©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a 38-kDa Heparin-binding Glycoprotein (gp38k) in Differentiating Vascular Smooth Muscle Cells as a Member of a Group of Proteins Associated with Tissue Remodeling (*)

Lisa M. Shackelton (1), David M. Mann (2), Albert J. T. Millis (1)(§)

From the (1) Center for Cellular Differentiation, Department of Biological Sciences, University at Albany, State University of New York, Albany, New York 12222 and the (2) Department of Biochemistry, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cultured aortic smooth muscle cells (SMC) exhibit morphological and phenotypic modulation characterized by a change from a substrate attached monolayer culture to a multilayered nodular cell culture in which SMC are imbedded into the extracellular matrix. Associated with nodule formation is a change in the pattern of SMC gene expression including increased expression of a well characterized marker of smooth muscle cell differentiation, SM -actin, and a 38-kDa glycoprotein (gp38k). gp38k has sequence homology with proteins reported to be correlated with tissue remodeling. To characterize the gp38k mRNA we designed degenerate oligonucleotides based on partial polypeptide sequencing to select a cDNA encoding the full-length gp38k. Southern analysis indicates that porcine gp38k is present as a single copy gene. Northern analysis indicates that the increase in gp38k is correlated with an increase in the steady state level of gp38k mRNA; and is present in cultures that have initiated the formation of multilayered foci and nodules. The correlation between SMC differentiation and gp38k expression is further established by using culture conditions that facilitate SMC differentiation. Cultures seeded onto reconstituted extracellular matrix show rapid formation of nodules and increased expression of gp38k mRNA. Comparison of the gp38k and cDNA sequences with nucleotide and protein sequences available through GenBank and SwissProt data banks revealed that molecules homologous to gp38k were present in human, mouse, bovine, and Drosophila tissues, suggesting that the gp38k may be a member of a gene family. Although a function for gp38k has not been identified, this report represents the first report of its correlation with a specific process important in phenotypic and morphological modulation of vascular SMC.


INTRODUCTION

The principal function of aortic medial smooth muscle cells (SMC)() is to maintain the vessel wall tension and contractility (1, 2) . Medial SMC are embedded in an interstitial extracellular matrix and in response to endothelial injury migrate into the intima, proliferate, and synthesize components of the extracellular matrix (3, 4, 5, 6, 7) . The phenotypic modulation of medial SMC from a quiescent differentiated phenotype to a migrating synthetic phenotype is considered to be a key event in the development of atherosclerotic plaques in response to injury (8, 9, 10) .

In culture, explanted SMC also appear to modulate from a contractile to a synthetic phenotype under the influence of serum factors, cytokines, and extracellular matrix components (11, 12, 13) . In post-confluent and density inhibited cultures, SMC initiate phenotypic changes such as increased expression of the smooth muscle (SM) specific genes SM -actin and SM-myosin heavy chains (14, 15, 16, 17, 18, 19) . In the appropriate environment, cultured SMC can express proteins that appear to be related to cell differentiation, including clusterin (20, 21) and bone morphogenetic protein (22) .

After subcultivation in serum containing medium, medial SMC initially form a confluent cell monolayer. Within the monolayer multicellular foci form which subsequently develop into morphologically and molecularly distinct nodules that contain differentiated non-proliferating SMC embedded in a carbohydrate-rich extracellular matrix (22, 23, 24) . In contrast to representative cells in monolayer culture, cells in the nodule contain myofilaments and dense bodies. A comparison of the proteins synthesized and secreted by nodular SMC cultures with those secreted by monolayer SMC cultures revealed increased expression of two glycoproteins: clusterin (20, 21, 25) and a heparin-binding glycoprotein of 38-kDa (gp38k) (26, 27) . The factors regulating differential expression of those genes, and SMC modulation, in general, are not presently known. However, it is known that cultured SMC respond to soluble factors including cytokines, growth factors, and extracellular matrix macromolecules (28, 29, 30, 31, 32, 33, 34, 35, 36) . It is possible that SMC modulation is mediated by proteins secreted by SMC in response to unidentified signals (23, 29) .

In order to characterize the porcine SMC gp38k and begin to develop an understanding of its role in SMC modulation, we used degenerate oligonucleotides, based on polypeptide sequence information to isolate a cDNA encoding full-length gp38k from a nodular SMC cDNA library. The deduced amino acid sequence of the gp38k cDNA indicates that it encodes a protein composed of 383 amino acid residues, has a putative signal sequence, contains a single consensus sequence for N-linked glycosylation, and a heparin-binding consensus sequence. The deduced sequence reveals it to be a member of a family of related proteins whose expression is not limited to SMC. Homologues have been sequenced from human, bovine, murine, and Drosophila cDNAs and all of the family members show partial sequence homology to a group of proteins which cleave the invertebrate matrix polysaccharide chitin (37-40).

Expression of gp38k mRNA is correlated with multilayered SMC growth and nodule formation and the steady state level of the mRNA is increased during conditions that facilitate nodule formation. Addition of nodular cell culture conditioned medium or seeding SMC onto a preformed reconstituted extracellular matrix promote expression of gp38k and mRNA. Although the role of this protein in phenotypic modulation of SMC remains to be elucidated, this report provides strong support for the conclusion that gp38k is a marker of SMC differentiation by demonstrating that its expression parallels that of SM -actin (18, 19) and demonstrates for the first time that gp38k expression can be regulated by components of the extracellular matrix.


EXPERIMENTAL PROCEDURES

Materials

Medium 199 was obtained from Life Technologies, Inc. (Grand Island, NY); fetal bovine sera from HyClone Laboratories, Inc. (Logan, UT); [P]dCTP(3000 Ci/mmol) from Amersham, Inc.; Klenow fragment of DNA polymerase from U. S. Biochemical Corp. (Cleveland, OH); GeneScreen plus from Biotech System (Boston, MA); electrophoresis reagents from Bio-Rad; and TRI reagent from Molecular Research Center, Inc. (Cincinnati, OH).

Cell Culture

Porcine aortic smooth muscle cell cultures were initiated using explants of the luminal face of the thoracic aorta and cultivated in medium 199 supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 3% CO, 97% air as described previously (21, 23) . Cells were routinely subcultivated by treatment of confluent monolayer cultures with EDTA and trypsin in isotonic saline. At each passage the cell number was determined using a Coulter counter (Coulter Electronics, Hialeah, FL) and the cultures replated at 2 10 cells/cm in fresh serum containing medium. Nodular cell cultures were generated by replacing the medium of confluent cultures with fresh medium 199 supplemented with 5% fetal bovine serum. Under these conditions, nodules begin to form after 5-9 days and well nodulated cultures (>70 nodules/cm of surface) are present within 2-3 weeks.

Substrate Preparation

Freshly harvested cells were mixed with equal volumes of soybean trypsin inhibitor, centrifuged for 5 min at 1000 rpm, and immediately suspended in M199 containing 10% serum. These cells were seeded at a density of 7 10cells/cm on various substrata. Reconstituted basement membrane gel, Matrigel (Collaborative Biomedical Products, Bedford, MA), was used after 1:1 dilution, on ice, in cold serum-free medium M199. Collagen gel-coated cluster dishes were also prepared on ice using a collagen I solution consisting of 8 parts rat tail collagen I (Collaborative Biomedical Products), 1 part 0.01 M NaOH, 1 part 10 phosphate-buffered saline and the pH brought to 7.4 with 0.1 M NaOH. Each well of a 6-well cluster dish (Falcon, Becton Dickinson, Bedford, MA) was coated with 400 µl of either gel solution and allowed to gel for 30 min at 37 °C before addition of SMC (21) .

Culture morphology was monitored using a Videoscope imaging camera attached to an Olympus CK2 inverted microscope fitted with a 2 objective and the image processed with Metamorph imaging software (Universal Imaging Corp., West Chester, PA). The numbers of nodules are calculated by the software directly from the recorded image.

Preparation of Conditioned Media

Serum-free conditioned media from monolayer and nodular smooth muscle cell cultures were prepared by washing cultures 3 with Hank's balanced salt solution before incubation for 24 h in serum-free medium 199. The medium was collected into 1 mM phenylmethylsulfonyl fluoride and 1% aprotinin (Trasylol; Meloy Labs, NY), centrifuged for 15 min at 37,000 g, and either used immediately or stored frozen (23, 26) .

Gel Electrophoresis

For polyacrylamide gel electrophoresis the conditioned medium was precipitated with 15% trichloroacetic acid at 4 °C for 1 h. Greater than 95% of the total protein was precipitated by this procedure (as estimated from Bio-Rad Protein assays) and all of the immunodetectable gp38k was precipitated (trichloroacetic acid-soluble fractions were concentrated in Amicon ultrafiltrations units and examined by immunoassay). Electrophoresis grade reagents were obtained from Bio-Rad.

Polyacrylamide gel electrophoresis was performed using a 10% separating gel and a 5% stacking gel containing 1% SDS. Gels were prepared using the system of Laemmli (41) and run at a constant current of 16 mA for 1.5-2 h. Molecular weight standards were myosin (200,000), phosphorylase b(97, 400) , bovine serum albumin (69,000), carbonic anhydrase (30, 0) , trypsin inhibitor (21, 500) , and lysozyme (14, 300) . Gel lanes were loaded with either equal volumes of conditioned medium or equal amounts of protein (Bio-Rad protein assay).

Antibody Preparations and Immunoblotting

Polyclonal antibodies to rat clusterin (Sertoli cell clusterin) were generously provided by Drs. Michael Griswold and Steven Sylvester, Washington State University (42) . Polyclonal antibodies to gp38k were prepared in this laboratory as described previously (27) . Antibodies to gp38k were affinity purified by adsorption to purified gp38k fixed to nitrocellulose membrane (43, 44) . Proteins were separated on minigels and transferred from the gel onto nitrocellulose paper (Schleicher & Schuell) in 192 mM glycine, 20% methanol, 25 mM Tris-HCl, pH 8.3, using a Genie electroblotter (Idea Scientific). Following transfer at 25 V for 1 h the blot was incubated in a blocking agent consisting of 5% nonfat dried milk (Carnation, Los Angeles, CA). After incubation with primary antibody (1:2000 dilution of anti-SGP2 or anti-38K protein in rabbit sera) for 2 h at room temperature on a rocker, the membrane was washed and incubated with horseradish peroxidase-conjugated donkey anti-rabbit whole antibody (1:10,000 dilution; Amersham) for 60 min at room temperature. After washing, immobilized antigens were detected using an enhanced chemiluminescence assay (ECL: Amersham). Following immunotransfer, each gel was stained with Coomassie Blue to evaluate the efficiency of transfer.

Purification and Amino-terminal Sequencing of gp38k

gp38k was purified from nodular SMC conditioned medium by heparin affinity chromatography (Pharmacia, Inc., Pitscataway, NJ), followed by high performance liquid chromatography. Samples were purified using conditions to minimize amino-terminal blocking and further purified via 5-20% linear gradients of polyacrylamide (44) . The gel bands were visualized by brief staining with Ponceau S. Amino-terminal sequencing was performed on protein eluted from gel band slices by soaking overnight at 37 °C in 0.5 ml of 100 mM Tris, 0.1% SDS, pH 8.0, or by electroeluting in 0.1% SDS, 0.05 M NHHCO. This material was loaded onto the hydrophobic portion of a biphasic Hewlett-Packard sequencer column and washed with 0.2% trifluoroacetic acid, and 10% acetonitrile to remove the Tris and glycine. Sequencing was performed using automated Edman degradation chemistry on a Hewlett-Packard G1000A Protein Sequencing system.

To obtain internal sequences, samples were concentrated in 50 mM NHHCO, using an ISCO electroelution protein concentrator. Each sample was diluted with 5 volumes of 50 mM NHHCO and subsequently precipitated with 8 volumes of acetone at -20 °C. The pellet was then washed 3 times with 95% ethanol at -20 °C. The pellet was resuspended in 30 µl of Lys-C digestion buffer composed of 25 mM Tris-HCl, pH 8.5, 0.01% SDS, 1 mM EDTA and allowed to dissolve overnight at 4 °C. This preparation was digested for 24 h with 5 µg of Lys-C (Boehringer Mannheim, Indianapolis, IN) and fractionated on a C reverse phase column (Brownlee RP300) prior to sequencing.

From this analysis we obtained a single amino-terminal sequence and 5 internal sequences. Degenerate oligonucleotides were synthesized based on conserved regions in gp38k and mammalian homologues and porcine codon usage (47) . Degeneracy was reduced by substituting inosine at the third position of the triplet coding for leucine in the sense primer. To allow directional cloning, the sense primer was designed with a BamHI adaptor at the 5` end followed by TTYGAYGGNYTIGAYYT-3 and the antisense primer with an EcoRI adaptor at the 5` end followed by TCRTCRTANCCNACCCA-3`. The primers were synthesized and trityl-specific purified by Ransom Hill Bioscience (Ramona, CA). Amplification of the region between these two primers included nucleotide sequences encoding three of the polypeptide sequences that we obtained by direct amino acid sequencing of purified gp38k.

Selection of Clone Expressing gp38k cDNA (pBS38K)

To determine the cDNA sequence of gp38k, a strategy employing reverse transcription-PCR and cDNA library screening was used.

Reverse Transcription

RNA isolated from cultured porcine aorta nodular smooth muscle cells was reverse transcribed as follows: 2-4 µg of RNA was brought up to a final volume of 16.5 µl and incubated at 65 °C for 5-7 min. After heating, the RNA was quenched on ice for 2 min and centrifuged briefly. A reverse transcription reaction mixture (final volume of 13.5 µl) containing 250 mM Tris-Cl, pH 8.3, 50 mM MgCl, 300 mM KCl, 50 mM dithiothreitol, 50 mg/ml oligo(dT) (Pharmacia, Upsala, Sweden), 0.5 mM dNTP mixture (U. S. Biochemical Corp.), 1 mg/ml bovine serum albumin, 667 units/ml RNasin (Promega, Madison, WI), and 900 units/ml AMV Reverse Transcriptase (Molecular Genetic Resources, Tampa, FL) was added to the tube containing RNA and the reaction was incubated at 42 °C for 1 h. After incubation, 20 µl of double distilled HO was added to the resulting cDNA before using in subsequent PCR reactions.

Polymerase Chain Reaction

Polymerase chain reaction was performed in 50-µl samples containing 500 mM KCl, 100 mM Tris-Cl, pH 8.3, 25 mM MgCl, 200 µM dNTP mixture, 4 mM each of the sense and antisense degenerate primers, 80 units/ml Taq DNA polymerase (Perkin Elmer), and 10 µl of cDNA reverse transcribed under the conditions listed above. Each 50-µl reaction was overlaid with 1 drop of DNase-free, RNase-free mineral oil (Sigma), denatured for 5 min at 94 °C, and cycled 30 times using the following parameters; 60 s at 94 °C, 90 s at 48 °C, and 60 s at 72 °C in an Eppendorf Microcycler E (Eppendorf, Fremont, CA)

Subcloning and Sequencing of PCR Product

EcoRI and BamHI (40 units) were added to the reaction mixture containing the PCR product and incubated at 37 °C for 2.5 h. After electrophoresis in 1% agarose and treatment with ethidium bromide, an amplification product of approximately 600 bp was observed and excised from the gel. The excised DNA was purified and then ligated between the EcoRI and BamHI sites of the polylinker region of pBluescript SK- (Stratagene, La Jolla, CA). Xl-1 Blue bacteria (Stratagene) were transformed with pBluescript containing the insert and incubated overnight at 37 °C on LB plates containing ampicillin.

White colonies were picked and grown overnight at 37 °C in LB medium containing 50 µg/ml ampicillin. Minipreps were performed to isolate plasmid DNA, and the DNA was displayed on a 1% agarose gel next to 2 µg of pBluescript SK- without an insert. The insert was partially sequenced using the chain termination method (45) with [S]dATP (Amersham) and a Sequenase 2.0 DNA Sequencing Kit (U. S. Biochemical Corp.) and double stranded plasmid DNA that was prepared by the alkaline lysis procedure (45) .

cDNA Library Screening

A Uni-Zap XR cDNA library was prepared using poly(A) RNA isolated from nodular SMC (20) . XL-1 Blue Escherichia coli (Stratagene) cells were infected with 10 plaque forming units of the Uni-Zap cDNA library and plated onto 100 15-mm LB Agar plates. The plates were incubated at 37 °C for 8 h.

Each plaque lift was transferred onto nitrocellulose (Schleicher & Schuell, Inc.) and denatured by submerging in 1.5 M NaCl, 0.5 M NaOH for 2 min. After neutralization, the DNA was cross-linked to the filters for 30 s using a Stratalinker UV cross-linker (Stratagene).

Filters were prehybridized at 42 °C for 2 h in a solution containing 50% deionized formamide, 0.5% SDS, 2 Pipes buffer, pH 6.5, and 100 mg/ml denatured sonicated salmon sperm DNA. A cDNA probe was prepared using the 600-bp EcoRI/BamHI restriction fragment of pBS600 excised from a low melting point agarose gel. The probe was labeled with [P]dCTP (Amersham) using a MultiPrime DNA Labeling System (Amersham). Hybridization was accomplished using 1 10 counts per filter at 42 °C for 16 h. To identify positive clones, filters were washed, and then exposed to Kodak X-Omat film overnight at -70 °C in a cassette including an intensifying screen. Positive clones were isolated, replated, and prescreened until single isolates were obtained.

In Vivo Excision of Potential Clones

Clonal isolates were excised in vivo, from the Uni-Zap vector following the procedures described by the manufacturer and used to transform XL-1 Blue bacteria and plated onto LB plates containing 50 µg/ml ampicillin coated with isopropyl-1-thio--D-galactopyranoside and 5-bromo-4-chloro-3-indoyl -D-galactoside. Plates were grown overnight at 37 °C. Single white colonies were picked and grown overnight at 37 °C in LB broth containing 50 µg/ml ampicillin.

DNA sequencing utilized new primers after approximately every 200 nucleotides. During the course of sequencing, the BLAST e-mail server at the National Center for Biotechnology Information was used to search for homologous sequences in various data bases (48) . In addition, the PC GENE package (Intelligenetics, Mountain View, CA) and FASTA were used to search for homologies against various data bases.

Southern Blotting

Genomic DNA was isolated from nodular porcine SMC using TRI Reagent, according to the manufacturer's instructions. Genomic DNA (12 µg) was digested with 40 units of BamHI, EcoRI, or HindIII for 20 h at 37 °C. Each sample was loaded onto a 0.7% agarose gel containing 1 mg/ml EtBr and run at 60 V until the markers (1 kilobase pair molecular weight ladder; Life Technologies, Inc., Grand Island, NY) were well separated (approximately 5 h).

After denaturation in 0.4 M NaOH containing 0.6 M NaCl the gel was incubated twice for 15 min in a neutralization solution (0.5 M Tris-HCl, pH 7.8, 1.5 M NaCl). Size fractionated DNA fragments were transferred onto nylon membrane (GeneScreen, DuPont) using downward capillary transfer for 16 h (49) . The membrane was then washed in neutralization solution and the DNA cross-linked to the membrane using a Stratalinker (Strategene, La Jolla, CA).

The membrane was prehybridized in buffer containing 5 SSC, 5 Denhardt's solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA at 65 °C for 4 h. A 600-bp cDNA (excised from pBS600) was random primer labeled with [P]dCTP and added to the prehybridization solution at an activity of 1.2 10 counts/ml. Hybridization occurred at 65 °C for 20 h. Subsequently the membrane was washed 4 times for 30 min each at 65 °C in 0.5 SSC, 0.1% SDS before exposure to X-Omat film.

Northern Analysis

Total cellular RNA was isolated from smooth muscle cells using TRI Reagent which is based on guanidine-thiocyanate-phenol-chloroform extraction procedure (45, 46) . Each sample was denatured in 10% glyoxal and 50% formamide by heating to 65 °C for 5 min. After electrophoresis in 1% agarose the gels were treated for 30 min with 200 mM NaOH, vacuum transferred to GeneScreen Plus (DuPont NEN) membranes, and neutralized. The 28 S and 18 S RNA species were examined on each membrane to evaluate the consistency of sample loading and transfer. After UV cross-linking the nucleic acid on the membrane (Stratalinker; Stratagene), the membrane was prehybridized in 8% Denhardt's solution (1% Ficoll, polyvinyl pyrrolidine, and Pentax fraction V of bovine serum albumin), 43% formamide, 0.69 M NaCl, 0.09% Na pyrophosphate, 0.9% SDS, 0.7% dextran sulfate, 1.74 mg/ml heparin, and 86 mg/ml denatured salmon sperm DNA for 2 h overnight at 42 °C. Random primer P-labeled cDNA (10 cpm/ml) was added to the same buffer, and the membrane hybridized at 42 °C overnight. The membrane was then washed twice in 0.2% SDS, 1 SSC for 15 min at room temperature and once in 0.2% SDS, 0.1 SSC at 50 °C. After incubation at -70 °C for 15-48 h the membrane was analyzed by autoradiography. Quantitation of radioactive signals was achieved using a Betascope 603 (Betagen, Intelligenetics Inc., Mountain View, CA). The gp38k mRNA signal was detected using either the full-length cDNA encoding porcine gp38k cloned in this laboratory and described in this manuscript for Northern assays or a 600-bp PCR product for the Southern assay. The Northern blot was stripped and subsequently reprobed with a mouse 600-bp polymerase chain reaction product encoding human glyceraldehyde-3-phosphate dehydrogenase and a 500-bp polymerase chain reaction product encoding human -actin (provided by Dr. S. Kumar, University at Albany).

Photography

Phase-contrast microscopy was performed using a Diaphot-TMD inverted microscope (Nikon, Tokyo, Japan) using a 10 objective.


RESULTS

A 38,000-Da glycoprotein (gp38k) was previously isolated and identified as a heparin-binding glycoprotein secreted by cultured vascular smooth muscle cells that had undergone conversion from monolayer to nodular cell culture (23, 26, 27) . To determine if gp38k was also present in the nodule layer we stained well developed nodules with antibodies to gp38k and antibodies to the smooth muscle specific isoform of -actin (SM -actin), an antigen expressed in differentiated SMC (18, 19) , and observed that both antigens co-localize in the nodule indicating that both are present in the nodular structures (data not presented).

Substrate Effects on gp38k Expression

The time required for nodule formation is significantly reduced in SMC cultures seeded on a reconstituted gelatinous matrix (21) . Therefore we examined the effects of two reconstituted matrices, Matrigel and a collagen gel on gp38k expression. Fig. 1shows the nodules formed on each of these substrates at 24-36 h after seeding monolayer cells. Panels A-C show SMC, growing in medium 199 supplemented with 5% fetal bovine serum, on untreated plastic (A) or Matrigel (B and C) substrates at 24 h after seeding. Panel A shows that on plastic the cells are attached and spread but have not reorganized to form nodules as shown in the photograph in panel B. Panel C shows a lower power image recorded by a video imaging system and reveals a density of 81 nodules/cm. Panel D shows nodule formation in cultures seeded onto collagen gel and recorded by video imaging. This image was recorded after 36 h since, as we previously reported, nodule formation is slower on this substrate (21) . The number of nodules in the collagen gel image was 78 nodules/cm.


Figure 1: Effects of reconstituted matrix on SMC nodule formation. Cultures were seeded onto plastic (A), Matrigel (B and C), or collagen gel (D) and incubated for 24 h (A-C) or 36 h (D). Panels A and B were photographed using a Diaphot-TMD inverted microscope and a 10 objective. The images in panels C and D were obtained using video imaging microscopy with a 2 objective, as described under ``Experimental Procedures.''



Cultures seeded onto collagen gel or Matrigel were examined for gp38k expression as shown in Fig. 2. In both cases gp38k is detected in the cultures seeded on the reconstituted matrices. Western immunoassay was used to determine if either Matrigel or collagen gel contained gp38k and none was detected (data not presented).


Figure 2: Western analysis of cultures seeded on reconstituted matrix. Cultures were seeded onto plastic (A and C), collagen gel (B), or Matrigel (D) and incubated for 24 h. Conditioned medium was collected from each culture, fractionated by SDS-polyacrylamide gel electrophoresis, and probed with anti-gp38k antibody (38 kDa protein).



gp38k Full-length Sequence

To identify the complete amino acid sequence of gp38k, gp38k protein was isolated from nodular cell conditioned medium via heparin affinity chromatography followed by elution with a step gradient of NaCl (27) . The gp38k-containing fractions were further purified by polyacrylamide gel electrophoresis (48). Partial amino acid sequence was obtained from non-digested and Lys-C-digested gp38k yielding the underlined sequences shown in Fig. 3.


Figure 3: cDNA sequence of pBS38kDa and conceptual translation of the amino acid sequence. A 1733-bp cDNA insert encodes an open reading frame of 383 amino acids beginning with an ATG codon at nucleotide position 66. The conceptual translation indicates a putative signal sequence including amino acid residues 1-21 (bold type). Single underlined regions mark sequences identified by direct amino acid sequencing of purified SMC gp38k. The position of a putative N-linked glycosylation site is indicated by a vertical arrow and a consensus heparin binding site by a double underline (residues 144-149). The sequences used to design degenerate oligonucleotides are bracketed and in bold type at residues 132-137 and 325-330.



A cDNA clone encoding gp38k was selected from a nodular SMC cDNA library using a PCR product described under ``Experimental Procedures.'' As shown in Fig. 3, the gp38k cDNA insert is 1733 bp, contains a single open reading frame beginning with the ATG at base pair 67, and can encode a polypeptide sequence 383 amino acids in length with a calculated molecular mass of 42,440 Da. That this sequence corresponds correctly to the 38-kDa heparin-binding protein secreted by nodular SMC cultures is confirmed by sequence identity with five internal peptide sequences which were obtained by direct amino acid sequencing of the purified protein. The sequences of these 5 polypeptides, generated by digesting the purified secreted protein with Lys-C, and their positions within the cloned cDNA are shown in Fig. 3. Previous reports (26, 27) indicated that the gp38k protein radiolabels when SMC are metabolically labeled with [H]glucosamine, is sensitive to endoglycosidase F treatment, and is synthesized with a reduced apparent M when SMCs are grown in the presence of tunicamycin, an inhibitor of N-linked glycosylation. Consistent with these properties is the occurrence of a consensus sequence for asparagine-linked glycosylation which occurs at amino acid 60 encoded by the cDNA (Fig. 3).

The protein encoded by this cDNA has a calculated theoretical isoelectric point of 9.28, contains 6 cysteines, and a cluster of basic amino acids, RRDKRH, located at positions 144-149, which represent the heparin binding consensus sequence reported for a number of other heparin-binding molecules (58) . The cDNA sequence predicts a secreted protein with a conventional signal sequence corresponding to the first 21 amino acids. The mature protein is predicted to begin with the YKLVCYYTSWQYRE sequence and this is in agreement with the results obtained from direct amino acid sequencing of the 38-kDa protein secreted by SMC cultures.

gp38k Is Homologous to Proteins in Other Species

A comparison of the cDNA encoded sequence with the SwissProt, GenBank, and Protein Inquiry Resource (PIR) data bases, using the internet BLAST and FASTA e-mail servers (described under ``Experimental Procedures''), revealed sequence homology with cDNAs isolated from human cartilage, bovine oviduct, and mouse macrophage (36, 37, 38, 39) as shown in Fig. 4 . The human and porcine homologues are 84% similar over the length of the 383-amino acid polypeptide. The bovine and mouse homologues show less homology over the length of the proteins, but there are regions that are highly conserved between all of the homologues. The mouse homologue is 401 amino acids and the bovine homologue 546 amino acids in length. Recently a homologous sequence was discovered in Drosophila and that sequence encodes a 455-amino acid protein (40) .


Figure 4: Alignment of gp38k with homologous sequences from human, murine, Drosophila, and bovine species. Amino acid sequences of gp38k, human HC gp39K (GenBank M80927), mouse macrophage (GenBank M94584), bovine oviduct glycoprotein (D16639), and Drosophila are compared with chitinase (SwissProt P29030). Alignment was achieved using the Waterman algorithm in the MacDNASIS Pro-analysis system. Consensus positions are highlighted and insertions indicated by dashed lines.



gp38k Is Encoded by a Single Gene

In order to estimate the number of gene copies in porcine genomic DNA, Southern blot analysis was performed with samples restricted with BamHI, EcoRI, or HindIII enzymes as shown in Fig. 5 . Using stringent hybridization conditions we detected a single hybridization band in the HindIII restricted sample suggesting that gp38k is encoded as a single copy gene. The multiple bands present in the BamHI and EcoRI restricted samples likely resulted from internal restriction sites.


Figure 5: Southern blot analysis of porcine genomic DNA extracted from SMC. A Southern blot containing 12 µg of BamHI (A), EcoRI (B), and HindIII (C) restricted genomic DNA was probed with a random-primer labeled 600-bp PCR product. Hybridizations and washes were done using high stringency conditions as described under ``Experimental Procedures.''



Expression of gp38k mRNA in SMC

The steady state expression of gp38k mRNA was compared in total RNA preparations isolated from monolayer and nodular SMC cultures. Fig. 6shows a Northern assay of total RNA probed for gp38k, SM -actin, and -actin mRNAs. In nodular cell cultures the expression of gp38k mRNA is increased >10 as detected by Betagen analysis. The Northern analysis establishes that the pBS38k cDNA insert hybridizes with an mRNA of 1.8 kilobase, and that this mRNA is expressed at higher levels in nodular than in monolayer SMC cultures. The expression of a smooth muscle isoform of -actin (SM -actin) has been reported to be correlated with differentiated SMC while the -actin isoform is expressed more broadly (18, 19, 50, 51, 52) . Fig. 6 shows that the gp38k mRNA level is greater in cultures that express an increased level of SM -actin and a decreased level of -actin mRNA and may represent an additional marker for differentiated SMCs.


Figure 6: Northern assay of gp38k and actin mRNA. Total RNA (8 µg/lane) from confluent monolayer (M) and 14-day nodular (N) cell cultures were probed with random primer labeled cDNA to gp38k (38 kDa protein), SM -actin, -actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).



Time Course of gp38k Expression

Previously we reported that nodules form in SMC culture in multilayered regions that begin to form after 4 days in culture and continue to form after that time (21) . By day 14 the number of nodules has increased to >65 nodules/cm. To examine the correlation between nodule formation and gp38k expression we analyzed conditioned medium and cell extracts from SMC at intervals from 2 to 14 days of culture. The steady state levels of gp38k protein and mRNA levels were examined by immunoblot (Fig. 7A) and Northern (Fig. 7B) analysis, respectively, to evaluate the correspondence between the time of nodule formation and gene expression. The antigen is detected after 4 days of culture and the level is increased in 12-14-day cultures (>50 nodules/cm). Northern assays indicate that gp38k mRNA is also detectable at days 4-6 and the level of expression increased after day 10. These results indicate that nodulating SMC express more mRNA for gp38k and accumulate more gp38k protein in their culture medium as the density of nodules increases.


Figure 7: Time course of gp38k expression. Conditioned medium (A) was collected from SMC at 24-h intervals during a 2-14 day time period (lanes 2-14) and equal quantities were probed with anti-gp38k antibodies. Lane C contains 0.5 µg of heparin-purified gp38k probed with anti-gp38k antibodies. Total RNA (B) was extracted from SMC cultures during the same 2-14-day time period and 8 µg loaded into each lane and probed with cDNA to gp38k and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).




DISCUSSION

The process of SMC phenotypic modulation in vitro is correlated with increased expression and secretion of a heparin-binding glycoprotein with M = 38,000 (gp38k) (23, 26, 29, 53) . The level of gp38k expression is 0.01 µg/ml in monolayer SMC cultures and increases to 1.9 µg/ml in SMC cultures that have formed nodules (26) . Previous reports demonstrated that representative cells in nodules are morphologically differentiated and express some of the characteristics of medial SMC (13, 23, 24) . In contrast, SMC in monolayer cell culture express characteristics representative of migrating proliferating SMC.

Recent studies indicate that the pattern of gene expression is modified in nodular cell cultures. Clonal populations of human and bovine nodular cells derived from aortic media were reported to express bone morphogenetic protein-2a and osteocalcin (22) . Porcine SMC nodules derived from aortic media also show an altered pattern of gene expression that includes an increase in clusterin and gp38k (21, 23, 25, 53, 54) . In order to investigate the expression of gp38k mRNA and its correlation with SMC differentiation we have now cloned and sequenced a full-length cDNA that encodes the porcine gp38k and report that its expression is correlated with that of SM -actin (18, 19) .

The deduced amino acid sequence of gp38k includes a putative signal sequence of 21 amino acid residues, a single consensus sequence for N-linked glycosylation, and a putative heparin binding site. Previously we demonstrated that gp38k is a heparin-binding glycoprotein that is eluted from heparin-Sepharose by 400 mM NaCl (27) . Presently it is not known if the interaction with heparin is related to a functional interaction with glycosaminoglycan components of the extracellular matrix. gp38k contains 6 cysteine residues, however, our previous work indicates that they are not involved in disulfide bonding since the apparent molecular weight of gp38k is similar in reduced and nonreduced samples examined by SDS-polyacrylamide gel electrophoresis (27) . It is not known if its homology with chitinase has any functional significance since chitinase activity has not been detected in two of the gp38k homologues (37, 40) .

SMC nodulation, in vitro, is facilitated by the presence of a collagenous extracellular matrix (26) . Medial SMC, in vivo, are embedded in a three-dimensional extracellular matrix and we postulated that nodules mimic the media by containing SMC embedded in an extracellular matrix which is elaborated by the cell cultures. During the formation of the multilayered regions that give rise to nodules, gp38k synthesis is initiated. The time required for nodules to form in standard tissue culture conditions is greater than 9 days. However, that time requirement is reduced to 24-36 h when SMC are seeded onto a reconstituted extracellular matrix, composed of collagen gel or Matrigel (21, 55, 56) . Other laboratories have reported that SMC cultured on Matrigel exhibit an altered pattern of gene expression suggestive of SMC differentiation (35) . In this report, we demonstrate that gp38k mRNA expression is induced by each of the reconstituted matrices and gp38k expression correlates well with nodule formation and SM -actin expression.

Recently genes homologous to gp38k have been cloned from three mammalian species and Drosophila(36, 37, 38, 39) and the mRNA identified in tissues other than SMC. The human homologue, HC gp39k has been identified in articular chondrocytes and synovial cells (37) . A bovine oviduct-specific glycoprotein, with sequence homology to gp38k, appears to correlate with changes during ovulation (39) . An oviduct specific homologue in baboon is larger than the porcine gp38k and more heavily glycosylated (57) . The occurrence of gp38k homologues in several species and in different tissues suggests that it may be a member of a larger protein family. To address that question we used Southern transfer analysis done under high stringency conditions to estimate the number of genes for gp38k in porcine SMC genomic DNA. Restriction digestion with HindIII and hybridization with a 600-bp cDNA revealed a single band suggesting that porcine gp38k is present as a single gene.

The occurrence of gp38k in SMC undergoing morphological reorganization and the gp38k homologues in other tissues undergoing remodeling suggests the possibility that gp38k has a regulatory role in that process in vascular as well as other tissues. For example, in experimental animal models of restinosis medial SMC develop a morphology and pattern of cytoskeletal protein expression, including change in the predominant actin iosform, suggesting a de-differentiated phenotype. At later times after injury the SMC show partial re-differentiation including increased amounts of vimentin, desmin, tropomyosin, and -actin (59) . Although the mechanism of gp38k action is not known, the presence of heparin binding activity and consensus sequence suggests the possibility that it may interact with glycoprotein or proteoglycan components of the extracellular matrix or cell surface to modify cell adhesion, cell motility, or gene expression; and consequently influence the phenotypic state of the SMC during the process of recovery from injury. The data presented here represents the first report of its correlation with a specific process important in phenotypic and morphological modulation of vascular SMC.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant HL40417 (to A. M.) and the American Red Cross (to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The gp38k sequence is available as GenBank U19900.

§
To whom correspondence should be addressed. Tel.: 518-442-4361; Fax: 518-442-4767; E-mail, millis@cnsunix.albany.edu.

The abbreviations used are: SMC, smooth muscle cells; SM, smooth muscle; gp38k, 38-kDa glycoprotein; PCR, polymerase chain reaction; bp, base pair(s); Pipes, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Dr. Greg Lnenicka (Department of Biology, University at Albany) for advice on video imaging and providing access to the Video Imaging Facility at the University at Albany.


REFERENCES
  1. Campbell, G. R., and Campbell, J. H.(1985) Exp. Mol. Pathol. 42, 139-162 [Medline] [Order article via Infotrieve]
  2. Ross, R.(1993) Nature 362, 801-809 [CrossRef][Medline] [Order article via Infotrieve]
  3. Mosse, P. R., Campbell, G. R., Wang, Z. L., and Campbell, J. H.(1985) Lab. Invest. 53, 556-562 [Medline] [Order article via Infotrieve]
  4. Fingerle, J., Johnson, R., Clowes, A. W., Majesky, M. W., and Reidy, M. A.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8412-8416 [Abstract]
  5. Dilley, R. J., McGeachie, J. K., and Prendergast, F. J.(1987) Atherosclerosis 63, 99-107 [Medline] [Order article via Infotrieve]
  6. Schwartz, S. M., Campbell, G. R., and Campbell, J. H.(1986) Circ. Res. 58, 427-444 [Abstract]
  7. Ross, R., and Klebanoff, S. J.(1971) J. Cell Biol. 50, 159-171 [Abstract/Free Full Text]
  8. Clowes, A. W., Reidy, M. A., and Clowes, M. M.(1983) Lab. Invest. 49, 327-333 [Medline] [Order article via Infotrieve]
  9. Nobuyoshi, M., Kimura, T., Ohishi, H., Horiuchi, H., Nosaka, H., Hamasaki, N., Yokoi, H., and Kim, K.(1991) J. Am. Coll. Card. 17, 433-439 [Medline] [Order article via Infotrieve]
  10. Johnson, D. E., Hinohara, T., Selmon, M. R., and Braden, L. J.(1990) J. Am. Coll. Card. 15, 419-425 [Medline] [Order article via Infotrieve]
  11. Chamley-Campbell, J. H., Campbell, G. R., and Ross, R.(1981) J. Cell Biol. 89, 379-383 [Abstract]
  12. Raines, E. W., and Ross, R.(1993) Br. Heart J. 69, (suppl.) S30-S37
  13. Gimbrone, M. A., and Cotran, R. S.(1975) Lab. Invest. 33, 16-27 [Medline] [Order article via Infotrieve]
  14. Rovner, A. S., Murphy, R. A., and Owens, G. K.(1986) J. Biol. Chem. 261, 14740-14745 [Abstract/Free Full Text]
  15. Frid, M. G., Shekhonin, B. V., Koteliansky, V. E., and Glukhova, M. A. (1992) Dev. Biol. 153, 185-193 [Medline] [Order article via Infotrieve]
  16. Birukov, K. G., Frid, M. G., Rogers, J. D., Shirinsky, V. P., Koteliansky, V. E., Campbell, J. H., and Campbell, G. R.(1993) Exp. Cell Res. 204, 46-53 [CrossRef][Medline] [Order article via Infotrieve]
  17. Glukhova, M. A., Koteliansky, V., Fondacci, C., Marotte, F., and Rappaport, L.(1993) Dev. Biol. 157, 437-447 [CrossRef][Medline] [Order article via Infotrieve]
  18. Shanahan, C. M., Weissberg, P. L., and Metcalfe, J. C.(1993) Circ. Res. 73, 193-204 [Abstract]
  19. Holycross, B. J., Blank, R. S., Thompson, M. M., Peach, M. J., and Owens, G. K.(1992) Circ. Res. 71, 1525-1532 [Abstract]
  20. Diemer, V. D., Hoyle, M., Baglioni, C., and Millis, A. J. T.(1992) J. Biol. Chem. 267, 5257-5264 [Abstract/Free Full Text]
  21. Thomas-Salgar, S., and Millis, A. J. T.(1994) J. Biol. Chem. 269, 17879-17885 [Abstract/Free Full Text]
  22. Bostrom, K., Watson, K. E., Horn, S., Wortham, C., Herman, I. M., and Demer, L. L.(1993) J. Clin. Invest. 91, 1800-1809 [Medline] [Order article via Infotrieve]
  23. Brennan, M. J., Millis, A. J. T., and Fritz, K. E.(1982) J. Cell. Physiol. 112, 284-290 [Medline] [Order article via Infotrieve]
  24. May, J. F., Paule, W. J., Rounds, D. E., Blankenhorn, D. H., and Zemplenyi, T.(1975) Virchows Arch. B. Cell Pathol. 18, 205-211
  25. Rosenberg, M. E., Dvergsten, J., and Correa-Rotter, R.(1993) J. Lab. Clin. Med. 121, 205-214 [Medline] [Order article via Infotrieve]
  26. Millis, A. J. T., Hoyle, M., Reich, E., and Mann, D. M.(1985) J. Biol. Chem. 260, 3754-3761 [Abstract]
  27. Millis, A. J. T., Hoyle, M., and Kent, L.(1986) J. Cell. Physiol. 127, 366-372 [Medline] [Order article via Infotrieve]
  28. D'Amore, P. A., and Smith, S. R.(1993) Growth Factors 8, 61-75 [Medline] [Order article via Infotrieve]
  29. Brennan, M. J., Millis, A. J. T., Mann, D., and Fritz, K. E.(1983) Dev. Biol. 97, 391-397 [Medline] [Order article via Infotrieve]
  30. Hedin, U., and Thyberg, J.(1987) Differentiation 33, 239-246 [Medline] [Order article via Infotrieve]
  31. Thyberg, J., Palmberg, L., Nilsson, J., Ksiazek, T., and Sjoelund, M. (1983) Differentiation 25, 156-167 [Medline] [Order article via Infotrieve]
  32. Kunzelmann, U., and Dartsch, P. C.(1992) Cell. Physiol. Biochem. 2, 49-56
  33. Majack, R. A.(1987) J. Cell Biol. 105, 465-471 [Abstract]
  34. Owens, G. K., Geisterfer, A. A. T., Yang, Y. W-H., and Komlriya, A. (1988) J. Cell Biol. 107, 771-780 [Abstract]
  35. Pauly, R. R., Passaniti, A., Crow, M., Kinsella, J. L., Papadopoulous, N., Monticone, R., Lakatta, E. G., and Martin, G. R.(1992) Circulation 86, Suppl. III, 68-73
  36. Davidson, J. M., Zoia, O., and Liu, J-M.(1993) J. Cell. Physiol. 155, 149-156 [Medline] [Order article via Infotrieve]
  37. Hakala, B. E., White, C., and Recklies, A. D.(1993) J. Biol. Chem. 268, 25803-25810 [Abstract/Free Full Text]
  38. Chang, N. C. A., Liu, C. H., and Chang, A. C.(1993) Accession #S27879, submitted to data bank June, 1993
  39. Sendai, Y., Abe, H., Kikuchi, M., Satoh, T., and Hoshi, H.(1994) Biol. Reprod. 50, 927-934 [Abstract]
  40. Kirkpatrick, R. B., Matico, R. E., McNulty, D. E., Strickler, J. E., and Rosenberg, M.(1995) Gene (Amst.) 153, 147-154 [CrossRef][Medline] [Order article via Infotrieve]
  41. Laemmli, U.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  42. Collard, M. W., and Griswold, M. D.(1987) Biochemistry 26, 3297-3303 [Medline] [Order article via Infotrieve]
  43. Matsudaira, P. T.(1989) A Practical Guide to Protein and Peptide Purification for Microsequencing, pp. 1-129, Academic Press, San Diego, CA
  44. Hunkapillar, M. W., Lujan, E., Ostrander, F., and Hood, L. E.(1983) Methods Enzymol. 91, 227-236 [Medline] [Order article via Infotrieve]
  45. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.(1987-1994) Current Prototcols in Molecular Biology, Vols. 1 and 2, John Wiley and Sons, New York
  46. Chomczynski, P.(1993) Manufacturer's Protocol: TRI Reagent-RNA/DNA/Protein Isolation Reagent, Molecular Research Center, Cincinnati, OH
  47. Wada, K-N., Aota, S-i., Tsuchiya, R., Ishibashi, F., Gojobori, T., and Ikemura, T.(1990) Nucleic Acids Res. 18, (suppl.) 2367-2411 [Medline] [Order article via Infotrieve]
  48. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  49. Chomczynski, P.(1992) Anal. Biochem. 201, 134-139 [Medline] [Order article via Infotrieve]
  50. Corjay, M. H., Thompson, M. M., Lynch, K. R., and Owens, G. K.(1989) J. Biol. Chem. 264, 10501-10506 [Abstract/Free Full Text]
  51. Strauch, A. R., Min, J. C., Reeser, H., Yan, H., Foster, D. N., and Berman, M. D.(1992) J. Cell. Biochem. 50, 266-278 [Medline] [Order article via Infotrieve]
  52. Demouliere, A., Rubbia-Brandt, A., Abdiu, T., Waltz, A., Macieira-Coehlo, A., and Gabbiani, G.(1992) Exp. Cell Res. 201, 64-73 [Medline] [Order article via Infotrieve]
  53. Sell, C., Held, P., and Janakidevi, K.(1992) Cell Biol. Int. 16, 221-233
  54. Jenne, D. E., and Tschopp, J.(1992) Trends Biochem. Sci. 17, 154-159 [CrossRef][Medline] [Order article via Infotrieve]
  55. Kubota, Y., Kleinman, H. K., Martin, G.. R., and Lawley, T. J.(1988) J. Cell Biol. 107, 1589-1598 [Abstract]
  56. Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., and Reddi, A. H.(1992) Exp. Cell Res. 202, 1-8 [Medline] [Order article via Infotrieve]
  57. Donnelly, K. M., Fazleabas, A. T., Verhage, H. G., Mavrogianis, P. A., and Jaffe, R. C.(1991) Mol. Endocrinol. 5, 356-364 [Abstract]
  58. Cardin, A., and Weintraub, H.(1989) Arteriosclerosis 9, 21-32 [Abstract]
  59. Kocher, O., Gabbiani, F., Gabbiani, G., Reidy, M. A., Cokay, M. S., Peters, H., and Huttner, I(1991) Lab. Invest. 65, 459-470 [Medline] [Order article via Infotrieve]

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