *, p < 0.05). In the
bar chart, the first unfilled bar denotes cells
with 100% re-growth, whereas the second bar
(T0) shows freshly wounded, maximally denuded
cells. All experiments were repeated at least three times and shows a
representative example. WB, Western blot. MBP,
myosin basic protein.
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Fig. 5.
Down-regulation of Ras protein using
antisense reagents reduces o-HA-induced signal transduction and
angiogenesis in BAEC. A, BAEC exposed to Ras
antisense oligonucleotides (left panel, lane 2,
10 µM, 72 h) showed a reduction in Ras protein
expression compared with control sense-treated cells (right
panel). B, cells stimulated with o-HA (1 µg/ml, 5 min) after Ras antisense treatment showed a reduction in
activation of Ras protein (left panel, lane 3).
-Actin protein loading controls are also shown. C shows a
reduction in pERK1/2 expression in cells treated as in
B above (lane 4). D, antisense
treatment was sufficient to significantly reduce o-HA (1 µg/ml,
72 h)-induced cell proliferation, and E, o-HA (0.5 µg/ml, 18 h)-induced wound recovery (lane 5) (*,
p < 0.05, in both cases). In the bar chart
(E), the first unfilled bar denotes cells with
100% re-growth and the second bar
(T0) shows freshly wounded, maximally denuded
cells. All experiments were repeated at least three times and shows a
representative example. WB, Western blot.
i/o/t/z, G subunit (after o-HA
treatment 1 µg/ml, 5 min), Gs/olf and
Gq/11 subtypes (Fig.
6A). Antibodies treated with
peptide inhibitors (+) served as negative controls to confirm
expression of the protein and specificity of the antibodies.
Cytoplasmic loading of either anti-Gi/o/t/z or anti-G
(5 µg/ml as described earlier) inhibited o-HA (1 µg/ml, 5 min)-induced pERK1/2 expression (66 and 62% and 74 and
68%, respectively) and ERK activity by 70 and 75%, respectively (Fig.
6B), whereas anti-Gs/olf and
Gq/11 had no inhibitory effect. Activation of G
proteins causes their translocation from the plasma membrane to the
cytoplasm together with formation of both G and G subunits,
the G subunits remaining plasma membrane-bound (51).
Gi/o/t/z proteins were detected in the cytoplasmic
fraction and G subunits in the membrane fraction, within 2 min, in
o-HA (1 µg/ml)-treated BAEC (Fig. 6C). Pretreatment of
BAEC with the specific Gi/o protein inhibitor pertussis toxin (52) (100 ng/ml, 6 h), reduced o-HA (1 µg/ml, 5 min) induced cytoplasmic expression of Gi/o/t/z proteins
by 65%, and appearance of G subunits in the membrane fraction by ~95% (Fig. 6D). No cellular cytotoxicity was found at
this concentration. Pretreatment of BAEC with pertussis toxin markedly
reduced o-HA (1 µg/ml, 5 min) induced PKC, -1, and -2
membrane translocation (92, 55, and 77%, respectively, Fig.
7A), pERK1/2
expression (50 and 55%, respectively) and activity (51%) (Fig.
7B). Pertussis toxin at the same concentration significantly inhibited both proliferation (72 h) and wound recovery (18 h) of BAEC
(66%, p < 0.05, Fig. 7C and 47%,
p < 0.05, Fig. 7D, respectively), compared
with vehicle-only treated cells. o-HA-induced Ras activation was not
affected in pertussis toxin-treated cells (Fig. 7E),
suggesting it acts on a separate pathway. When the previous experiments
were repeated using the specific Gq inhibitor GP ant-2A
(53), there was no demonstrable effect on o-HA-induced mitogenic or
wound healing responses (data not shown). These results suggest that activation of Gi/o proteins in o-HA-treated BAEC is at
least in part responsible for control of tyrosine kinase-associated events resulting in mitogenesis and wound healing. We next performed co-precipitation studies to identify possible binding partners for
Gi/o proteins and G() subunits. There was no
association between Gi/o/t/z and G subunit
immunoprecipitates from o-HA (1 µg/ml, 2, 5, and 10 min)-treated
cells, and PLC1, -2, or -3 (data not shown). However, G
subunits co-precipitated with PLC1 within 2 min (Fig.
8A), and this was reduced by
80% after preincubation of BAEC with pertussis toxin (Fig.
8B). Tyrosine phosphorylation of PLC1 was also partially
inhibited by 64% following preincubation with pertussis toxin in
o-HA-treated cells (Fig. 8C). Finally, preincubation with
the Src kinase inhibitor PP2 (100 nM, 24 h), reduced
PLC1 association with G subunits (90%) in G
immunoprecipitates (Fig. 8D). These results suggest a role for G protein regulation independent of Ras in a distinct signal transduction pathway.
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Fig. 6.
o-HA induces activation of pertussis
toxin-sensitive G-proteins in BAEC. A, expression of
G i/o/t/z, Gs/olf, Gq/11,
and G subunits (after o-HA treatment 1 µg/ml, 5 min) in BAEC was
demonstrated by Western blotting (WB). Confirmation of
specificity is shown by the disappearance of the protein band in
specific inhibitory peptide-treated antibody mixtures (shown as +,
lane 2). B, cell cytoplasm loading of antibodies
to either Gi/o/t/z (lane 3) or G subunits
(lane 4) inhibited o-HA-induced pERK1/2
expression and ERK activity. C shows appearance of G
subunits in the membrane fraction (right panel) and
Gi/o/t/z in the cytoplasmic fraction (left
panel) within 2 min in o-HA (1 µg/ml)-treated cells (lanes
2-4). D, preincubation of cells with pertussis toxin
(P.toxin) (100 ng/ml, 6 h) inhibited o-HA-induced (1 µg/ml, 5 min) redistribution and separation of G-proteins (lane
3). All experiments were repeated at least three times and a
representative example is shown. MBP, myosin basic
protein.
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Fig. 7.
Pertussis toxin inhibits o-HA-induced signal
transduction and mitogenesis. A, BAEC pretreated with
pertussis toxin (P.toxin) (100 ng/ml, 6 h) reduced
plasma membrane translocation of PKC , 1, and 2 isoforms in the
presence of o-HA (1 µg/ml, 5 min) (lane 3). B,
pretreatment of BAEC with pertussis toxin also inhibited o-HA-induced
pERK1/2 expression and ERK activity (lane 3), as
well as, C, cell proliferation after 72 h (*,
p < 0.05). D, o-HA (0.5 µg/ml, 18 h)-induced wound recovery was significantly reduced in the presence of
the toxin (lane 5, *, p < 0.05). In the
bar chart, the first unfilled bar denotes cells
with 100% re-growth, and the second bar
(T0) shows freshly wounded maximally denuded
cells. E, shows the inability of pertussis toxin
to inhibit Ras activity in o-HA (1 µg/ml, 5 min)-treated cell lysates
processed as described previously (lane 3). All experiments
were repeated at least three times and the above shows a representative
example. WB, Western blot. MBP, myosin basic
protein.
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Fig. 8.
G subunits
co-precipitate with PLC: sensitivity to both
pertussis toxin and PP2. A, immunoprecipitated G
subunits co-precipitated with PLC1 within 2 min of o-HA (1 µg/ml)
treatment (lanes 2-4). B, preincubation of BAEC
with pertussis toxin (P.toxin) (100 ng/ml, 6 h) reduced
o-HA (1 µg/ml, 5 min)-induced association of G subunits with
PLC1 in G immunoprecipitates (lane 3). C,
the same treatment also reduced o-HA (1 µg/ml, 5 min)-induced
phosphorylation of PLC1 (lane 3). D,
preincubation of cells with PP2 (100 nM/24 h) also
inhibited o-HA (1 µg/ml, 5 min)-induced G subunit co-precipitation
with PLC1 (lane 3), measured in G immunoprecipitated
cell lysates. All experiments were repeated at least three times and
the shows a representative example. WB, Western blot.
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Fig. 9.
o-HA induces activation of Src family kinase
which is sensitive to PP2. A shows expression of the
Src family kinase in BAEC (lane 2), compared with a positive
control cell lysate (lane 1). B, o-HA (1 µg/ml)-induced phosphorylation and activation of Src within 5 min of
treatment (lanes 2-4). Total Src expression is shown as a
loading control. C shows PP2 (100 nM, 24 h)
inhibition of o-HA (1 µg/ml, 5 min)-induced activation and tyrosine
phosphorylation of Src (lane 3). E shows that
preincubation of BAEC with pertussis toxin (P.toxin) (100 ng/ml, 6 h) had no effect on o-HA (1 µg/ml, 10 min)-induced Src
activation or tyrosine phosphorylation of proteins (lane 3).
All experiments were repeated at least three times and shows a
representative example. WB, Western blot.
subunits, or PLC1 (data not
included).
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Fig. 10.
PP2 inhibits o-HA-induced signal
transduction and mitogenesis in BAEC. A, PP2 (100 nM, 24 h) significantly inhibited o-HA (1 µg/ml)-induced cell proliferation after 72 h (*,
p < 0.05). B, PP2 (100 nM for
24 h) significantly inhibited o-HA (0.5 µg/ml, 18 h)-induced wound recovery (lane 5, *, p < 0.05). In the bar chart, the first unfilled bar
denotes cells with 100% re-growth and the second bar
(T0) shows freshly wounded maximally denuded
cells. C, PP2 at the same concentration inhibited
by o-HA (1 µg/ml, 5 min)-induced Ras activation (lane 3),
as well as D, pERK1/2 formation and ERK activity
(lane 3). All experiments were repeated at least three times
and shows a representative example. WB, Western blot.
MBP, myosin basic protein.
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Fig. 11.
o-HA induces Src-dependent mobilization of
Shc and its association with Sos. A, expression of Shc
(p60/p66) is shown in BAEC lysates, with (+, lane 2) or
without ( , lane 1) specific inhibitory antibody blocking
peptide. B, tyrosine phosphorylation of Shc was increased
within 2 min treatment with o-HA (1 µg/ml) in Shc immunoprecipitates
(lanes 2-4). C, PP2 (100 nM, 24 h) reduced o-HA (1 µg/ml, 5 min)-induced Shc tyrosine phosphorylation
(lane 3). D, shows co-precipitation of Src within
2 min of treatment with o-HA (1 µg/ml) following Western blotting of
Shc immunoprecipitates (lanes 2-4). E, o-HA (1 µg/ml, 5 min)-mediated co-precipitation of Src with Shc was inhibited
in the presence of PP2 (100 nM/24 h) (lane 3).
F, expression of Sos 2 (p170) in BAEC lysates. Specificity
of the antibody was shown following disappearance of the band after
treatment with epitope binding inhibitory peptide (+, lane
2). G, co-precipitation of Shc with Sos 2 occurred
within 2 min of o-HA treatment in Sos immunoprecipitates (lanes
2-4). All experiments were carried out at least three times and a
representative example is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, which is the
natural activator of PKC (56). Cytoplasmic incorporation of
anti-PLC1 antibodies resulted in a notable reduction in ERK1/2
activation, suggesting a role for PLC1 in regulation of mitogenesis.
PLC 1 and 2 isoforms, although transiently translocated, had no
effect on ERK1/2 activity. Other studies have shown that mobilization
of PLC isoforms could be associated with heterotrimeric G-protein
involvement in the signaling process (57).
s/olf or Gq/11 had no effect on
o-HA-induced ERK1/2 activity, suggesting that adenyl cyclase-associated
and pertussis toxin-insensitive pathways were not involved (57).
Furthermore, although the specific Gq/11 enzyme
inhibitor, GP ant-2A (53), attenuated bombesin-induced pERK1/2 expression in our cell system, it had no effect on
o-HA-induced signaling or mitogenesis.2 We found a rapid
increase in expression of G subunits in the plasma membrane and
Gi/o/t/z in the cytoplasmic fraction of o-HA-treated cells. Cytoplasmic introduction of antibodies to either of these proteins reduced ERK1/2 activity, suggesting the involvement of pertussis toxin-sensitive G-proteins. Formation of G() subunits as well as their cell compartmentalization is a feature of their activation (50), and can result in complex formation and activation of
multiple isoforms of PLC (57). Other studies have shown the ability of
G dimers to activate phosphoinositol-3-kinase resulting in cell
proliferation and survival, usually involving activation of the
transcription factor NF-B (58, 59). Although HA fragments can also
activate NF-B in some human carcinoma cell lines (30), our
unpublished data2 showed that phosphoinositol-3-kinase was
not activated by o-HA in BAEC, and furthermore, that wortmannin, a
specific inhibitor of phosphoinositol-3-kinase (60), had no effect on
o-HA-induced signaling events. We demonstrated co-precipitation of G
subunits with PLC1 after o-HA treatment, and also that inhibition of
Gi/o proteins using pertussis toxin was sufficient to
inhibit phosphorylation of PLC1, plasma membrane translocation of
PKC isoforms, and ERK1/2 activity. Cell proliferation and wound
recovery were also attenuated suggesting that the association of G
with PLC1 has an important role in o-HA-induced cell signaling.
Other studies have described an association between PLC1 and
Gi proteins in BAEC resulting in PLC1 activation
(61). This association usually occurred in the presence of another
tyrosine kinase-activated co-precipitant such as Src kinase. For
example, in human embryonic intestinal epithelial cells, leukotriene
D4 induced co-precipitation of G subunits with
PLC. This interaction was blocked by PP2, suggesting that physical
association of Src with PLC after agonist stimulation could result
in PLC·G complex formation (62). Similarly, agonist
activation of Src kinase in a variety of cell lines was sensitive to
pertussis toxin indicating a role for Gi-coupled receptors (63). In this study, we showed a rapid increase in tyrosine
phosphorylation (within 2 min), and activation (within 5 min) of the
Src family kinase in o-HA-treated cells. The importance of Src
activation was demonstrated by a significant reduction in
o-HA-induced proliferation and wound recovery in PP2-treated cells,
whereas preincubation with PP2 reduced o-HA-induced G·PLC1 complex formation, suggesting that Src might be a necessary component of PLC1 activation. We could not, however, show direct association of Src with PLC1. Furthermore, pertussis toxin did not inhibit activation of Src, suggesting its activation was independent of heterotrimeric G-proteins.
-dependent manner in human T24 bladder carcinoma
cells, has previously been demonstrated (30). We show an o-HA-induced
increase in Ras activity within 2 min, whereas ERK1/2 activity, cell
proliferation, and wound recovery were inhibited by the Ras
farnesyltransferase inhibitor FTI-277 and Ras antisense reagent.
Activation of Ras was independent of heterotrimeric G proteins and PKC,
because neither pertussis toxin nor Go 6976 were inhibitory. Also, the
direct PKC activator phorbol 12,13-dibutyrate was unable to activate
Ras.2
subunits from
pertussis toxin-sensitive heterotrimeric G-proteins can also result in
tyrosine phosphorylation of Shc and induction of Grb2·Sos complex
formation (52) or Shc·Grb2·Src complex formation (57). In this
study, Shc became tyrosine phosphorylated within 2 min of treatment
with o-HA, and furthermore, co-precipitated strongly with Src. PP2 treatment was sufficient to reduce Shc tyrosine phosphorylation, complex formation between Src and Shc, and subsequent Ras activity. Finally, we demonstrated co-precipitation of Shc with Sos, suggesting that o-HA activates Ras through Src-dependent activation of
Shc-Grb2-Sos. This mechanism of Ras activation has previously been
described following treatment of human epidermoid carcinoma cells
(A431) with (S)-12- hydroxyeicosatetraenoic acid (54).
, 1, 2, and were important in formation of pERK1/2 and cell mitogenesis (31). We have examined the
function of these isoforms in more detail. Activation of ERK1/2 and
mitogenesis occurred mainly through PKC, whereas PKC1/2 had a
much weaker effect but was necessary for efficient wound recovery in
o-HA-treated cells. Other authors have highlighted the importance of
PKC in mediating cell proliferation induced by phorbol esters in rat microvessel EC (67), and in vascular endothelial growth
factor-treated human umbilical vein EC (68). Similarly, a role
for PKC in regulation of cell migration, in human coronary smooth
muscle cells induced by high glucose (69), and in porcine vascular smooth muscle cells treated with insulin-like growth factor (70), has
been described. Promotion of cell movement through activation of PKC
may involve its localization to specific microtubule-organizing centers
and microtubules in the cytoskeleton (71). HA-induced motility of
Ras-transformed 10T1/2 (C3) fibroblasts in a PKC-dependent manner that was also associated with rapid uptake of HA by both CD44
and RHAMM receptors (72). Our own data2 has shown that
inhibitors of tyrosine phosphorylation (genistein) and Mek-1 (PD98059)
were sufficient to reduce wound recovery, suggesting these signaling
pathways are also important.
()
subunits seem to be important in mediating PLC1 activation of PKC,
whereas separately, Src family kinases activate Ras through
Shc·Grb2·Sos complex formation. The pathways may overlap because
PP2 inhibited G association with PLC1. Identification of the
interactions between HA receptors and their primary second messengers
should be the focus of further studies. More than one potentially
important HA cell surface receptor has been described in vascular EC.
We have previously shown expression of the CD44 receptor in BAEC and
its role in promotion of o-HA-induced tyrosine phosphorylation (31).
Other studies have reported a similar response to o-HA in human glioma
cells (73), whereas association of CD44 with Src was shown in human
neutrophils (74) and human ovarian tumor cells (53) after treatment
with o-HA. Expression of RHAMM was demonstrated on the cell surface of
a variety of primary human vascular EC (33). Both native HA and in
particular o-HA treatment, resulted in RHAMM-dependent
tyrosine phosphorylation and MAP kinase activation, suggesting that
this was the functional HA receptor. Src activity has also been
associated with RHAMM-mediated cell motility in mouse fibroblasts (26). Other EC-specific receptors such as white fat-HA binding protein, detected in human aorta and atherectomy samples (76), have also been
identified, although their importance in mediation of HA-induced signal
transduction has not been determined. Our results leave open the
possibility that more than one o-HA-specific receptor may be present on
the surface of BAEC. The mechanism through which o-HA (F3 fragments)
binds to receptors and activates angiogenesis in both our in
vivo and in vitro models should be examined in detail.
Whereas other authors have demonstrated the ability of native HA to
weakly activate signal transduction activity associated with
proliferation in vascular EC of different origin (33), different sized
fragments of HA may elicit specific responses in individual cells for a
variety of reasons. For instance, it may depend on the types of
receptor expressed, the operational signal transduction pathways
available (which may work in synergy as described in this work), and
also on the ability of the molecule to specifically interact with the
appropriate link module binding site on the receptor (77). The data
presented here may be useful in the identification of potential targets
for modulation of angiogenesis in a variety of diseases associated with
abnormal EC growth.
View larger version (26K):
[in a new window]
Fig. 12.
Schematic diagram showing the signal
transduction pathways through which o-HA induces vascular
endothelial cell mitogenesis and wound recovery. Previous work has
shown the ability of o-HA to induce vascular EC proliferation through
the CD44 receptor (78). Similarly, signaling through RHAMM can also
activate proliferation via ERK activation (33). In depth examination of
signaling intermediates has not been studied. We show that o-HA induces
mobilization of G i/o/t/z resulting in G-dependent
activation of PLC1. PKC induced mitogenesis through ERK1/2
nuclear translocation and activation of early response genes.
Activation of PKC and to a lesser extent PKC was important for
wound recovery. o-HA also activated Src family kinase, which bound to
Shc and activated Ras following Shc·Grb2·Sos complex formation.
This pathway was important for both wound healing and proliferation.
G·PLC1 complex formation was also dependent on Src suggesting
an important interaction between these two pathways. In other cell
types, HA binding to CD44 results in association and activation of
c-Src, Ras, and Grb2 resulting in cell activation (30, 54, 66)
(highlighted in green), similarly RHAMM can initiate
proliferation via activation of the platelet-derived growth factor
receptor and ERK (75) (highlighted in red). Our findings
suggest that a similar pattern of receptor-second messenger interaction
may occur in BAEC. In conclusion, our work demonstrates the possibility
that more than one receptor can modulate o-HA-induced angiogenesis in
BAEC. The precise interactions between o-HA receptors and second
messengers should be the focus of further study. Dotted
lines represent possible interactions between primary second
messengers and o-HA receptors, whereas multiple arrowheads
indicate the likely presence of unidentified intermediates.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 G. Cao, R. C. Savani, M. Fehrenbach, C. Lyons, L. Zhang, G. Coukos, and H. M. DeLisser Involvement of Endothelial CD44 during in Vivo Angiogenesis Am. J. Pathol., July 1, 2006; 169(1): 325 - 336. [Abstract] [Full Text] [PDF]
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 T. Shi, Z.-H. Duan, R. Papay, E. Pluskota, R. J. Gaivin, C. A. de la Motte, E. F. Plow, and D. M. Perez Novel {alpha}1-Adrenergic Receptor Signaling Pathways: Secreted Factors and Interactions with the Extracellular Matrix Mol. Pharmacol., July 1, 2006; 70(1): 129 - 142. [Abstract] [Full Text] [PDF]
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 J. Milan, C. Charalambous, R. Elhag, T. C. Chen, W. Li, S. Guan, F. M. Hofman, and R. Zidovetzki Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration Am J Physiol Cell Physiol, July 1, 2006; 291(1): C155 - C164. [Abstract] [Full Text] [PDF]
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K. Kumari, B. A. Baggenstoss, A. L. Parker, and P. H. Weigel Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size J. Biol. Chem., April 28, 2006; 281(17): 11755 - 11760. [Abstract] [Full Text] [PDF]
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 M. Zareie, A. S. De Vriese, L. H. P. Hekking, P. M. ter Wee, C. G. Schalkwijk, B. A. J. Driesprong, I. L. Schadee-Eestermans, R. H. J. Beelen, N. Lameire, and J. van den Born Immunopathological changes in a uraemic rat model for peritoneal dialysis Nephrol. Dial. Transplant., July 1, 2005; 20(7): 1350 - 1361. [Abstract] [Full Text] [PDF]
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Y. Takahashi, L. Li, M. Kamiryo, T. Asteriou, A. Moustakas, H. Yamashita, and P. Heldin Hyaluronan Fragments Induce Endothelial Cell Differentiation in a CD44- and CXCL1/GRO1-dependent Manner J. Biol. Chem., June 24, 2005; 280(25): 24195 - 24204. [Abstract] [Full Text] [PDF]
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 V. B. Lokeshwar, W. H. Cerwinka, and B. L. Lokeshwar HYAL1 Hyaluronidase: A Molecular Determinant of Bladder Tumor Growth and Invasion Cancer Res., March 15, 2005; 65(6): 2243 - 2250. [Abstract] [Full Text] [PDF]
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 N. T. Seyfried, C. D. Blundell, A. J. Day, and A. Almond Preparation and application of biologically active fluorescent hyaluronan oligosaccharides Glycobiology, March 1, 2005; 15(3): 303 - 312. [Abstract] [Full Text] [PDF]
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K. R. Taylor, J. M. Trowbridge, J. A. Rudisill, C. C. Termeer, J. C. Simon, and R. L. Gallo Hyaluronan Fragments Stimulate Endothelial Recognition of Injury through TLR4 J. Biol. Chem., April 23, 2004; 279(17): 17079 - 17084. [Abstract] [Full Text] [PDF]
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 R. F. Thorne, J. W. Legg, and C. M. Isacke The role of the CD44 transmembrane and cytoplasmic domains in co-ordinating adhesive and signalling events J. Cell Sci., January 22, 2004; 117(3): 373 - 380. [Abstract] [Full Text] [PDF]
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P. L. DeAngelis, L. C. Oatman, and D. F. Gay Rapid Chemoenzymatic Synthesis of Monodisperse Hyaluronan Oligosaccharides with Immobilized Enzyme Reactors J. Biol. Chem., September 12, 2003; 278(37): 35199 - 35203. [Abstract] [Full Text] [PDF]
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K. N. Sugahara, T. Murai, H. Nishinakamura, H. Kawashima, H. Saya, and M. Miyasaka Hyaluronan Oligosaccharides Induce CD44 Cleavage and Promote Cell Migration in CD44-expressing Tumor Cells J. Biol. Chem., August 22, 2003; 278(34): 32259 - 32265. [Abstract] [Full Text] [PDF]
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