From the Cardiovascular Biology Laboratory, Harvard
School of Public Health, Boston, Massachusetts 02115, the
¶ Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115, the
Cardiovascular and
** Pulmonary Divisions, Brigham and Women's Hospital,
Boston, Massachusetts 02115, and
Cardiovascular Drug Discovery, F12-01,
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton,
New Jersey 08543
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The ~4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vascular smooth muscle cells (VSMCs)1 are the predominant component of the blood vessel wall, where their principal function is to regulate vascular tone (1). Although VSMCs normally exist in a differentiated state, they can dedifferentiate and proliferate in response to certain stimuli. Activation of VSMCs from a contractile and quiescent state to a proliferative and synthetic state contributes to several disease processes, including arteriosclerosis (2). Defining effectors that modulate VSMC function and identifying marker proteins that characterize a given VSMC phenotypic state will contribute to our understanding of the mechanisms regulating VSMC differentiation (1).
The origins of VSMCs during embryonic development are diverse (reviewed
in Refs. 1, 3, and 4). During development, VSMCs derive from many cell
types, such as local mesodermal precursors and neural crest cells (3,
5). Despite the fact that they express a similar set of smooth muscle
cell marker genes, these cell populations can differ in morphology and
respond in a lineage-dependent manner to factors such as
transforming growth factor-1 (6). An understanding of the complex
regulation of smooth muscle cell differentiation requires the
identification of proteins involved in this response.
In a search for potential markers and regulators of smooth muscle cell growth and differentiation, we identified a novel gene product termed aortic carboxypeptidase-like protein (ACLP). ACLP contains a signal peptide, a repeating motif, a discoidin-like domain, and a domain with homology to the carboxypeptidases. ACLP is expressed highly in adult aortic smooth muscle cells, as detected by Northern blotting, Western blotting, and in situ hybridization. Also, expression of ACLP increases in cultured aortic smooth muscle cells after serum starvation. Using a recently developed in vitro system that allows the differentiation of multipotential mouse neural crest cells into smooth muscle cells, we show that ACLP is up-regulated dramatically. These results suggest that ACLP may play a role during development in the acquisition by VSMCs of the differentiated phenotype.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Lines, Cell Culture, and Reagents-- Rat aortic smooth muscle cells (RASMCs) and mouse aortic smooth muscle cells (MASMCs) were isolated by the method of Gunther et al. (7) from the thoracic aortas of adult male Sprague-Dawley rats and C57Bl/6 mice. Human aortic smooth muscle cells (HASMCs) were purchased from Clonetics (San Diego, CA), and rat A7r5 smooth muscle cells and C2C12 mouse myoblasts were purchased from the ATCC (Rockville, MD). The mouse neural crest cell line Monc-1 was provided by David Anderson (Pasadena, CA). Monc-1 cells were cultured on fibronectin-coated plates as described (8), with minor modifications (9). RASMCs, MASMCs, and A7r5 cells were cultured in Dulbecco's modified Eagle's medium with 3.7 g/liter glucose (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 4 mM L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, and 10 mM HEPES (pH 7.4). C2C12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, 4 mM L-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin. HASMCs were cultured in M199 medium (Life Technologies, Inc.) supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin. Cells were grown at 37 °C in a humidified incubator containing 5% CO2.
Cloning and Sequencing of Human and Mouse ACLP--
A
recombinant E47 fusion protein (N3-SH[ALA]) containing the basic
helix loop helix domain of hamster shPan-1 (amino acids 509-646, with
mutations R551A, V552L, and R553A) and a heart muscle kinase
recognition sequence and FLAG epitope was expressed and purified as
described (10, 11). The fusion protein was phosphorylated with heart
muscle kinase in the presence of [-32P]ATP and then
used to screen a human aorta
gt11 cDNA expression library
(1.5 × 106 pfu; CLONTECH, Palo
Alto, CA) by interaction cloning (10, 11). A 1450-base pair (bp)
cDNA clone that resulted from this interaction cloning was
radiolabeled by random priming and used to isolate an ~2.8-kilobase
(kb) cDNA clone from the same human aorta
gt11 cDNA library.
Because Northern blotting revealed that the latter was also a partial
cDNA clone, we isolated additional 5' sequences from HASMC RNA by
5' rapid amplification of cDNA ends (Life Technologies, Inc.).
Northern Blot Analysis-- Total RNA was obtained from mouse organs by using RNAzol B according to manufacturer's instructions (Tel-Test, Inc., Friendswood, TX). RNA from cultured cells was isolated by guanidinium isothiocyanate extraction and centrifugation through cesium chloride (13). RNA was fractionated on 1.2% agarose (6% formaldehyde) gels and transferred to nitrocellulose filters (NitroPure, Micron Separations, Westboro, MA). The filters were hybridized with random-primed, 32P-labeled cDNA probes as described (13, 14). Equal loading was verified by hybridizing the filters to a 32P-labeled oligonucleotide complementary to 18S ribosomal RNA (15). Blots were exposed to x-ray film and a phosphor screen, and radioactivity was measured on a PhosphorImager running the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and normalized to 18S.
Cellular Localization of ACLP--
To construct a c-myc-tagged
ACLP expression plasmid (pcDNA3.1/ACLP-Myc-His), we amplified the
open reading frame of mouse ACLP with the Expand Long Template PCR
System (Boehringer Mannheim). We used a 5' primer containing an
EcoRI site (5'-CGGAATTCAGTCCCTGCTCAAGCCCG-3') and a 3'
primer containing a HindIII site
(5'-CGAAGCTTGAAGTCCCCAAAGTTCACTG-3') to delete the endogenous
termination codon. The PCR product was digested with EcoRI
and HindIII restriction enzymes and ligated into the
EcoRI and HindIII sites of
pcDNA3.1()/Myc-His A (Invitrogen). Cells were transiently
transfected with pcDNA3.1/ACLP-Myc-His by the DEAE-dextran method
with minor modifications (16). Twenty-four hours after transfection,
cells were trypsinized, plated onto chamber slides (Nunc, Naperville,
IL), and grown for an additional 24 h. Cells were fixed with
4% paraformaldehyde in phosphate-buffered saline and immunostained as
described (17) with a monoclonal anti-c-myc primary antibody (9E10
Ab-1; Oncogene Research Products, Cambridge, MA) and a
rhodamine-conjugated goat anti-mouse IgG secondary antibody. Nuclei
were counterstained with Hoechst 33258 (1 µg/ml) and visualized
with a fluorescence microscope.
Antibody Production and Western Blot Analysis--
To produce a
polyclonal anti-ACLP antibody, we subcloned a BamHI to
EcoRI fragment of mouse ACLP (encoding amino acids
615-1128) into the pRSET C bacterial expression vector (Invitrogen).
The plasmid was transformed into BL21(DE3)pLysS-competent bacteria (Stratagene), and protein expression was induced with 1 mM
isopropyl -D-thiogalactopyranoside for 3 h.
Bacteria were sonicated in lysis buffer (50 mM
NaH2PO4, 10 mM Tris, pH 8, 100 mM NaCl) containing the protease inhibitors aprotinin,
leupeptin, and phenylmethylsulfonyl fluoride. Lysates were clarified by
centrifugation at 10,000 × g for 15 min, and the
pellet was resuspended in lysis buffer supplemented with 8 M urea. His-tagged proteins were purified with Talon resin (CLONTECH) and eluted in lysis buffer containing 8 M urea and 100 mM ethylene diamine tetraacetic
acid. Proteins were dialyzed against water and measured with the
Bio-Rad (Hercules, CA) protein assay reagent, and 100 µg was used to
immunize New Zealand White rabbits. Antiserum was collected, titered
against the recombinant protein, and used for immunoblot analysis as
described below. Specificity of the antiserum was determined by using
preimmune serum and by competition with a recombinant protein.
In Situ Hybridization-- Adult male Sprague-Dawley rats were perfused with 4% paraformaldehyde, and their organs were removed and sectioned (19). Probe was prepared, and in situ hybridization was conducted as described (19, 20). ACLP mRNA was detected with a [35S]UTP-labeled antisense riboprobe synthesized with SP6 RNA polymerase from a linearized 0.7-kb fragment of ACLP cDNA in pCR2.1. As a control, a sense RNA probe was synthesized with T7 RNA polymerase from a linearized ACLP cDNA fragment in pCR2.1.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and Characterization of Human and Mouse ACLP cDNAs-- To identify proteins interacting with products of the E2A gene (E12/E47) in VSMCs, we screened a human aorta expression library with a 32P-labeled E47 fusion protein. One truncated clone isolated from this screen (number 11) led to the full-length ACLP clone characterized here. Using in vitro binding assays, we determined that proteins derived from clone 11, but not from the full-length protein, bound to E12 and E47 (data not shown). The 3935 bp, full-length human ACLP cDNA contains an open reading frame of 1158 amino acids (Fig. 1A) and a Kozak consensus sequence for initiation of translation (GCCATGG) (21) preceded by an in-frame stop codon. The protein has a calculated molecular mass of 130 kDa and an estimated pI of 4.8, and it contains a putative signal peptide sequence (22, 23), an 11 amino acid lysine- and proline-rich motif repeated four times at the N terminus, a domain with 30% amino acid identity to the slime mold adhesion protein discoidin I, and a C-terminal domain with 39% identity to carboxypeptidase E (Fig. 1B).
|
Characterization of ACLP-- To confirm the putative open reading frame of mouse ACLP, we performed in vitro transcription and translation reactions with the mouse cDNA used as template. Translated products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a prominent band of ~175 kDa was detected (Fig. 2A). To identify the endogenous ACLP, a C-terminal fragment of mouse ACLP was expressed in bacteria, purified, and used to raise antibodies in rabbits. By Western blot analysis, this antibody detected a single band with an apparent mobility of ~175 kDa in MASMC extracts (Fig. 2B). The similar migration of the endogenous ACLP and the protein transcribed and translated in vitro indicates that we isolated a full-length cDNA clone.
|
|
Tissue Expression of Mouse ACLP-- Although the ACLP cDNA was cloned originally from aortic smooth muscle cells, we also wanted to examine its mRNA and protein expression in other tissues. As expected, levels of ACLP mRNA were high in the whole aorta (including adventitia) (Fig. 4A). Also, ACLP message was present in other tissues, including the colon and the kidney (Fig. 4A). To examine expression of ACLP, we subjected extracts from mouse tissues to Western blot analysis. ACLP was expressed abundantly in the mouse aorta (without adventitia) but not in the adventitia, heart, liver, skeletal muscle, or kidney (Fig. 4B). The presence of ACLP mRNA in the kidney (Fig. 4A) but absence of protein may indicate translational regulation. To identify cell types expressing ACLP in the adult, we performed in situ hybridization on adult rat aorta and skeletal muscle. The antisense riboprobe detected specific ACLP expression in the smooth muscle cells of the aorta (Fig. 5A), whereas the control, sense probe did not (Fig. 5B). As expected, neither the sense nor the antisense probe hybridized to skeletal muscle cells (Fig. 5, C and D).
|
|
ACLP Expression in Cultured Smooth Muscle Cells-- Because ACLP expression was high in the differentiated smooth muscle cells of the aorta (Fig. 3B), we examined the effect of VSMC growth and differentiation on ACLP expression. MASMCs were cultured for 3 days in 0.4% calf serum containing medium that induces quiescence. RNA and protein extracts were then prepared from the cells and analyzed. ACLP mRNA was more abundant (~2-fold) in serum-starved MASMCs than in growing controls (Fig. 6A). In RASMCs, ACLP mRNA was ~3-fold more abundant in quiescent cells than in their actively proliferating counterparts (Fig. 6A). ACLP was also elevated in quiescent MASMCs (Fig. 6B). Although these changes in message and protein levels are modest, they are consistent with increases in VSMC differentiation-specific markers observed in other systems (24, 25).
|
ACLP Expression in Smooth Muscle Cell Differentiation--
Our
laboratory recently developed an in vitro system for
differentiating smooth muscle cells from Monc-1 cells, a mouse line derived from the neural crest (9). Monc-1 cells differentiate into
smooth muscle cells when medium supplemented with chick embryo extract
is replaced with differentiation medium (9). To examine ACLP expression
during the conversion of undifferentiated Monc-1 cells to smooth
muscle, we measured the time course of ACLP expression. ACLP mRNA
was nearly undetectable in undifferentiated Monc-1 cells (Fig.
7A). As the cells
differentiated, however, ACLP expression increased until it became
marked at days 4 and 6 after the start of differentiation (Fig.
7A). Under these conditions, induction of ACLP appeared to
lag behind that of smooth muscle -actin, a marker for smooth muscle
cells. To compare the level of ACLP in cells treated similarly, we
prepared protein extracts from undifferentiated Monc-1 cells and cells
allowed to differentiate for 6 days (Fig. 7B). ACLP was not
detectable in undifferentiated Monc-1 cells (day 0) but was expressed
highly (day 6) under conditions that promote Monc-1 cell
differentiation into smooth muscle cells. The abundance of ACLP in
these cells was similar to that in MASMCs.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have cloned a novel cDNA from human aortic smooth muscle cells, termed ACLP, and its mouse homologue. Notable features of the protein include a predicted signal peptide sequence at the N terminus, a lysine- and proline-rich 11-amino acid repeat, a discoidin-like domain, and a large C-terminal carboxypeptidase-like domain (Fig. 1B).
The screen that led to the identification of ACLP was performed to identify binding partners of the E2A proteins. The products of the E2A gene, E12 and E47, serve as heterodimerization partners for tissue specific transcription factors that regulate growth and differentiation in several cell types. Although the E2A gene products are expressed ubiquitously (26), a vascular smooth muscle specific heterodimerization partner or transcription factor has not been identified. We cloned the C-terminal portion of human ACLP (amino acids 793-1158) by using a labeled E47 protein probe and verified its binding to E47 by in vitro assays (data not shown). However, the full-length ACLP, because of its predicted signal peptide sequence (Fig. 1) and nonnuclear subcellular localization (Fig. 3), probably does not function as a heterodimerization partner for E47 in vivo.
GenBankTM searches indicated high homology between the C terminus of human ACLP and the mouse AEBP1 described by He et al. (12). To determine the relation between ACLP and AEBP1, we cloned the mouse ACLP cDNA. By sequence comparison, AEBP1 was found to be identical to mouse ACLP, beginning at ACLP methionine 410 (Fig. 1A). We then determined that ACLP is a single-copy gene in the mouse and cloned the region corresponding to the 5' end of AEBP1 from genomic DNA.2 Analysis of the genomic clone confirmed that the AEBP1 sequence is missing a G residue 11 bases 5' to the identified ATG. The presence of this G residue in ACLP would eliminate the in frame stop codon proposed by He et al. (12) and extend the open reading frame.
The 2.5-kb AEBP1 cDNA is unlikely to code for an authentic protein. Probes derived from AEBP1 and both the 5' and 3' ends of ACLP detected a single, ~4-kb band by Northern blot analysis, which is consistent with the size of the human as well as the mouse ACLP cloned cDNAs. Because the AEBP1 cDNA contains a putative polyadenylation signal and a poly(A) tail, the difference between the AEBP1 cDNA and mRNA is ~1.5 kb. This missing 1.5 kb of sequence is present in the 5' end of the ACLP cDNA. Also, the anti-ACLP antibody generated for these studies was raised from the C terminus of ACLP, which is identical to AEBP1. The antibody detected only a single band of ~175 kDa by Western blotting in several tissues examined (Fig. 4B), which is consistent with the mobility of ACLP transcribed and translated in vitro (Fig. 2). We also detected a single band of identical mobility in protein extracts from several cell lines in culture, including 3T3-L1 preadipocytes. ACLP was expressed in 3T3-L1 preadipocytes at substantially lower levels than in MASMCs or differentiated Monc-1 cells (data not shown). Thus, AEBP1 appears to be a truncated clone of mouse ACLP. AEBP1 is missing the ACLP signal peptide, repeat domain, and part of the discoidin domain.
ACLP has a prominent carboxypeptidase-like domain of about 500 amino acids at its C terminus (Fig. 1B). This domain is 39% identical to carboxypeptidase E. Despite this high sequence similarity, however, we3 and others (27) have been unable to demonstrate that this domain of ACLP has any catalytic carboxypeptidase activity. These results may reflect the divergence of specific residues in ACLP from sequences of the carboxypeptidase family (27). For example, a histidine involved in zinc binding in carboxypeptidases is replaced by an asparagine (amino acid 763) in human ACLP. Catalytically important tyrosine and glutamic acid residues in the carboxypeptidases are substituted by asparagine (amino acid 852) and tyrosine (amino acid 874) in human ACLP, respectively. Also, the positively charged arginine residue in the substrate recognition pocket of the carboxypeptidases that stabilizes the C-terminal carboxyl group of the substrate is replaced by a negatively charged glutamic acid residue (amino acid 700) in human ACLP. Although catalytically inactive, ACLP may interact with other proteins via this carboxypeptidase-like domain, as evidenced by our initial isolation of the ACLP cDNA from an expression library screened with a 32P-labeled protein probe. Carboxypeptidase E also serves as a sorting receptor in the secretory pathway (28), implicating functions other than catalysis for the carboxypeptidase domain.
The second important motif in ACLP is a discoidin-like domain (Fig. 1B), which has been identified in coagulation factors V and VIII (29-31), milk fat globule membrane proteins (32, 33), the discoidin domain tyrosine kinase receptor (34), the endothelial cell protein del-1 (35), and the A5/neuropilin protein (36-38). Discoidin is a lectin produced by the slime mold Dictyostelium discoideum and is thought to facilitate cellular aggregation and migration by functioning as fibronectin does in vertebrates (39). ACLP and many other proteins containing a discoidin-like domain lack the RGD motif important to the function of both discoidin and fibronectin (40). The discoidin-like domain may be important for cell-cell recognition, or it may be involved in cell migration mediated through homotypic and heterotypic interactions (36, 39). The discoidin domain tyrosine kinase receptors are activated by collagen, although the receptor domain involved in this interaction has not been identified (41, 42). The discoidin-like domain may also bind to phospholipids (33, 43). As ACLP lacks a predicted transmembrane-spanning domain (Fig. 1A), the discoidin-like domain may mediate the interaction of ACLP with the cell membrane.
Although ACLP is not expressed in neural crest cells, it is induced markedly during Monc-1 cell to smooth muscle cell differentiation (Fig. 7). This induction of ACLP during Monc-1 differentiation, in conjunction with the preferential expression of ACLP in VSMCs in vivo (Fig. 4), links ACLP expression to the development of the VSMC lineage. Moreover, induction of ACLP by culture medium that confers a differentiated VSMC phenotype (Fig. 6) further suggests a role for ACLP in the differentiation of this cell type.
![]() |
ACKNOWLEDGEMENTS |
---|
The Monc-1 cell line was kindly provided by D. J. Anderson (Pasadena, CA). We thank T. McVarish for editorial assistance and B. Ith for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute and by National Institutes of Health Grants HL03194 (to M. A. P.) and GM53249 (to M.-E. L.).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) AF053943 and AF053944 for mouse and human ACLP, respectively.
§ Contributed equally to this work.
§§ To whom correspondence should be addressed: Cardiovascular Biology Laboratory, Bldg. 2, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4994; Fax: 617-432-0031; E-mail: lee{at}cvlab.harvard.edu.
1 The abbreviations used are: VSMC, vascular smooth muscle cell; ACLP, aortic carboxypeptidase-like protein; RASMC, rat aortic smooth muscle cell; MASMC, mouse aortic smooth muscle cell; HASMC, human aortic smooth muscle cell; bp, base pair(s); kb, kilobase(s); AEBP1, adipocyte enhancer-binding protein 1; PCR, polymerase chain reaction.
2 M. D. Layne and M.-E. Lee, unpublished observations.
3 W. O. Endege and M.-E. Lee, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|