Cloning and Characterization of Alternatively ... - Semantic Scholar

THE JOURNAL OF BIOLOOICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 269,No. 32,Issue of August 12,pp. 20757-20763, 1994 Printed in U.S.A.

Cloning and Characterization of Alternatively SplicedIsoforms of Rat Tenascin PLATELET-DERIVED GROWTH FACTOR-BB MARKEDLYSTIMULATES EXPRESSION OF SPLICED VARIANTS OF TENASCIN mRNA IN ARTERIAL SMOOTH MUSCLE CELLS* (Received for publication, March 16, 1994, and in revised form, May 9, 1994)

David. W. LaFleur, James.A. FaginS, James.S. Forrester, Stanley A. RubinO, and Behrooz G. Sharifin From the Division of Cardiology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048

of attachments with the underlying substratum. In vivo, ASMC To understand the alteration of extracellular matrix compositionevokedbychemotacticfactors,wehave are stationary and embedded in an ECM. To migrate, cells studiedtheexpression ofadhesive(fibronectin)and must alter their interactions with the surrounding matrix. A anti-adhesive (tenascin) proteins in response to plateletnumber of studieshaveshown a biphasicrelationship bederived growth factor-BB (PDGF-BB), a potent chemoat- tween the migration speedof human ASMC and murine skeltractantforrataorticsmoothmusclecells (ASMC). etal myoblasts and the composition of ECM components. The PDGF-BB markedly induced two major tenascin mRNA strength of thecell-substratumattachmentincreaseswith transcripts, whereas fibronectin mRNA levels did not surface concentration of absorbedfibronectin, collagen type change. The results of immunoprecipitation studies paralleled Northern blot data. Since alternative splicing is IV, and laminin, whereas the mean cell movement speed and mean persistence time reach maximal values at intermediate responsibleforthegenerationofmultipletenascin mRNAs in othercelltypes, we studiedtheeffect of concentrations (2, 3). chemotactic factorson the relative abundance of tenas- Cell-ECM interaction is mediated by a number of attachment cin isoforms.The alternatively spliced region of ASMC- and detachment factors. The initial attachment of cells to a derived rat tenascin was amplified and the identity of substrate is followed by a reorganization of cytoskeletal comthe products confirmed by sequencing. Three major poponents, resulting in a flattening and spreading of cell body lymerase chain reaction products were detected a 1727- (see Ref. 4, forreviews).Subsequently,specialized adhesive base pair unspliced form which was maximal2 h atand structures, suchas focal adhesions, areoften formed(4). In vivo 636- and 362-base pair products which were more abunthe adhesion process must be carefully regulated, and in cerdant at 8 h after treatment with PDGF-BB or angioten- tain situations (i.e. during mitosis and migration) the cells sin 11. Functional studies showed that the unspliced isomust detach from their adhesive structure inan orderly fashform of human tenascin inhibited attachment of both ion. The molecular mechanismsmediatingthis process are human and ratASMC to fibronectin. These resultssugpoorly understood. Previous studies have shown that certain gest that PDGF-BB markedly up-regulates the expresECM components such as tenascin may regulatecell adhesion sion of tenascin variants, which may lead to destabilization of cell-matrix interactions and promotion of cell by interfering with the attachment of cells to a number of adhesive ECM proteins (5). migration. Tenascin is an oligomeric glycoprotein (reviewed in Ref. 6). Human, mouse, and chicken tenascin contain at their amino Migration of ASMC’ across the internal elastic lamina is a termini a cysteine-rich segment, followed by epidermal growth key step in the development of atherosclerotic or restenotic factor-like repeats, fibronectin type I11 repeats (FNIII), and a plaques (1).Cell movement is a complex and highly dynamic globular carboxyl terminus, homologous to fibrinogen (6). The first five and the last three FNIII domains are constitutively phenomenon, involving the continuous formation and breakage expressed, and the other FNIII repeats are either included or * This work was supported in part by Grant-in-aid GI-1 from the skipped. Tenascin inhibits the attachment of fibroblasts and American Heart Association of the greater Los Angeles area and Na- endothelial cells to various adhesive proteins (5).The functional Institutes of Health Grant HL43802. The costsof publication of tional activity of tenascin inASMC is unknown. this article were defrayed inpart by the payment of page charges. This In an effort to provide insight into the mechanismof ASMC article must thereforebe hereby marked “aduertisement” in accordance migration,particularlythe chemotacticfactor-induced cell with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper hasbeen submitted movement, we have focused on the alterationof ECM makeup to the GenBankTMIEMBL Data Bank with accession number(s) U09361 by ASMC in response to chemoattractants.We postulated that (pl71), U09400 (~921, and U09401 (p123). chemotactic factors may promote cell migration, in part, by $ Recipient of an Established Investigator Award of the American inducing ASMC to alter the composition of ECM to one whichis Heart Association and Bristol-Myers Squibb. 0 Present address: VA Medical Center (llOOC), 5901 East 7th St., conducive to cell movement. In this paper, we examined the Long Beach, CA 90822. regulation of fibronectin and tenascin synthesis in response to 11 To whom correspondence should be addressed: Cedars-SinaiMedi- PDGF-BB and cloned the alternatively spliced region of rat cal Center, Davis Bldg. 1016, 8700 Beverly Blvd., Los Angeles, CA tenascin, theregion t o which anti-adhesive activityof tenascin 90048. Tel.: 310-855-7621;Fax: 310-652-8131. ‘The abbreviations used are: ASMC, aortic smooth muscle cells; has been attributed. ECM, extracellular matrix;FN, fibronectin; PDGF-BB,platelet-derived growth factor-BB; DMEM, Dulbecco’s modified Eagle’s medium; PBS, EXPERIMENTAL. PROCEDURES phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin;PCR, polymerase chain reaction; bp,base Materials-Rat fibronectin was from Telios Pharmaceuticals, San pair(s). Diego, CA. Protein A-agarose was from Repligen,Cambridge, MA.

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Regulation of Tenascin Synthesis by PDGF-BB

Fluoro-Hancewasobtained from ResearchProductsInternational, (200,000/well) were added to the culture dish followed by incubation at Mount Prospect,IL. Dulbecco's modified Eagle's medium (DMEM/F-12) 37 "C for 45 min. The nonadherent cells were removedby washing with and trypsin/EDTA were from Irvine Scientific, Irvine, CA. Fetal calf binding buffer, the adherent cells were dissolved by NaOH, and the serum and lactalbumin hydrolysate were from Life Technologies, Inc. radioactivity was measured by scintillation counting. Guanidinethiocyanatewasobtained from BoehringerMannheim. cDNA Probes-Rat fibronectin cDNA p-rlf-1, corresponding to the Prestained protein standards were from Bio-Rad. Collagenase, elastase, COOH-terminal portion of the gene (ll),was a gift from R. 0. Hynes saralasin, and other chemicals were reagent grade and were obtained (Massachusetts Institute of Technology). The human tenascin cDNA from Sigma. was a gift from K. Steffansson(University of Chicago). We useda Cell Culture-Rat ASMC were isolated by enzymatic digestionof rat 2-kilobase pair EcoRYBamHI subfragment of p31-24, a region corre(Sprague-Dawley) aorta, and the culture conditions have been described sponding t o the 3' ends of both the 6.2- and 7.8-kilobase tenascin tranpreviously (8). Baby hamster kidney cells transfected with full-length scripts. Human glyceraldehyde phosphate dehydrogenasecDNA probe human tenascin cDNA were provided by H. P. Erickson (Duke Univer- was obtained from CLONTECH, Palo Alto, CA. sity). These cells were grown inDMEM/F-12 media (Life Technologies, Quantification of RNA Leuels-Cultured cells were grown toconfluInc.) supplemented with 10% fetal bovine serum (Hyclone Laboratories, ence and growth-arrested as described above. Total cellular RNA was Logan, UT). After reaching confluence, cells were transferred to 1% isolated from control (serum-free media) and PDGF-BB-treated culserum, and the conditioned media were collected for purification of tured cells. Cell monolayers were lysed with a solution of 4 M guanidine tenascin. thiocyanate, 25 mM sodium citrate, pH 6, 0.5% Sarkosyl, and 0.72% Antibodies-Rabbit polyclonal antibodiesdirectedagainst rat fi- 2-mercaptoethanol, followed by centrifugation through a 5.7 M cesium bronectin and laminin were obtained from Telios Pharmaceuticals, and chloride cushion (11). The RNA was quantified by AaBOnm, and equal anti-tenascin antibodies werea gift from Dr. Erickson. Thespecificity of amounts were denatured, separated by electrophoresis on 1.0% agathese antibodies was evaluatedby Western blot and immunoprecipita- rose-formaldehyde gels, and transferred x SSC by capillary action in 20 tion as described previously (7). (3 M sodium chloride, 0.3 M sodium citrate, pH 7.0) to anylon membrane Radiolabeling of Cell Cultures-Confluent cultures of ASMC were (Hoefer). Ethidium bromide staining of 18 and 28 S ribosomal bands growth arrested in serum-free media as described above. The media were confirmed that equal amounts of RNA were applied to each lane. To removed and replaced with complete DMEM/F-12 media containing further establish the loading uniformity, the blots were reprobed with PDGF-BB and 100 pCi/ml Trar~~~S-labeled (70% ~-[~~Slmethionine, 15% glyceraldehyde phosphate dehydrogenase cDNA. ~ - [ ~ ~ S l c y s t e i1104 n e , mCi/mmol, ICN, Costa Mesa, CAI. After the indiThe cDNA probes were radiolabeled with L3'P1dCTP using the rancated time periods, the media were removed, adjusted t o 0.04% sodium dom priming technique as described by the manufacturer (Amersham azide, 0.2% Tween 20, 1mM phenylmethylsulfonyl fluoride, centrifuged Corp.). Unincorporated nucleotides were removed by centrifugation of to remove cell debris, and usedfor immunoprecipitation studies. the reactionmixturethrougha G50 Sepharose column (Boehringer Immunoprecipitation-Quantitativeimmunoprecipitationwasperof 5% Mannheim). The blots were prehybridized overnighta solution in formed as described (7). Briefly, aliquots (100 p1) of the media were sodium dodecyl sulfate (SDS), 1%BSA, and 40 mM sodium phosphate, diluted 1 : l with 50 mM Tris, pH 7.5, 150 mM NaCI, 0.05%Tween 20. pH 7.2 (NaPP) at 60 "C. Labeled probe was added (1 x lo6 c p d m l of Rabbit antibodies to fibronectin, laminin, tenascin, or nonspecific rabbit hybridization buffer), and the hybridization was continued overnight. mM40 NaPP, 5%SDS at 60 "C for antibodies (10 pl) were mixed with the diluted media and 50 pl of The blots were washed three times in 25 min. The blots wereexposed to Kodak XAR-5 film with two intensiprotein A-agarose was addedthe to mixture. The mixture was incubated overnight at 4 "C with gentle mixing, washed two times with PBS, 0.1% fying screenat -70 "C. The relative intensitiesof the RNA bands were Triton X-100,0.5%deoxycholate, 0.1% SDS, and finally with m 20 M Tris quantified by scanning densitometry. To strip off the hybridized probe, buffer. To prevent variation due to the presence of residual buffer in the the blots wereboiled in a solutionof 1x SSPE (2.9M NaCl, 0.19 M NaPP, pellet, the samples were dried on a Savant speed vacuum after the last 25 mM EDTA, pH 7.4) for 15 min. PCR a n d Cloning-Total RNA was extractedfrom ASMC treated for wash. The immunoprecipitated proteins were solubilized in 50 p1of 2 and 8 h with angiotensin 11, which we have shown previously to reducing SDS samplebuffer, and the%-labeled proteins wereresolved increase tenascin mRNA abundance (7). First strand cDNA synthesis by6% SDS-PAGE. The gels were treated with Fluoro-Hance, dried under vacuum, and exposed to film for autoradiography. Bands of in- was performed on 2 pg of total RNA, primed with 50 pmol of random terest were quantifiedby scanning densitometry and integration of the hexamers (Boehringer Mannheim). Reverse transcription was carried out at 42 "C for 1 h, using 200 units of reverse transcriptase (Life resultant peaks. The high molecular mass prestained markers used were myosin (200 kDa), P-galactosidase (116.5 kDa), bovine serum al- Technologies, Inc.)/20-ml reaction volume. PCR amplification was performed on 5 ml of the reverse transcription reaction, using 1unit ofVent bumin (80 kDa), and ovalbumin (49.5 kDa). Proteins in the supernapolymerase, 250 mM dNTP, 10 pl of 10 x reaction buffer (allfrom New tants, which remained after the overnight immunoprecipitation step, England Biolabs, Beverly MA). The primers, 5"GGAATTCAGAAAGwere first adjusted to 10% trichloroacetic acid,then washed twice with GCAGACACAAGAGCAAG-3' and 5"GGAATTCTGAGTCNGTGATGTice-cold acetone, dried, and analyzedby SDS-PAGE. Purification of Tenascin-Tenascin was purified from conditioned TGGCTNTCA-3' (Operon Technologies, Alameda CA), were designed 5' end and were used a t a concentration of 1m media of baby hamster kidney cells transfected with full-length human with EcoRI sites at the in the 100-pl reaction volume. Thirty-five cycles were performed using tenascin cDNA (unspliced variant). Briefly, proteins contained in the conditioned media (600 ml) were concentrated by addition of ammo- 95 "C for denaturation for 1min, 65 "C annealing for 1 min, and 72 "C nium sulfate to a final saturation of 40%. The solution was centrifuged,extension for 3 min. ThePCR products were size fractionatedon a 1% agarose gel, cut out, digested with EcoRI, and ligated into the pBlueand the pellet was resuspended in 50 mM Tris, pH 7.6, 700 mM NaCI. The insoluble material was removedby centrifugation, and the super- script I1 SK+ (Stratagene, La Jolla, CAI. Transformation of HBlOl natant was applied t o a Sephacryl s-500 gel filtration column previ- competent cells (Life Technologies, Inc.) was followed by selection on plates containing 100 mg/ml ampicillin, spread with 40pl of 5-bromoously equilibrated with the TrisLN'aCI buffer. Since tenascin is a very (20 mg/ml in dimethylformamide) and 4 large molecule with low coefficients of diffusion, the flow rate was ad- 4-chloro-3-indolyl-~-galactoside pl of isopropylthio-D-galactoside(200 mg/ml). White colonies were justed to 0.2mVmin to increase the resolutionof the column. Fractions positive clones were (2 ml) were collected, and the protein content was measured by spec- grownup for restriction enzyme analysis, and trophotometry. The puntyof the proteins was evaluated by SDS-PAGE sequenced using Sequenase (U. S. Biochemical Corp.). Sequence analWisconsin Sequence Analysis Package (Genetfollowed by silver staining as described (8).The identityof the proteins ysis was done using the was confirmed by Western blotting using specific antibodies tofibronec- ics Computer Group, Madison,WI). RNase ProtectionAssay-Based on our sequence data, PCR primers tin, laminin, and tenascin as described(7). Fractions containing tenawere designed t o flank fibronectin type I11 repeat 12 (FN121; 50 bases scin were pooled and the protein content measuredas described (9). from Adhesion Assay-ASMC were labeled with50 mCi/ml PHIthymidine upstream from the beginning of FN12 and 130 bases downstream for 72 h. Radiolabeled cells were removed by brief trypsinization, fol- its end. Using thecloned cDNA as template,we obtained a 450-bp PCR I1 SK+, using theEcoRI and lowed by several washes with PBS containing 3 mg/ml bovine serum product. This was ligated into pBluescript KpnI restriction sites which had been designed into the upstream and albumin (BSA) and 0.5 mg/ml trypsin inhibitor. Specific activity was determined by counting number of cells with Coulter counter, and ra- downstream primers, respectively. To confirm its identity, we have sequenced the riboprobe and have established a complete homology bedioactivity measured by scintillation counting. Tissue culture plates were coated with a solution of fibronectin (10 pg/ml in PBS) overnight tween our probe and the rat sequences. T7 polymerase was used to at 4 "C, and nonspecific sites wereblocked by a solution of non-fat dry transcribe an antisenseriboprobe for use in the ribonuclease protection milk as described (10). RGD peptide or purified tenascin were prepared assay. Hybridization was performed with 300,000 cpm probe/20 pg of sample RNA for 16 h at 37 "C (Promega, Madison WI). by dilution with the binding buffer to the desired concentration. Cells

Regulation of Tenaacin Synthesis by PDGF-RR

20759 0

A I PDGF-BB+

d

CVdQoCu

SERUM

+

0.01

0.1

1

A

d

r C u d Q o ( U

200kDa

-

B

200kDa

- "w-4

was modestly influenced by PDGF-RR, thr exprrssion ofthc. intermediate size variant polypeptide. 220 kDa. was markcdly increased, and the s.vnthesis of small isoform polypeptidc. 210 kDa, was unaffected (Fig. 2.4 I . In contrast to tenascin synthesis, fibronectin is constitutively cxpressrd under basal condition. and PDGF-RR had little.if any. effect on fibronectin levels (Fig. 2R ). To examine the potential rolc of mc.tabolic inhihitors on tenascin induction, ASMC were treated with PDGF-AR in t h r presence and absence of actinomycin D and cyclohc~ximidr.Simultaneousaddition of actinomycin D and PDGF-RR comFIG.1. Effect of PDGF-RR on tenascin and fibronectin mRNA pletely hlocked thc induction of trnascin mRNA (Fig. 3 1 . In abundance. ASMC ww"' trcwtrd with 1 nxl PI)