|
Originally published In Press as doi:10.1074/jbc.M104162200 on July 10, 2001
J. Biol. Chem., Vol. 276, Issue 36, 34175-34181, September 7, 2001
Cloning of a Novel Retinoid-inducible Serine Carboxypeptidase
from Vascular Smooth Muscle Cells*
Jiyuan
Chen,
Jeffrey W.
Streb ,
Kathleen M.
Maltby,
Chad M.
Kitchen, and
Joseph M.
Miano§
From the Center for Cardiovascular Research, University of
Rochester Medical Center, Rochester, New York 14642
Received for publication, May 8, 2001, and in revised form, May 21, 2001
 |
ABSTRACT |
Retinoids block smooth muscle cell
(SMC) proliferation and attenuate neointimal formation after vascular
injury, presumably through retinoid receptor-mediated changes in gene
expression. To identify target genes in SMC whose encoded proteins
could contribute to such favorable biological effects, we performed a
subtractive screen for retinoid-inducible genes in cultured SMC. Here,
we report on the cloning and initial characterization of a novel retinoid-inducible serine
carboxypeptidase (RISC). Expression of RISC is low in
cultured SMC but progressively increases over a 5-day time-course
treatment with all-trans-retinoic acid. A near full-length
rat RISC cDNA was cloned and found to have a 452-amino acid open
reading frame containing an amino-terminal signal sequence, followed by
several conserved domains comprising the catalytic triad common to
members of the serine carboxypeptidase family. In vitro
transcription and translation experiments showed that the rat
RISC cDNA generates a ~51-kDa protein. Confocal
immunofluorescence microscopy of COS-7 cells transiently transfected
with a RISC-His tag plasmid revealed cytosolic localization of the
fusion protein. Western blotting studies using conditioned medium from
transfected COS-7 cells suggest that RISC is a secreted protein. Tissue
Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues
analyzed; high level RISC expression was also observed in human kidney.
In situ hybridization verified the localization of RISC to
medial SMC of the adult rat aorta. Interestingly, expression in kidney
was restricted to proximal convoluted tubules; little or no expression
was observed in glomerular cells, distal convoluted and collecting
tubules, or medullary cells. Radiation hybrid mapping studies placed
the rat RISC locus on chromosome 10q. These studies reveal a novel
retinoid-inducible protease whose activity may be involved in vascular
wall and kidney homeostasis.
| |
INTRODUCTION |
Vascular smooth muscle cell
(SMC)1 activation is a
salient feature of several pathological conditions including
atherosclerosis, hypertension, vein graft failure, restenosis, and
transplant arteriopathy (1, 2). These conditions are
characterized by the proliferation and subverted differentiation of SMC
with consequent neointimal formation and possible plaque instability.
Thus, much effort has been directed toward the identification and
testing of interventions that inhibit SMC growth (2) as well as
elucidating the transcriptional program of SMC differentiation (3).
Considerable progress has been made in the latter case with the cloning
and characterization of several SMC-restricted promoters whose utility
will likely be appraised in the context of SMC-specific gene targeting
for vascular disease. It must be stressed, however, that although many
pharmaceutical approaches have proved effective in animal models
of vessel disease, their translation into clinical efficacy has been
disappointing. Thus, new interventional strategies should be directed
toward numerous aspects of vessel wall disease rather than targeting
any one pathway or protein.
Retinoids are natural and synthetic derivatives of vitamin A (4)
that have myriad effects on cellular growth and differentiation processes (5). Retinoids have been used clinically for the successful
management of several diseases, most notably certain cancers (6).
Given the similar events underlying the pathogenesis of neoplasia and
such vascular disorders as atherosclerosis and post-injury restenosis
(i.e. exuberant cell growth and subverted cellular
differentiation), numerous studies have been undertaken to demonstrate
the utility of retinoids in conferring a desirable SMC or vascular wall
phenotype. For example, retinoids can antagonize growth
factor-stimulated SMC proliferation in vitro (7-12),
inhibit SMC migration (12, 13), and promote a more differentiated SMC
phenotype (12, 14-16). In a series of complementary in vivo studies, retinoids were shown to minimize vascular narrowing following injury to the vessel wall (16-22). In a very recent report, rexinoids were demonstrated to reduce atherosclerosis in the apolipoprotein knockout mouse (23). Thus, there is mounting evidence to support a
possible role for retinoids and rexinoids in the treatment of vascular
disease. Importantly, the mechanisms through which these agents exert
their effects on SMC and the vessel wall are not known.
Many retinoids bind and activate retinoid receptors, which are members
of the steroid receptor superfamily of ligand-activated transcription
factors (24). As such, activated retinoid receptors modulate a cell's
transcriptome and hence its phenotype. Cultured SMC and aortic tissue
express five of the six retinoid receptors and display retinoid
receptor activity, at least in vitro (8). We recently
proposed that the identification of retinoid response genes in
SMC could provide new avenues of research to understand the mechanisms
underlying retinoid-induced changes in SMC and vessel wall phenotype
(25). Accordingly, we performed a subtractive screen for
retinoid-inducible genes in cultured SMC and reported on the cloning of
a novel retinoid response gene set in vascular SMC (26). Here, we
describe the nucleotide and amino acid sequence, expression, and
chromosomal mapping of a novel retinoid-inducible gene that appears to
be a member of the serine carboxypeptidase family of proteins.
| |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Cultured rat aortic SMC (RASMC) were derived
from aortas of male Harlan Sprague-Dawley rats by a combined
explant-enzymatic digestion procedure as described previously (27).
These cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and antibiotics. Human coronary artery SMC
(hCASMC) were obtained from a commercial vendor and grown in SmGM-2
media (Clonetics, San Diego, CA). Experiments were performed with
cultured COS-7, PAC-1 SMC, and RASMC between passages 10 and 20 or
hCASMC between passages 5 and 10. Unless indicated otherwise, cells
were grown on 100-mm dishes and treated with either
2×10-6 mol/liter all-trans-retinoic acid
(atRA) or an equal volume of dimethyl sulfoxide (Me2SO) for
various times when they were 70-80% confluent.
Suppression Subtractive Hybridization and Differential
Screening--
Suppression subtractive hybridization (SSH) was
performed using a PCR-SelectTM cDNA subtraction kit according to
the manufacturer's instructions (CLONTECH
Laboratories, Inc., Palo Alto, CA). We pooled two time points (12 and
72 h) of total RNA (2 µg/time point) from RASMC treated with
either 2 × 10 6 mol/liter atRA ("tester"
cDNA pool) or an equal volume of Me2SO ("driver"
cDNA pool). Hybridizing excess driver cDNA to the tester cDNA pool resulted in a subtracted library of clones containing atRA-induced transcripts. Following transformation in TOP10F
Escherichia coli, individual clones were randomly selected
for differential screening using the PCR-SelectTM differential
screening kit (CLONTECH). Briefly, cloned inserts
were PCR-amplified using nested primers as specified by the
manufacturer. PCR products were then arrayed in duplicate on 48-well
slot-blot nylon membranes. One membrane was hybridized to radiolabeled
tester cDNA and the other to radiolabeled driver cDNA. A third
membrane was used to back-hybridize cloned cDNAs in order to reduce
the number of duplicates. Positively identified clones were sequenced
on both strands (University of Rochester Core Nucleic Acid Laboratory)
and analyzed with the Genetics Computer Group (GCG) suite of software
programs (version 10.1, GCG, Madison, WI). Here, we report on the
sequence and characterization of one of our novel retinoid-inducible genes.
cDNA Cloning, Sequencing, and Bioinformatic Analysis--
A
RASMC cDNA library was screened with a 405-nt fragment of RISC to
isolate longer fragments of the RISC cDNA. 5'-Rapid amplification of cDNA ends (RACE) was performed using a 5'-RACE kit (Life
Technologies, Inc.) with gene-specific primers. 3'-Untranslated (UTR)
sequences were obtained by reverse transcriptase polymerase chain
reaction (RT-PCR) (First Strand Synthesis Kit, Amersham Pharmacia
Biotech) of atRA-stimulated RASMC using oligo(dT) and RISC-specific
primers. Phage clones and RACE products were sequenced on both strands (University of Rochester Core Nucleic Acid Laboratory) and analyzed with the GCG suite of software programs (version 10.1) and BLASTN at
the National Center for Biotechnology Information website
(www.ncbi.nlm.nih.gov/BLAST/). RISC peptide sequence comparison was
performed using BlastP. Molecular weight and pI were determined using
ProtParam (expasy.cbr.nrc.ca/tools/protparam.html). The PROSITE
pattern, PROSITE profile, BLOCKS, ProDom, PRINTS, and Pfam data bases
were scanned using MOTIF (motif.genome.ad.jp/). Signal peptide
prediction was performed using SignalP V2.0
(www.cbs.dtu.dk/services/SignalP-2.0/). Multiple sequence alignments
were performed using the pileup command in GCG. Serine carboxypeptidase
active site consensus motifs were obtained from PROSITE. Finally, the
Online Mendelian Inheritance in Man Morbid Map was consulted to
determine whether human RISC was associated with a known genetic
disease (www.ncbi.nlm.nih.gov/Omim/searchmorbid.html).
Analysis of the rat RISC cDNA using BLAST revealed numerous mouse
expressed sequence tags (ESTs) with similarity to rat RISC. ESTs were
aligned using the pileup command in the GCG Wisconsin package, with the
rat cDNA as the template. A consensus sequence was determined using
the pretty command with the pileup file as the input. The consensus
sequence was determined from at least five overlapping ESTs when
possible. A region between 600 and 900 nt was underrepresented (<4
ESTs) in the EST pool, and thus no perfect consensus was reached. Here
ambiguous nucleotides were edited using rat RISC as a guide to preserve
the reading frame. The ESTs used to assemble the mouse RISC cDNA
are AA612086, AA726687, AI118955, AI854112, AI875629, AI957256, AW107478, AW744471, BE850553, BE852371, BF016593, BF182152, C89145, and
W10703.
In Vitro Transcription/Translation--
RISC protein was
in vitro translated with the TNT T7 quick coupled
transcription/translation system (Promega, Madison, WI). A fragment
corresponding to the full-length open reading frame of RISC was
generated by RT-PCR of total RNA isolated from RASMC treated with atRA
for 96 h using restriction enzyme-clamped (underlined sequence)
RISC-specific primers (forward primer,
5'-GATACGTCGACCTGAGGCGGGGTTTTCATC-3' and reverse primer,
3'-GATACGATATCTGTGATGGAGCCGAGGATGC-3'). Template cDNA
was obtained by subcloning the PCR-amplified product into pBluscriptII
SK+ (Stratagene). A Luciferase T7 cDNA was used as a positive
control. In vitro transcription-translation was carried out
with 1 µg of plasmid DNA in 50 µl of reaction mixture supplemented with 50 µCi of [35S] methionine (Amersham Pharmacia
Biotech) and various amounts of TNT T7 Quick Master Mix (Promega) for
90 min at 30 °C. 10 µl of the products were separated by 10%
SDS-polyacrylamide gel electrophoresis, and dried gels were analyzed by autoradiography.
Confocal Microscopy of RISC-His-tagged Fusion
Protein--
RT-PCR was performed using the ProSTAR Ultra HF RT-PCR
System (Stratagene) employing a pair of RISC-specific primers (same forward primer as above, reverse primer,
5'-GATACTCTAGACTCCTGCTGAGTAACCAG-3'). The amplification
product, corresponding to the open reading frame of RISC less the stop
codon at nt 1376-1378, was subcloned into pEF1/V5-His (Invitrogen,
Carlsbad, CA) to generate RISC-His. Subcellular localization of
RISC-His fusion protein was appraised by confocal immunofluorescence
microscopy. Briefly, COS-7 cells were grown to 60-80% confluence on
four-well chamber slides and transfected with RISC-His using
LipofectAMINE Plus reagents. The vector without insert was also
transfected in parallel as a mock control. The cells were then allowed
to grow for 24 or 48 h in complete medium. After washing with cold
PBS, the cells were fixed in cold methanol/acetone (1:1) mixture at
 20 °C for 10 min. The slides were then incubated at room
temperature for 1 h in the presence of anti-His monoclonal antibody (Invitrogen) diluted 1:200 in PBS, washed with PBS, and finally incubated for 1 h in the presence of fluorescein
isothiocyanate-conjugated secondary antibody (Pierce) diluted 1:100 in
PBS. Cells were then stained with the DNA fluorochrome, TO-PRO-3 iodide
(Molecular Probes, Eugene, OR) to view the cell nucleus. Slides were
coverslipped under aqueous mounting medium and viewed with a Fluoview
FV 300 confocal microscope (Olympus, Melville, NY). Voltage settings were kept constant between RISC-His and mock-transfected cells during
imaging. For each experiment, primary and secondary antibody controls
were also included. Final images were captured and processed with Adobe Software.
Western Blotting--
The secretory property of RISC was
examined by Western blotting. COS-7 cells were grown in six-well plates
to 60-80% confluence and then transfected with RISC-His using
LipofectAMINE Plus reagents. 24 h after transfection, the cells
were re-fed with 0.1% fetal bovine serum or serum-free medium and
incubated for another 24 or 48 h. The conditioned medium and
extracts prepared from transfected cell were analyzed for expressed
RISC-His protein by Western blotting using an anti-His monoclonal
antibody (Invitrogen). The proteins from the medium (non-concentrated)
and cell lysates were separated on a 10% SDS-polyacrylamide gel,
transferred onto nitrocellulose membrane, and immunoblotted with
anti-His monoclonal antibody diluted 1:2000. Immunocomplexes were
detected with a secondary antibody conjugated to horseradish peroxidase
(Pierce) and visualized with SuperSignal West Pico Luminol/enhancer
solution (Pierce).
Northern Blotting Studies--
10- 20 µg of total RNA from
RASMC, PAC-1 SMC, hCASMC, or rat tissues was fractionated on 1.1%
agarose gel in the presence of 0.66 mol/liter formaldehyde, transferred
to nylon membrane, and hybridized with a 405-nt RISC cDNA probe
labeled with [ -32P]dCTP. For human tissue Northern
blotting, a human multiple tissue Northern blot was obtained from
CLONTECH and hybridized with a human RISC DNA
probe, cloned from a human bacterial artificial chromosome
(AC007114; obtained from Research Genetics, Inc.) by PCR.
Hybridizations were carried out in Rapid-hyb buffer (Amersham Pharmacia
Biotech) or Expresshyb hybridization solution
(CLONTECH) containing labeled probe
(~2×106 cpm/ml) at 62-68 °C for 1-2 h or overnight,
depending on the probe used. The blots were then washed under stringent
conditions and exposed to Kodak XAR films for different lengths of
time. Housekeeping genes used as internal controls for equal RNA
loading included glyceraldehyde-3-phosphate dehydrogenase, 18 S rRNA, and  -actin.
In Situ Hybridization Studies--
Selective rat tissues were
used to determine RISC mRNA expression in vivo. The
original 405-nt rat RISC cDNA fragment obtained from the SSH screen
was cloned into pBluescript SK and then linearized with either
BamHI (for antisense riboprobe) or XhoI (for
sense riboprobe). [33P]UTP-labeled riboprobes were
synthesized by in vitro transcription using MAXIscript
in vitro transcription Kit (Ambion). Both antisense and
sense riboprobes were assayed and the latter used as a control for
specificity of the signal. Paraffin-embedded, formaldehyde-fixed tissue
sections were deparaffinized and treated with 5 µg/ml proteinase K at
37 °C for 6 min. Hybridization with 3×107 cpm of
probe/ml of hybridization solution was performed overnight at 52 °C
in a humidified chamber. Slides were washed to remove unbound probe,
treated with RNase A, dehydrated with ethanol, air-dried, and dipped in
emulsion (Kodak NTB2). After 1 week, slides were developed in Kodak D19
developer. Darkfield and brightfield images were taken with an Olympus
digital camera and processed in Adobe Photoshop. The final composite of
images was assembled in FreeHand (Macromedia, San Francisco, CA).
Chromosomal Mapping of Rat RISC--
The location of RISC in the
rat genome was determined with a rat/hamster radiation hybrid (RH)
panel (Research Genetics, Huntsville, AL) using rat-specific primers.
The sequence of the forward primer was 5'-CTCTTCTTCCCGACTCTACCAT-3';
the sequence of the reverse primer was 5'-GAACTTGTGATGGAGCCGAGG-3'. All
RH clones except numbers 1, 20, 35, 38, 60, 90, and 101-106 were used.
PCR products were separated on a 1.2% agarose gel, and a vector was
obtained by scoring resolved PCR products as negative = 0, positive = 1, or ambiguous = 2. The resulting vector was
submitted to the Rat RH Map Server at the Rat Genome data base
(rgd.mcw.edu/RHMAPSERVER/) to obtain the chromosomal location of
RISC.
| |
RESULTS |
Differential Cloning of a Novel Retinoid Response Gene in Vascular
SMC--
Previously, we reported on the cloning and expression of
several retinoid response genes whose sequences matched known cDNAs (26). During the characterization of this gene set, we found a cDNA
clone that was not found in any of the genomic data bases but showed
significant homology to several serine carboxypeptidase ESTs. We refer
to the clone characterized in this report as RISC for
retinoid-inducible serine
carboxypeptidase. Low stringency Southern blotting of rat
genomic DNA suggests that RISC is a single-copy gene (data not shown).
Fig. 1A shows the induction of
a ~2.1-kilobase RISC transcript beginning 3 h after atRA
administration. RISC mRNA levels increased progressively over a
5-day time course in which fresh atRA was applied daily (Fig.
1A). A similar course of RISC mRNA induction was
observed in cells treated with only one application of atRA (Fig.
1B). We have also observed increases in RISC mRNA
following atRA stimulation of PAC-1 SMC and hCASMC (data not
shown).
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
Induction of RISC mRNA expression by atRA
in a time-dependent manner. Total RNA isolated from
RASMC treated with 2.0 µM atRA for 0, 3, 6, 12, 24, 48, 72, 96, or 120 h with fresh atRA used every 24 h
(A) or over 5 days with one application of the same dose of
atRA (B) were analyzed by Northern blotting with a rat RISC
cDNA probe. Blots were also probed with glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as a control for equal loading for
each lane. Data shown are representative of two independent
experiments.
|
|
To obtain additional sequence data, we screened a rat aortic SMC
cDNA library with the 405-nt SSH-derived fragment and sequenced 12 independent clones. Assembly of these clones, coupled with 5'-RACE and
3'-RT-PCR, led to the deduced RISC sequences shown in Fig.
2A. The rat RISC cDNA is
at least 2,046 nt in length, containing a 452-amino acid open reading
frame (nt 19-1375) with a calculated molecular mass of 51.2 kDa and a
predicted pI of 5.37. An in-frame stop codon upstream of a Kozak
methionine residue precedes a putative signal sequence (28) having a
possible cleavage site at Ala-28 (boxed amino acids in Fig.
2A). A high homology domain that represents the substrate
binding site of serine carboxypeptidases is found 45 amino acids
downstream from the putative cleavage site of the signal sequence
(first underlined sequence in
Fig. 2A). Three additional domains that comprise the
catalytic triad of serine carboxypeptidases are found at
amino acids 163-170, 365-373, and 421-437 (second through
fourth underlined sequences in Fig.
2A). Finally, several putative N-linked
glycosylation sites are found in the RISC primary amino acid sequence
(circled residues in Fig. 2A). Fig. 2B
shows the amino acid sequence and spacing homology of the substrate
recognition domain and catalytic triad of mammalian RISC (domains
I-IV) to several evolutionarily distant serine carboxypeptidases.
View larger version (63K):
[in this window]
[in a new window]
|
Fig. 2.
Rattus norvegicus
RISC nucleotide and deduced amino acid sequence.
Uppercase letters represent coding sequence;
lowercase letters represent 5'- and 3'-UTR. The
boxed amino acids at the NH2 terminus represent
the putative signal peptide; the cleavage site is predicted between
amino acids 28 and 29. The four heavy
underlined regions of the amino acid sequences
are conserved motifs common to serine carboxypeptidases; the
italicized letters in each are putative residues
of the Ser-Asp-His catalytic triad. The lightly
underlined region in the 3'-UTR is a consensus
polyadenylation sequence. B, sequence comparison of RISC's
putative SC substrate binding (I) and catalytic domains
(II-IV) compared with five known SCs. The five known SCs
are Aedes aegypti VCP (P42660), human protective protein
(NP_000299), Caenorhabditis elegans F41C3.5 (U23521),
Hordeum vulgare carboxypeptidase C (Y09604), and
Saccharomyces cerevisiae carboxypeptidase Y (NP_014026).
C. elegans F22E12.1 (T21275), and Drosophila
melanogaster CG3344 (AAF47405) are two putative SCs with homology
to RISC. Boxed residues represent invariant amino acids in
the conserved domains. Asterisk (*) indicates critical
residues in the serine carboxypeptidase Ser-Asp-His catalytic triad.
Numbers between domains represent length of intervening amino
acids.
|
|
We have assembled several mouse RISC ESTs into a cDNA sequence
(GenBankTM accession no. AF330052), which is 93% homologous to the
open reading frame of rat RISC (92% amino acid sequence identity/similarity). While this report was in preparation, the human
ortholog of rat RISC (named human serine carboxypeptidase precursor 1)
was deposited in GenBankTM (accession no. AF282618). The rat RISC
cDNA is 80.9% homologous to the open reading frame of human RISC
(with 82% amino acid identity).
In Vitro Translation and Intracellular Localization of
RISC--
In vitro transcription-translation of rat RISC
resulted in a protein with a molecular mass close to the predicted mass
of 51.2 kDa (Fig. 3). To determine the
cellular localization of RISC, we performed confocal immunofluorescence
microscopy of COS-7 cells transfected with a RISC-His tag fusion
protein. These results, shown in Fig.
4A, reveal a perinuclear
distribution of the fusion protein that extends into the cytosol.
Little or no nuclear accumulation of RISC was observed (Fig.
4A). Consistent with the presence of a putative
NH2-terminal signal sequence, RISC-His is secreted from
transfected COS-7 cells (Fig. 4B). Although the predicted RISC-His fusion protein is ~55 kDa, the secreted and intracellular forms of the protein migrate at a molecular mass greater than 60 kDa
(Fig. 4B). This could be due to post-translational
modifications such as N-linked glycosylation (see Fig.
2A).
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
In Vitro transcription and
translation of RISC cDNA. RISC cDNA fragment corresponding
to the coding sequence was cloned into pBluescript and in
vitro transcription and translation of the resulting circular
plasmid was performed with [35S]methionine as described
under "Experimental Procedures." Molecular size markers are
indicated to the left. A luciferase cDNA was included as
a control and point of reference.
|
|
View larger version (45K):
[in this window]
[in a new window]
|
Fig. 4.
Expression of a rat RISC-His-tagged fusion
protein in COS-7 cells. A, confocal
immunofluorescence micrographs of mock-transfected (left)
versus RISC-His-transfected (right) COS-7 cells.
Note the perinuclear accumulation of RISC-His in the right
panel. B, Western blotting results of COS-7 cells
transfected transiently with RISC-His (lanes 1 and 3) or mock-transfected with empty His plasmid
(lanes 2 and 4). CM refers
to the conditioned medium, and CL denotes cellular lysate.
Note the shift in size of the immunoreactive products (as compared with
in vitro translated RISC in Fig. 3) due to the His residues
and probable post-translational modifications (e.g.
N-linked glycosylation).
|
|
RISC mRNA Is Expressed in a Tissue-restricted Manner--
To
determine the tissue distribution of RISC mRNA, we performed
Northern blotting on panels of rat and human tissues. The results
depicted in Fig. 5A show that
RISC mRNA is highly expressed in rat aorta, bladder, and kidney
with lower levels in several other tissue types. A human
poly(A)+ tissue blot (Fig. 5B) shows a similar
restricted pattern of expression with highest levels in the kidney and
heart (the latter is likely due to contaminating aortic tissue).
Consistent with the kidney expression data, at least 10 ESTs with
homology to RISC were obtained from various kidney cDNA libraries
(data not shown). These results indicate that high level RISC mRNA
expression is tissue-restrictive, suggesting that it may have functions
specific for cells found in aorta, bladder, and kidney.
View larger version (50K):
[in this window]
[in a new window]
|
Fig. 5.
Tissue-restricted expression of RISC
mRNA. Total RNA isolated from various normal rat tissues
(A) or poly(A)+ RNA from human tissues
(B) were subjected to Northern hybridization using a rat or
human RISC cDNA probe. The transcript for rat RISC was detected in
aorta, bladder, and kidney with low level expression in heart, lung,
spleen, and stomach. 18 S ribosomal RNA was also probed and bands used
as controls for equal loading of total RNA for each tissue. The human
RISC transcript was highly enriched in kidney and heart. -Actin
mRNA demonstrates relatively equal mRNA loading in the human
blot. The lower band obtained with the -actin probe in skeletal
muscle probably represents cross-hybridization to the skeletal muscle
-actin mRNA. PBL represents peripheral blood
leukocyte mRNA.
|
|
Spatial Localization of RISC mRNA in Rat Tissues--
In
situ hybridization studies were carried out to determine the
spatial distribution of RISC mRNA in various rat tissues (Fig. 6). Rat RISC mRNA is modestly
elevated in the rat aorta (Fig. 6A), which may reflect the
reduced cellularity of this tissue as compared with others and/or the
heterogeneity that exists between SMC lineages of the aorta (29). RISC
expression was localized to the transitional epithelium of the bladder
(Fig. 6C). In the kidney, RISC showed expression throughout
the renal cortex with little or no hybridization signal in the renal
medulla (Fig. 6E). Careful examination of the cortical
expression of RISC revealed that the transcript was confined largely to
the epithelium of the proximal convoluted tubules (Fig. 6, G
and H). Glomerular cells, distal convoluted tubules,
collecting ducts, juxtaglomerular cells, peritubular capillaries, and
larger blood vessels showed only background hybridization signals (Fig.
6 (G and H) and data not shown). Consistent with
the Northern blotting data, heart, liver, spleen, skeletal muscle, and
brain showed only background RISC hybridization (data not shown).
View larger version (121K):
[in this window]
[in a new window]
|
Fig. 6.
In Situ hybridization analysis of
RISC mRNA expression in rat tissues. Sections obtained from
aorta (A and B), bladder (C and
D), and kidney (E-H) were prepared for in
situ hybridization with antisense (A, C,
E, G, and H) or sense (B,
D, and F) RISC riboprobes. Panels
A-F represent darkfield microscopic images. Sense RISC
exhibited only background hybridization. A modest increase in RISC
mRNA is observed in the tunica media of the rat aorta
(A). Expression of RISC appears to be enriched in the
transitional epithelium of the bladder (C). Note the
restricted expression of RISC to the renal cortex with little or no
signal in the underlying medulla (E). The punctate regions
of the cortex devoid of hybridization signal in panel
E represent glomeruli. High magnification brightfield
microscopy (G and H) shows that RISC is
restricted to the cuboidal epithelium of the proximal convoluted
tubules. m, renal medulla; d, distal convoluted
tubule; g, glomerulus; p, proximal convoluted
tubule. Original magnifications, ×20 (for A-F) and ×600
(for G and H).
|
|
Rat RISC Maps to the Long Arm of Chromosome 10--
Using a
rat-hamster RH panel, we mapped RISC to 10q31-10q32.1 of the rat
genome (Fig. 7). This region of
chromosome 10q is syntenic to chromosome 17q23.1 in the human genome
(see GenBankTM accession number AC007114). In
silico analysis showed that the human ortholog of RISC is
comprised of at least 13 exons spanning >25 kilobase pairs of genomic
sequence (data not shown). Analysis of the Online Mendelian Inheritance
in Man Morbid Map did not reveal a disease phenotype mapping to the
region of human RISC.
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 7.
Chromosomal mapping of rat and human
RISC. Rat RISC was mapped to rat chromosome 10q using a Radiation
Hybrid panel. Numbers to the left side of the
map represent map distances in centi-Rays. This region of
rat chromosome 10q is syntenic with human chromosome 17q23.1.
|
|
| |
DISCUSSION |
Retinoids such as atRA have been shown to have desirable effects
on SMC growth, migration, and differentiation both in vitro and in vivo (25, 30). The underlying mechanisms of atRA's effects in SMC, however, are currently not well understood, although it
is presumed that much of the biological activity relates to retinoid
receptor-mediated changes in gene expression. Thus, identifying retinoid response genes in SMC represents a potentially fruitful endeavor toward understanding the biology of retinoids in both SMC and
the vessel wall. We recently reported on the cloning of several
retinoid-responsive genes in atRA-stimulated SMC (26). One of the genes
cloned, tissue transglutaminase, appears to mediate retinoid-induced
SMC apoptosis in vitro (31). Other genes cloned included
those associated with growth inhibition and SMC differentiation (26).
In the present report, we have identified a novel retinoid-inducible gene in vascular SMC that has significant sequence homology to several
critical domains found in serine carboxypeptidases. Accordingly, we
have named the novel gene retinoid-inducible serine carboxypeptidase or
RISC.
Several carboxypeptidases have been described in vascular SMC, where
they function to either activate or inactivate proteins involved with
vasomotion or growth (32-34). An aortic carboxypeptidase-like protein
was shown to be up-regulated during vascular SMC differentiation in vitro (35). Curiously, the latter protein does not
exhibit detectable carboxypeptidase activity, suggesting a unique
function in vascular SMC of the vessel wall (35).
Serine carboxypeptidases (SCs; EC 3.4.16.1) are a family of lysosomal
glycoproteins (45-75 kDa) that exhibit carboxyl-terminal proteolytic
activity at acidic pH (36). SCs share a number of structural features
including a signal sequence for intracellular trafficking and/or
secretion, multiple N-linked glycosylation sites, and four
evolutionarily conserved domains involved with substrate binding and
catalysis (see Fig. 2B). All such features are present in
the deduced amino acid sequence of RISC, although formal designation of
RISC as a SC will require an assessment of its activity in
vitro. The majority of SC are found in the plant kingdom, where
they function in the normal turnover of proteins and the cleavage of
amino acids for nutrition (36). A yeast SC, called KEX1,
functions as a membrane-associated protease involved in the processing
of precursors to secreted mature proteins (37). In mammals, one of the
more extensively studied SC is cathepsin A-like
protective protein (CAPP) (38, 39). CAPP (also
called lysosomal protective protein or protective protein) is involved in the lysosomal processing of -galactosidase and neuraminidase. Interestingly, CAPP is the same size as RISC (452 amino acids) and is a
secreted protein. In addition, expression studies show that, like RISC,
CAPP is highly expressed in the epithelium of the proximal convoluted
tubules with little or no expression in other renal cell types; no
studies examined its expression in SMC (40). However, CAPP and RISC
appear to have separable functions. Gene mutations in CAPP are the
basis for the neurodegenerative lysosomal storage disease
galactosialidosis, which is characterized by reduced activities of
-galactosidase and neuraminidase (40). Complete loss of CAPP
activity leads to perinatal death due to heart and kidney failure (40).
Apparently, RISC is unable to compensate for the loss in CAPP activity.
By RH mapping, we placed rat RISC on chromosome 10q. The human RISC
ortholog mapped to two bacterial artificial chromosomes from
human chromosome 17q21, both of which are syntenic with rat chromosome
10q. At the time of this writing, we were unable to assign this locus
to a known disease entity. Defining the substrates of RISC and deducing
its activity will be critical in the identification of a disease
phenotype mapping to the RISC locus.
In addition to its role in the lysosome, CAPP has also been shown to
inactivate endothelin, a potent SMC vasoconstrictor and mitogen (41,
42). Interestingly, a previous report demonstrated that atRA
antagonized endothelin-induced SMC mitogenesis through the attenuation
of ERK activity (9). Northern blotting studies indicate that CAPP is
also induced by atRA in SMC (data not shown). Thus, it is intriguing to
consider the possibility that one mechanism for atRA's antagonism of
endothelin-induced SMC growth is through the proteolytic cleavage of
endothelin by atRA-induced CAPP. It will be interesting to determine
whether RISC also displays substrate binding for endothelin or other
growth factors whose biological activities are attenuated by atRA. In
this regard, we recently cloned another cathepsin gene, cathepsin L,
whose activity has been linked to retinoid X receptor inactivation
(26). Again, it will be useful to have a fuller understanding of the
substrates that cathepsin L binds and catalytically modulates.
The induction of RISC mRNA by atRA occurs in a
time-dependent manner. In normally cultured RASMC, RISC
mRNA expression is low. 12 h following atRA stimulation, there
is a detectable increase in RISC mRNA. This increase appears to
peak around 4 days following stimulation. At least some of this
induction is dependent on de novo protein synthesis, as
cycloheximide blocked increased RISC expression 48 h following
atRA treatment (data not shown). No change in RISC expression was
observed 24 h following treatment, suggesting that the RISC
mRNA has a long half-life or increase in expression at this time is
independent of protein synthesis (data not shown). Analysis of the
tissue expression of RISC revealed that it is highly expressed in the
aorta, bladder, and kidney of the rat; high level expression was also
observed in human kidney. The restricted expression of RISC to cuboidal
epithelial cells of the proximal convoluted tubule is suggestive of a
function unique to these highly metabolic cells. These cells appear to be major sites of amino acid reabsorption. Interestingly, endothelin has been shown to be inactivated in the kidney by a protease with structural properties similar to RISC (43). It will be interesting to
determine whether this protease is in fact RISC. Given the physiological differences between SMC and the proximal convoluted tubule epithelium, RISC may very likely display disparate functions in
these two cell types. Future work will require the development of
antisera to RISC as well as specific assays to assess its function in
the injured vessel wall treated with retinoids.
| |
FOOTNOTES |
*
This work was supported in part by Scientist Development
Grant 9730145N (to J. M. M.) and by Postdoctoral Fellowship
Grant 0020233T from the American Heart Association (to J. C.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF330051 (rat RISC) and AF330052 (mouse RISC).
Recipient of National Institutes of Health Cardiovascular Research
Training Grant 1T32HL07949.
§
To whom correspondence should be addressed: Center for
Cardiovascular Research, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-1664; Fax:
716-273-1497; E-mail: joseph_miano@urmc.rochester.edu.
Published, JBC Papers in Press, July 10, 2001, DOI 10.1074/jbc.M104162200
| |
ABBREVIATIONS |
The abbreviations used are:
SMC, smooth muscle
cell;
RISC, retinoid-inducible serine carboxypeptidase;
RASMC, rat
aortic smooth muscle cell;
hCASMC, human coronary artery smooth muscle
cell;
SSH, suppression subtractive hybridization;
atRA, all-trans-retinoic acid;
RACE, rapid amplification of
cDNA ends;
UTR, untranslated region;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
GCG, Genetics Computer Group;
SC, serine carboxypeptidase;
EST, expressed sequence tag;
PBS, phosphate-buffered saline;
RH, radiation hybrid;
nt, nucleotide(s);
CAPP, cathepsin A-like protective protein.
| |
REFERENCES |
| 1.
|
Libby, P.,
Schwartz, D.,
Brogi, E.,
Tanaka, H.,
and Clinton, S. K.
(1992)
Circulation
86,
III-47-III-52
|
| 2.
|
Schwartz, S. M.,
deBlois, D.,
and O'Brien, E. R. M.
(1995)
Circ. Res.
77,
445-465
|
| 3.
|
Owens, G. K.
(1995)
Physiol. Rev.
75,
487-517
|
| 4.
|
Sporn, M. B.,
Dunlop, N. M.,
Newton, D. L.,
and Smith, J. M.
(1976)
Fed. Proc.
35,
1332-1338
|
| 5.
|
Nau, H.,
and Blaner, W. S.
(2000)
Handbook of Experimental Pharmacology
, Vol. 139
, Springer-Verlag, Berlin
|
| 6.
|
Hong, W. K.,
and Sporn, M. B.
(1997)
Science
278,
1073-1077
|
| 7.
|
Hayashi, A.,
Suzuki, T.,
and Tajima, S.
(1995)
J. Biochem. (Tokyo)
117,
132-136
|
| 8.
|
Miano, J. M.,
Topouzis, S.,
Majesky, M. W.,
and Olson, E. N.
(1996)
Circulation
93,
1886-1895
|
| 9.
|
Chen, S.,
and Gardner, D. G.
(1998)
J. Clin. Invest.
102,
653-662
|
| 10.
|
Pakala, R.,
and Benedict, C. R.
(2000)
J. Cardiovasc. Pharmacol.
35,
302-308
|
| 11.
|
Benson, S.,
Padmanabhan, S.,
Kurtz, T. W.,
and Pershadsingh, H. A.
(2000)
Mol. Cell Biol. Res. Commun.
3,
159-164
|
| 12.
|
Axel, D. I.,
Frigge, A.,
Dittmann, J.,
Runge, H.,
Spyridopoulos, I.,
Riessen, R.,
Viebahn, R.,
and Karsch, K. R.
(2001)
Cardiovasc. Res.
49,
851-862
|
| 13.
|
James, T. W.,
Wagner, R.,
White, L. A.,
Zwolak, R. M.,
and Brinckerhoff, C. E.
(1993)
J. Cell. Physiol.
157,
426-437
|
| 14.
|
Haller, H.,
Lindschau, C.,
Quass, P.,
Distler, A.,
and Luft, F. C.
(1995)
Circ. Res.
76,
21-29
|
| 15.
|
Gollasch, M.,
Haase, H.,
Ried, C.,
Lindschau, C.,
Morano, I.,
Luft, F. C.,
and Haller, H.
(1998)
FASEB J.
12,
593-601
|
| 16.
|
Wiegman, P. J.,
Barry, W. L.,
McPherson, J. A.,
McNamara, C. A.,
Gimple, L. W.,
Sanders, J. M.,
Bishop, G. G.,
Powers, E. R.,
Ragosta, M.,
Owens, G. K.,
and Sarembock, I. J.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
89-95
|
| 17.
|
Miano, J. M.,
Kelly, L. A.,
Artacho, C. A.,
Nuckolls, T. A.,
Piantedosi, R.,
and Blaner, W. S.
(1998)
Circulation
98,
1219-1227
|
| 18.
|
Neuville, P.,
Yan, Z.,
Gidlöf, A.,
Pepper, M. S.,
Hansson, G. K.,
Gabbiani, G.,
and Sirsjö, A.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1430-1436
|
| 19.
|
DeRose, J. J., Jr.,
Madigan, J.,
Umana, J. P.,
Prystowsky, J. H.,
Nowygrod, R.,
Oz, M. C.,
and Todd, G. J.
(1999)
Cardiovasc. Surg.
7,
633-639
|
| 20.
|
Chen, J.,
He, B.,
Zheng, D.,
Zhang, S.,
Liu, J.,
and Zhu, S.
(1999)
Chin. Med. J.
112,
121-123
|
| 21.
|
Lee, C. W.,
Park, S. J.,
Park, S. W.,
Kim, J. J.,
Hong, M. K.,
and Song, J. K.
(2000)
J. Korean Med. Sci.
15,
31-36
|
| 22.
|
Leville, C. D.,
Dassow, M. S.,
Seabrook, G. R.,
Jean-Claude, J. M.,
Towne, J. B.,
and Cambria, R. A.
(2000)
J. Surg. Res.
90,
183-190
|
| 23.
|
Claudel, T.,
Leibowitz, M. D.,
Fiévet, C.,
Tailleux, A.,
Wagner, B.,
Repa, J. J.,
Torpier, G.,
Lobaccaro, J.-M.,
Paterniti, J. R.,
Mangelsdorf, D. J.,
Heyman, R. A.,
and Auwerx, J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2610-2615
|
| 24.
|
Mangelsdorf, D. J.,
Umesono, K.,
and Evans, R. M.
(1994)
in
The Retinoids: Biology, Chemistry, and Medicine
(Sporn, M. B.
, Roberts, A. B.
, and Goodman, D. S., eds)
, pp. 319-349, Raven Press, New York
|
| 25.
|
Miano, J. M.,
and Berk, B. C.
(2000)
Circ. Res.
87,
355-362
|
| 26.
|
Chen, J.,
Maltby, K. M.,
and Miano, J. M.
(2001)
Biochem. Biophys. Res. Commun.
281,
475-482
|
| 27.
|
Miano, J. M.,
and Olson, E. N.
(1996)
J. Biol. Chem.
271,
7095-7103
|
| 28.
|
Stroud, R. M.,
and Walter, P.
(1999)
Curr. Opin. Struct. Biol.
9,
754-759
|
| 29.
|
Topouzis, S.,
and Majesky, M. W.
(1996)
Dev. Biol.
178,
430-445
|
| 30.
|
Neuville, P.,
Bochaton-Piallat, M. L.,
and Gabbiani, G.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
1882-1888
|
| 31.
|
Ou, H.,
Haendeler, J.,
Aebly, M. R.,
Kelly, L. A.,
Cholewa, B. C.,
Koike, G.,
Kwitek-Black, A. E.,
Jacob, H. J.,
Berk, B. C.,
and Miano, J. M.
(2000)
Circ. Res.
87,
881-887
|
| 32.
|
Takai, S.,
Sakaguchi, M.,
Jin, D.,
Yamada, M.,
Kirimura, K.,
and Miyazaki, M.
(2001)
Clin. Chim. Acta
305,
191-195
|
| 33.
|
Mentlein, R.,
and Roos, T.
(1996)
Peptides
17,
709-720
|
| 34.
|
Reznik, S. E.,
Salafia, C. M.,
Lage, J. M.,
and Fricker, L. D.
(1998)
J. Histochem. Cytochem.
46,
1359-1367
|
| 35.
|
Layne, M. D.,
Endege, W. O.,
Jain, M. K.,
Yet, S. F.,
Hsieh, C. M.,
Chin, M. T.,
Perrella, M. A.,
Blanar, M. A.,
Haber, E.,
and Lee, M. E.
(1998)
J. Biol. Chem.
273,
15654-15660
|
| 36.
|
Remington, S. J.,
and Breddam, K.
(1994)
Methods Enzymol.
244,
231-248
|
| 37.
|
Cooper, A.,
and Bussey, H.
(1989)
Mol. Cell. Biol.
9,
2706-2714
|
| 38.
|
Galjart, N. J.,
Gillemans, N.,
Harris, A.,
van der Horst, G. TJ.,
Verheijen, F. W.,
Galjaard, H.,
and d'Azzo, A.
(1988)
Cell
54,
755-764
|
| 39.
|
Hanna, W. L.,
Turbov, J. M.,
Jackman, H. L.,
Tan, F.,
and Froelich, C. J.
(1994)
J. Immunol.
153,
4663-4672
|
| 40.
|
Rottier, R. J.,
Hahn, C. N.,
Mann, L. W.,
del Pilar Martin, M.,
Smeyne, R. J.,
Suzuki, K.,
and d'Azzo, A.
(1998)
Hum. Mol. Genet.
7,
1787-1794
|
| 41.
|
Jackman, H. L.,
Morris, P. W.,
Deddish, P. A.,
Skidgel, R. A.,
and Erdös, E. G.
(1992)
J. Biol. Chem.
267,
2872-2875
|
| 42.
|
Itoh, K.,
Kase, R.,
Shimmoto, M.,
Satake, A.,
Sakuraba, H.,
and Suzuki, Y.
(1995)
J. Biol. Chem.
270,
515-518
|
| 43.
|
Deng, Y.,
Martin, L. L.,
DelGrande, D.,
and Jeng, A. Y.
(1992)
J. Biochem. (Tokyo)
112,
168-172
|
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:
|
|
|
|
 
T.-H. D. Lee, J. W. Streb, M. A. Georger, and J. M. Miano
Tissue Expression of the Novel Serine Carboxypeptidase Scpep1
J. Histochem. Cytochem.,
June 1, 2006;
54(6):
701 - 711.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Fraser, L. W. Rider, and C. Chapple
An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family
Plant Physiology,
June 1, 2005;
138(2):
1136 - 1148.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
|
Advertisement
Advertisement
|
TOP
ABSTRACT
INTRODUCTION