cDNA Cloning and Gene Expression of Chicken Osteopontin

THEJOURNAL OF BIOLOGICAL CHEMISTRY IC)

Vol. 266, No. 15, Issue of May 25, pp. 9944-9949,1991 Printed in U.S.A.

1991 by The American Society for Biochemistry and Molecular Biology, Inc

cDNA Cloning and Gene Expression of Chicken Osteopontin EXPRESSIONOFOSTEOPONTINmRNAINCHONDROCYTESISENHANCED OF CELLS*

BY TRYPSINTREATMENT

(Received for publication, December 3, 1990)

Patrizio CastagnolaS, Paola Bets, Rodolfo Quarto, Massimo Gennari, and Ranieri Cancedda From the Laboratorio di Differenziamento Cellulare, Zstituto Nazionale per la Ricerca sul Cancro, Genoua, Ztaly

A cDNA clone, pCP15, specific for the chicken 66kDa major bone phosphoprotein (osteopontin), was isolated from a subtracted library enriched in DNAs coding for mRNAs expressed in chicken differentiating chondrocytes. Northern blot analysis of RNAs extracted from several chick embryo tissues and organs, confirm and extend the observation that osteopontin mRNA expression is not restricted to tissues involved in phosphate metabolism. Osteopontin mRNA was detected in sternal resting chondrocytes at higher levels than in hypertrophic chondrocytes; therefore osteopontin gene transcription occurs in chondrocytes at many stages of differentiation. The steady state level of osteopontin mRNA was enhanced by trypsin treatment of cultured cells. An increased level of osteopontin mRNA in quail chondrocytes constitutively expressing v-myc oncogene is also shown.

A major event during endochondral ossification is the proteolytic degradationof calcified cartilage extracellular matrix and its substitution with the bone-specific extracellular matrix synthesized and organized byosteoblasts. Oneof the most abundant products of this cell type is osteopontin, a glycosylated phosphoprotein witha highnumber of acidic amino acid residues and containing acell binding site defined by the RGD sequence. The osteopontin-completenucleotide and derived amino acid sequences have been obtained from cDNA clones for rat (Oldberg et al., 1986), mouse (Craig et al., 1988), human (Kiefer et al., 1989), and pig (Wrana et al., 1990). In several species this proteinwas isolated from and/or detected in tissues involved in phosphate metabolism such as bone (Franzen and Heinegard, 1985; Fisher et al., 1986; Kubota et al., 1989; Gotoh et al., 1990; Zhang et al., 1990), kidney (Craig et al., 1988; Bruder et al., 1990), and gut (Bruder et al., 1990) but also neuronal tissue (Swanson et al., 1989), decidua and placenta (Nomura et al., 1988), and blood of patients with metastatic sarcomas and carcinomas (Senger et al., 1988).

* This work was supported by funds from Associazione Italiana Ricerca sul Cancro, Milano, Italy, and from the Italian Minister0 dell’ Universita e della Ricerca Scientifica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence($ reported in thispaperhas been submitted to theGenBankTM/EMBLDataBank with accession numberfs) X56772. $ To whom correspondence shouldbe addressed Lab. Differenziamento Cellulare, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10 16132 Genova, Italy. Tel.: 39-10 352958; Fax: 3910 352999. Recipient of a fellowship from the Dottorato di Ricerca in Biologia Umana: Basi Molecolari e Cellulari, University of Turin, Italy.

Osteopontin gene expression is modulatedby osteotropic hormones (Prince and Butler, 1987; Yoon et al., 1987; Noda et al., 1988; Oldberg et al., 1989) and growth factors (Noda and Rodan, 1987; Nomura et al., 1988; Kubota et al., 1989; Rodan et al., 1989). The presence of an RGD sequence and of a stretch of acidic amino acid residues in a single molecule led to thehypothesis that itcould act as a bridge between osteoblasts and apatitic mineral. Several reports showed that osteopontin is bound via anintegrintothe cell membrane (Somerman et al., 1987; Oldberg et al., 1988a, 1988b), and that it accumulates in the extracellular matrix concomitantly with mineral deposition (Gerstenfeldet al., 1990). Its binding ability for calcium (Andrews et al., 1967; Herring, 1972) and its immunolocalization to electron-denseregions of mineralization (Gerstenfeld et al., 1990) was shown, but so far there is no clear evidence of a link between the cells and the mineral phasemediated by osteopontin(Gerstenfeld et al., 1990). Osteopontin is proteolytically cleaved to smaller molecular in vivo and in uitro. weight fragments after its secretion, both A preferentialassociation of osteopontin proteolytic fragments to thecell membrane was demonstrated (Gotoh et al., 1990). Osteopontin synthesis by chondrocytes in the area of cartilage-to-bonetransition wasshown both by immunocytochemistry (McKee et al., 1990) and by in situ hybridization (Franzen et al., 1989). During our studies aimed at a better understanding of the chondrocyte phenotypemodulat.ion at the molecular level, we generated a subtracted cDNA library enriched inclones coding for differentiating chondrocyte-specific transcripts. From this library we isolated and sequenced a cDNA clone coding for chicken major bone phosphoprotein (osteopontin). This clone hasbeenusedtoinvestigatetheosteopontin gene expressionin several embryonictissuesand in “in vitro” differentiating and transformed avian embryochondrocytes. In this manuscript we report that the steady state level of osteopontin mRNA isgreatly enhanced by v-myc constitutive expression andby trypsin treatmentof cells. This observation will provide the basis for furtheranalysesonosteopontin mechanism of action and the role of proteolysis in itsactivity. EXPERIMENTALPROCEDURES

Cell Culture-Cell culture methods are extensively described elsewhere (Castagnola etal., 1986). Culture medium was Coon’s modified Ham’s F-12 (Ambesi-Impiombato et al., 1980) with the addition of 10% fetalcalf serum (Flow Laboratories, Imine, Ayrshire, Scotland). To establish primary cultures Hamburger and Hamilton stage2830 (Hamburger and Hamilton, 1951) chick embryo tibiae were removed, cleaned, washed in phosphated-buffered saline, pH 7.2, and digested for 15 min at 37 “C with 400 units/ml collagenase I (Worthington, Biochemical Corp., N J ) and 0.25% trypsin (GIBCO). After sedimentation the supernatant, containing tissue debris and perichondrium, was discarded and thepellet was digested foran additional

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Regulation Osteopontin of Chicken

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45-60 min with the above dissociation buffersupplemented with 1000 lated in a wheat germ translation system. Preparation, properties, units/ml of collagenase I1 (Worthington, Biochemical Corp.). Cells and composition of the incubation mix have been already described (Cancedda et al., 1985). ~-[4,5-'H]Leucine at thespecific activity of were grown inanchorage-dependentconditions onregular tissue culture dishes and transferred by trypsin treatment (0.25% in phos120-190 Ci/mmol and at the concentration of 1.6 mCi/ml was used to label the translation product. phate-bufferedsalinebuffer)toanchorage-independentconditions SDS-PAGE was performed as described (Laemmli, 1970) and on a 1%agarose layer. This change in culture conditions triggers the chondrocyte differ- modified (Bonatti and Descalzi-Cancedda, 1982). The concentration entiation program (Castagnola et al., 1986).Differentiating(clone of acrylamide was 9%. 956) and nondifferentiating (clone 957) cloned cell populations were obtained as in Quarto etal. (1990). Primary cultures of chick embryo RESULTS skin fibroblasts were obtained bydigestion of stage 38-39 chick Isolation of a ChickenOsteopontin cDNA Clone from a embryo skin in the same conditions used forisolation of chondrocytes. Normal and infected with the pM5 retrovirus quail chondrocytes Chondrocyte-subtracted cDNA Library-In order to identify were obtainedandculturedas described.'Briefly, normal QEC' cDNA clones specific for transcripts expressed during chonprimary cultures were obtained from stage 26-28 quail embryo tibiae drocyte differentiation, we synthesized a cDNA library from as described for CEC. To establish a QECcultureconstitutively which the clones coding for mRNAs expressed also by preexpressing v-myc oncogene,normal QECwere infected with retrovirus pM5, a deleted form in gaglmilof pM4 retroviral vector that resulted chondrogenic cells would be eliminated (subtracted). Due to in v-mil inactivation. This deletion has no effect on v-myc activity. a certain extent of cell heterogeneity in primary cultures,we pM5 infectedQEC, both in adherent and in nonadherent culture chose as mRNA sourcestwo cell clones. Both clones were conditions, are freezed in an early differentiation stage of the chon- obtained from single cells by enzymatic dissociation of chick drocyte differentiation pathway and neverbecome hypertrophic. embryo tibiae after cloning and expansion in chondrocyteIsolation of RNA, cDNA Cloning, and Nucleotide Sequencingconditioned medium. Cells from the clone 956 were able to RNA was extracted using guanidinium thiocyanate (Chomczynski express the typical markers of chondrocyte differentiation and Sacchi, 1987). Poly(A)' RNA was purified by oligo(dT)-cellulose chromatography (Maniatis etal., 1982). Two separatedcDNA samples when transferred to suspension culture (type I1 and type X were synthesized with poly(A)+ RNAs extracted, respectively, from collagens) and they reconstitute hypertrophiccartilage when clone 957 (nondifferentiating), grown in adherent conditions, and placed in the appropriate culture conditions. Cells from the from clone 956 (differentiating),after36 h fromthetransferto clone 957 did not proceed through the differentiationprocess nonadherent conditions. cDNAs were generated by oligo(dT) priming and continued to expressonly type I collagen. Full characterwith a kit from Amersham (Buckingamshire, England). After addition of BstXI adaptors (Invitrogen, SanDiego, CA) cDNAs were inserted ization of these cell clones is described elsewhere (Quarto et into the vector pCDNA I1 (Invitrogen). XLlBlue cells (Stratagene, al., 1990). Both cDNAs were cloned in a vector with an fl La Jolla,CA) were transformed with the plasmidsby electroporation origin, pcDNA I1 (Invitrogen), that allows the recovery of a with a Bio-Rad apparatus (Dower et al., 1988). The subtracted cDNA single-stranded DNA, following infection of the Escherichia library, enriched inclones coding for transcripts present incell clone coli host by a helper phage. The ssDNA molecules containing 956 and absent in the cell clone 957, was generated with the Invitrogen cDNAs of clone 957 t o be subtractedwere labeled with biotin, Subtractor kit. The library was screened, by differential hybridization, with '"P-labeled poly(A)+ RNAs, extracted from the two cell and, after liquid hybridization with unlabeled ssDNA moleclones, according to the Invitrogen instructions. The hybridization cules,specificfor the differentiated cellclone 956, hybrid and washing conditions were identicaltothose recommended by molecules were removed by the addition of streptavidin folAmersham for Hybond-N membranes. lowed by phenol andchloroform extraction. ssDNA molecules For single-stranded sequencing, restriction fragments of pCP15 in the aqueous phase, containing cDNA inserts that did not were subcloned into the XbaI and BamHI sites of M13 mp18 and labeled ssDNA pool, mp19. All sequencing reactions were carried out with Sequenase 2.0 have a complementing counterpart in the (U. S.Biochemical Corp.). Both strands of the cDNA insert were were used to transform E. coli cells and to generate a subcompletely sequenced. The primersused were the M13(-40) and seventracted plasmid cDNA library. Out of lo4 colonies screened internal oligos (see Fig. 2). by differential hybridization with "P-labeled mRNAs from All sequence analyses were performed with the"PC-gene" software thetwo cellclones, only 10' gave a hybridization signal package (Intelligenetics, Mountain View, CA). specific for the differentiated cell clone. These clones were Northern BlotAnalysis and DNA Labeling-For Northern blot further hybridized withcDNAprobes specific for type I1 analysis total RNA was extracted by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987) from culturedcells and from (Young et al., 1984) and type X (Castagnola et al., 1987) brain, lung, heart, liver, stomach, skin, and tibiaof stage 38-39 chick collagens, link protein (Kiss et al., 1987) and Ch21 protein embryos. Poly(A)' RNA was purified from chondrocyte clones and (Descalzi-Cancedda et al., 1990). The clones thatdidnot from caudal and cranial sterna of stage 43-44 chick embryos. Total hybridize with the above probes were further analyzed. The and poly(A)' RNA were electrophoresed through 1% agarose gels in characterization of the clone pCP15, that gave the strongest the presence of formaldehyde and blotted onto Hybond-N membranes. The blots were hybridized with the whole insert of pCP15 hybridization signal with the labeled RNA from clone 956, is labeled by random priming. Hybridization and washing conditions describedhere. T o verifyclone pCP15 specificity andto were performed as recommended by Amersham Corp. establishthemRNA size, we performeda Northern blot Densitometric scannings of different autoradiographic exposures were obtained with an LKBBiotechnology Inc. Ultrascan XL instru- analysis with poly(A)+ RNA extracted from cell clones 956 and 957. As shown in Fig. 1 a mRNA of about 1.4 kb was ment(Bromma,Sweden).The relative abundance of osteopontin mRNA was calculated by normalization on the bases of rRNA content detected only in clone 956 RNA. The cDNA insert of pCP15 in each lane determined by scanning of the autoradiographs of the plasmid was subcloned into M13 andsequenced. same filters probed with pXCR7 (Castagnola etal., 1987). Nucleotide and Deduced Amino Acid Sequencesof pCPl5Cell-free Translation of the RNA Transcribed frompCP15 and SDSThe nucleotide sequence of the 1164-bp cDNA insert (Figs. 2 PAGE of the Translated Protein-The plasmid pCP15 was digested with BamHI and incubated with theT 7 RNA polymerase (Promega, and 3) revealed an open reading frameof 792 bp followed by Madison, WI) to transcribe the cDNA insert, The mRNA was trans- three in-frame stopcodons, a 5"untranslated sequence of 107

' R.

Quarto, B. Dozin,C. Tacchetti, G. Robino, M. Zenke, G. Campanile, and R. Cancedda, submittedfor publication. 'The abbreviations used are: QEC, quail embryo chondrocytes; CEC, chicken embryochondrocytes;SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis; kb, kilobase(s);ssDNA, single-stranded DNA; bp, base pair(s).

bp and a 3"untranslated sequence of252 bp containing a polyadenylation addition signal, AATAAA, located 16 bp before the poly(A) tail. Computer-assisted homology search with the EMBL sequence data libraryrevealed a 51.7% identity of this sequence with the rat osteopontin mRNA coding sequence and a 50.5%

Regulation of Chicken Osteopontin

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I GGAGMAGCCAGAGCCTCACTCAGCCCGCAGTAGGAOTTOCGCCGGAGCC

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2 4 1 ACCACCAGGATCACGTGGA~TCAGTC~AGGAGCACCTOCAGCAGACACAGMTGACC

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361 C G G A C T T T C C T G A C A T n C T A G C M G A G C C M G A M i C C O l T G P D F P D I P S K S Q E A V D D D D D D 4 2 1 ACMTGATnCMTGACACTGACGAGTCTGATOMGTCGTCACAGAC~CCCCACAGAGG D N D S N D T D E S D E V V T D F P T E 481 CACCCGTGACACCTTTCMCCGTGGGGACMCGCAGGCCGGGGTGACAGTGTGGCATATG A P V T P F N R G D N A G R P D S V A Y

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5 4 1 GCTTCAGAGCCMAGCCCACGTGGTGMGGCGAGCMGCTCCGCAMGCTGCCAGGMGC U A K A H V V K A S K L R K A A R K 601 T C A ~ A G G A T G A C G C C A C C G C ~ A G G ~ G ~ A C A G C ~ G C T A G C G G G C C T C T G G m C L I E D D A T A E V G D S O L A G L w L 6 6 1 CCMGGAGAGCCGCGMCAGGACAGCCGTGAGCTGGCCCMCATCAGAGCGTAGAGMCG E S E S R E Q D S R E L A Q H Q S V E N 7 2 1 ACAGCCGGCCMGATTniACAGCCCTGAGGTGGACGGAG~GACAGCMGGCCAGTGCTG D S R P R F D S P E V D G G D S K A S A

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781 GGGTGGACAGCAGAGAGAGCCTGGCTAGCGCGTCCGCCGTCGACGCCAGCMCCAGACGC G V D S R E S L A S A S A V D A S N Q T

FIG.1. Northern blotof poly(A)+RNA extracted from clone 957 and 956. Lane I, clone 957 cultured in adherent conditions; lane 2, clone 956 after 36 h from the transfer to nonadherent conditions. On each lane 2 pg of poly(A)+ RNA were loaded. The probe used was the insert of pCPl5. Arrows refer to theposition of ribosomal RNAs. At the bottom the 18 S rRNA region of the same filter after rehybridization with the pXCR7 probe for rRNAs is shown.

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1021 TTAGGTCGAGGCGCCGTGGTGCCATTGAGCCGTACCGAGACGGGGCGAGCGCTGCCTGCG 1081 G G G A T G C T G C G C G T C C C G C T C T C C T G T G T A T A - T M C C O C 11I 1 ACAAAMCCTC-

FIG.3. Complete nucleotidesequence and conceptual trans-

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FIG. 2. Sequencing strategies of the pCP15 cDNA insert. The box indicates the pCP15 cDNA insert, continuous lines represent the vector pcDNA I1 poly-linker. Arrows starting from a full circle (0)indicate sequences determined with the M13-40 primer, whereas arrows starting from an empty circle (0)indicate sequences obtained with synthetic oligonucleotides.

lation product of the cDNA insert of pCP15. Amino acid residues (Gotoh et al., 1990) determined by automated Edman degradation of the chromatographically purified chicken 66-kDa phosphoprotein and three tryptic peptides of the same protein are underlined with a continuous line. Potential N-glycosylation sites are indicated with a square symbol (W); potential casein kinase I1 phosphorylation sites are indicated with an open circle (0);and the two potential binding sites are underlined with a broken line.

Determination of the Apparent Molecular Weight on SDSPAGE of the in Vitro Translated Osteopontin-In the culture medium of chick osteoblasts several bands were identified by Western blot analysis using specific antibodies against chick identity with human osteopontin mRNA sequence. Compari- osteopontin (Gerstenfeld et aL, 1990). On the contrary the son of the deduced amino acid sequence of pCP15 with those apparent molecular weighton SDS-PAGE of chick osteoponof rat, human, and porcine osteopontin showed an identity tin before its posttranslational modification has not been determined yet. In order to determine it, we translated the in of, respectively,37.5,39, and 39.4%. The amino acid sequence from pCP15 shows several fea- vitro transcribed RNA from pCP15 in a wheat germ extract tures conserved among osteopontins: high contents in acidic and ran the 3H-labeledproduct after reduction and alkylation amino acid residues, a signal peptide sequence 16 aminoacid on a 9% SDS-PAGE. As shown in Fig. 4, the migration of the residues long containing cysteinyl residues, the presence of a protein is much slower than the predicted migration for a potential cell binding site, an overall hydrophilic character, protein of about 27 kDa, since its apparent molecular mass is and thepresence of potential glycosylation and casein kinase about 50 kDa. Tissue Distribution of the Osteopontin mRNA-To identify I 1 phosphorylation sites. In particular the conceptual translation product of pCP15 has 21.2% acidic aminoacyl residues, tissues expressing osteopontin mRNA during chick embryoa stretch of 7 contiguous aspartyl residues, a low isoelectric genesis, we analyzed the RNAs extracted from several tissues point (4.2), four potential N-glycosylation sites, and five ca- of 12-day-oldchick embryo by Northern blot. As can be seen sein kinase I 1 potential phosphorylation sites (Fig. 3). The in Fig. 5a, a strong hybridization signal was detected in tibia sequences coding for two RGD are present in sequence. One and less intense signals were detected in stomach, liver, and RGD (defined by the GRGDS sequence), found downstream brain. By densitometric scanning we determined that the to a stretch of acidic aminoacyl residues, is conserved in amount of osteopontin mRNA is approximately 25, 40, and mammalian osteopontins, while the other one is only found more than 200 times less abundant, respectively, in stomach, in chicken deduced amino acid sequence and is located a few liver, and brain than in tibia. Since our subtracted library was obtained from cultured residues upstream from the conserved one. Weidentified this cDNA clone as coding for the chicken osteopontin on the differentiating chondrocytes and in order to verify whether basis of the homologies of its translation product with mam- chondrocytes express osteopontin gene also in vivo we anamalian osteopontins and identities of the deduced peptides lyzed poly(A)+ RNA purified from the third caudal portion, underlined in Fig. 3 with the previously reported amino acid constituted only by resting nonhypertrophic chondrocytes, sequences of three tryptic peptides and with the amino ter- and from the third cranial portion, mainly constituted by minal sequence of the chromatographically purified chick hypertrophic chondrocytes (Gibson et al., 1984) of stage 4344 chick sterna by Northern blot (Fig. 5b). osteopontin (Gotoh et al., 1990).

Regulation Osteopontin of Chicken 2

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FIG.4. Migration of osteopontin translatedin vitro on SDSPAGE.Cell-free translation product of the i n vitro transcript by the T 7 RNA polymerase of the pCP15 cDNA insert (lane 2). Numbers on the left refer to molecular weight markers (lane 1).

conditions, we observed thatthetransfertononadherent conditions induced expression of the osteopontin mRNA independently from the ability of the cell clone to differentiate (Fig. 6a). Additional Northern blot analyses were performed to establish whether the same effect could beobtainedin cultures of chondrocytes a t different stagesof development or in cultures of other mesenchymal cells. An increased level of hybridization to the probe (pCP15 insert)was observed also when dedifferentiated or hypertrophic chondrocytescultured in adherent conditionswere transferred to nonadherentconditions (Fig. 6b, lanes 1-4). To investigate the capability of a mesenchymal cell type noncorrelated with chondrocyte or osteoblast lineages to express mRNA for osteopontin,we cultured skin chick embryo fibroblastsinadherentconditions for 3 weeks and subsequentlytransferredthemtononadherent conditions. The Northern blot analysis of RNAs extracted from these fibroblasts showed a signal only when cells were transferred to nonadherent conditions, althougha t a much less extent than in CEC (Fig. 6b, lanes 5 and 6). To distinguish whether theincrease of osteopontin mRNA levels was due to the treatment of the cells with trypsin or to the change of the cell shape, we performedtransfer blot analyses of RNA extracted from hypertrophic chondrocytes continuously cultured in suspension for 3 weeks and from the same cells incubated with trypsin36 h before RNAextraction (Fig. 6c, lanes 1 and 2). A more than 25 times increase of osteopontinmRNA level aftertrypsintreatment was ob-

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FIG.5. Northern blot of RNAs extracted from embryonic tissues. a, total RNAs extracted from stages 38-39 of chick embryo

FIG.6. Induction of osteopontin gene expression in trypsin treated cells. a, Northern blot of total RNA extracted from the cell

clone 956, grown in adherent conditions (lane I ) , and from the cell clone 957, grown innonadherentconditionsafter36 h from the trypsin treatment (lane 2 ) . About 15 pg were loaded on each lane. Forcomparisonseealso Fig. 1. b, Northernblot of total RNA extracted from: dedifferentiated chondrocytes grown for 3 weeks in adherent conditions (lane I ); dedifferentiated chondrocytesgrown for 3 weeks in adherent conditions; and36 h in nonadherent conditions (lane 2 ) ; hypertrophic chondrocytes grown in adherent conditions (lune 3 ) ; hypertrophic chondrocytes grown in adherent conditions and 36 h in nonadherent conditions (lane 4 ) ; skin fibroblast grown in adherent conditions(lane 5 ) ;and skin fibroblastgrown in adherent conditions and 36h in nonadherent conditions(lune 6). About 20 pg Surprisingly, a three times stronger hybridization signal towere loaded on each lane. c, Northern blot of total RNA extracted the pCP15 insert probe was detected with thepoly(A)+ RNA from hypertrophic chondrocytes grown for 3 weeks in nonadherent conditions. RNA was extracted directly (lane I ) and following trypsin derived from the third caudal sterna than from the third treatment (lane 2). About 20 pg were loaded in eachlane. In all panels cranial sterna. the probe used was the insert of pCP15. Arrows refer to theposition Trypsin Digestion of Cultured Cells Strongly EnhancesOs- of ribosomal RNAs. At the bottom the 18 S rRNA ( a ) and the 37 S teopontin Gene Expression-When we checked the mRNA ( b and c) rRNAregions of the same filters afterrehybridization with level for osteopontin incell clone 957 cultured in nonadherent the pXCR7 probefor rRNAs areshown.

liver (lane I ) , lung (lane 2 ) , heart (lane 3 ) , skeletal muscle (lune 4 ) , skin (lane 5 ) , brain (lane 6), stomach (lane 7), and tibia (lane 8). About 15 pg of RNA were loaded on each lane. b, poly(A)' RNAs extracted from stages 42-44 of chick embryo sterna: third cranial portion (lane 1) and third caudal portion (lane 2). About 3 pg of RNA were loaded on each lune. The probe used was the insert of pCP15. Arrows refer to theposition of ribosomal RNAs. At thebottom the 18 S rRNA region of the same filter afterrehybridizationwiththe pXCR7 probe for rRNAs is shown.

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Regulation of Chicken Osteopontin

served. On the contrary, the treatment of adherent chondrocytes with 2 pg/ml of cytochalasin D for 24 h before RNA extraction didn’t induce any increase of osteopontin mRNA (data not shown). We also observed that after trypsin treatment thelevel of osteopontin mRNA resumes the initial basal level after about 2 weeks (data notshown). High Levels of Osteopontin mRNA Are Present inChondrocytes Constitutively Expressing v-myc-Constitutive expression ofmyc in differentiating chondrocytes prevents these cells from completing their maturationprocess to hypertrophy (Gionti et al., 1985).’ We examined whether a constitutive expression of v-myc in quail embryo chondrocytes has an effect also on osteopontin gene expression. Northern blot analysis ofRNA extracted from normal and pM5-infected QEC in adherent or in nonadherent conditions was performed (Fig. 7, lanes 1 and 2, respectively).pM5-infected QEC showed an eight times stronger signal than control normal cells both before and after trypsin treatment (Fig. 7, lanes 3 and 4 ) .

2 amino acid residues upstream the conserved GRGDS sequence. It was shown that chicken osteopontin promotes cell adhesion in vitro and that cell adhesion is specifically inhibited by peptides containing the GRGDS sequence (Oldberget al., 1986). The presence of two RGD on the protein raises the possibility that both RGD sites are used for cell binding and that they are bound by different integrin types or by the same integrin with different affinities. The cell-free translation product of the in vitro transcript containing the coding sequence for chickosteopontin on SDSPAGE shows an apparent molecular mass of about 50 kDa, 23 kDa higher than the calculated value from the deduced amino acid sequence. An anomalous slow migration on SDSPAGE was also reported for porcine osteopontin (Zhang et al., 1990). Northern blot analyses indicate expression of chick osteopontin gene also in tissues such as liver, stomach, and brain not involved in phosphate metabolism. The detection of the osteopontin mRNA in chick brain is in agreement with in situhybridization analysis in mouse (Swanson et al., 1989). DISCUSSION Although there areconflicting data about the hypothesis of a commonlineagebetween chondrocytes and osteoblasts We report here the isolation of a clone, pCP15, from a subtracted cDNA library enriched for cDNA clones specific (Holtrop, 1972; Hanoaka, 1976; Shimomura and Suzuki, for transcripts of differentiated chondrocytes. On the basis of 1984), there is evidence that chondrocytes can express some its nucleotide sequence and the comparison with EMBL se- of the osteoblast markers (Doty and Schofield, 1976;Termine quence data library and with available peptide sequences of et al., 1981; Oldberg et al., 1986; Descalzi-Cancedda et al., the chick major bonephosphoprotein we established that the 1988) and that chondrocytes can eventually differentiate to cDNA insert of pCP15 contains the complete open reading osteoblasts (Hall, 1968 and 1972; Kahn and Simmons, 1985; frame for chicken osteopontin. The identity percentage with Holtrop, 1972).For these reasons a more intricate relationship mammalian osteopontins is not very high, both at nucleotide between the two phenotypes is possible. To better investigate CEC ability of expressing the osteoand at the amino acidlevel,possiblydue to evolutionary divergency and to a different codon usage. Nevertheless, all pontin during their differentiation process, in vivo and in major features of the protein are conserved. Unique to the vitro, we performed several Northern blot analyses that chick protein is the finding of an extra RGD sequence located showed osteopontin expression in cultured hypertrophic chondrocytes, in agreement with previous results obtained in rat (Franzen etal., 1989);in addition we observed that resting 1 2 3 4 chondrocytes from the third caudal region of chick sterna have osteopontin mRNA levelsabout three times higher than hypertrophic chondrocytes from the third cranial region of sterna. Since v-myc expression can interfere with the differentiation program of avian chondrocytes, we analyzed the effect of v-myc constitutively expressed in QEC infected with pM5 virus on osteopontin mRNA level. pM5-infected QEC, having a phenotype of resting chondrocytes and unable to become hypertrophic, showed eight times higher osteopontin mRNA levels than uninfected cells. These results indicate a preferential expression of osteopontin gene by chondrocytes during early differentiation stage. When dedifferentiated chondrocytes are transferred to suspension or into collagen gels, they differentiate following a differentiation pathway very closeto theone observedin vivo (Castagnola et al., 1986; Castagnola et al., 1988; Solursh and Reiter, 1975); if the culture medium is supplemented with ascorbic acid they also organize a calcified matrix with lacunae (Tacchetti et al., 1987; Tacchetti et al., 1989). When we investigated osteopontin mRNAlevel in cultured differentiating chondrocytes we observed the expected osteopontin expression but we were surprised in observing a strong enhancement due to trypsin treatment of the cells. EnhanceFIG.7. Northern blot of total RNA. The RNA was extracted ment of osteopontin expression by trypsin treatment was from: normal quail embryo chondrocytes grown in adherent condi- observed also in pM5-infected QEC. Indeed,trypsin treatment tions (lane I); normal QEC grown for 1 week in nonadherent condi- induces osteopontin expression also in dedifferentiated chontions (lane 2 ) ;pM5-infected QEC grownin adherent conditions(lane drocyte clones incapable of developing to a chondrocyte phe3 ) ;pM5-infected QEC grown 1 week in nonadherent conditions ( l a n e notype when transferred to suspension culture and, although 4 ) . About 7.5 pg were loaded on each lune. The probe used was the insert of pCP15. Arrows refer to theposition of ribosomal RNAs. At to a much lesser extent than in skeletal cells, in chick skin the bottom the 18 S rRNA region of the same filter after rehybridi- fibroblasts. The maximum level was observedabout 36 h after trypsin treatment. To verify whether the activation of osteozation with the pXCR7 probe for rRNAs is shown.

Regulation Osteopontin of Chicken pontin mRNA expressionwas related to the cell shape change fromflattoroundedconsequenttotrypsintreatment we treated cell clone 957 with cytochalasin D. This drug interferes with the cytoskeleton organizationinduces and rounding of the cells (Zanetti and Solursh, 1984). No induction effect on osteopontin mRNAlevel was observed in these conditions. The fact that osteopontin induction is not due to cell shape change was confirmed by experiments in whicha 25-fold induction of osteopontin mRNAwas observed also aftertrypsin treatment of cells continuously grown in suspension culture. These results indicate that probably most the proteolysis of the pericellular matrix and/or cell membrane receptors triggers osteopontingene expression. The presence of intense proteolysis in the regions of calcified cartilageresorption during bone formation is well known (Mayne and von der Mark, 1983) the possibility that chondrocytes can beinduced also in vivo to accumulate osteopontin mRNA by the same proteolytic mechanism is suggested. The induction of osteopontin transcription by treating cells with trypsin could be the sign of a more profound modulationof the transcriptional activity. This phenomenon raises some caveat to the indiscriminate usage of cell culture systems for studying cell differentiation. Acknowledgments-We thankDr. G. Migliaccio for hisexpert advice, Drs. M. Romani and F. Molina for helping us with sequence analysis, and Drs. M. Sobel, F. Amaldi, and I. Kiss for DNA probes. REFERENCES Ambesi-Impiombato, F. S., Parks, L. A,, and Coon, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 3455-3459 Andrews, A. T., Herring, G. M., and Kent, P. W. (1967) Biochem. J. 1 0 4 , 705-715 Bonatti, S., and Descalzi-Cancedda, F. (1982) J. Virol. 4 2 , 64-70 Bruder, S., Caplan, A., Gotoh, Y., Gerstenfeld, L. C., and Glimcher, M. J. (1990) Calcif. Tissue Znt., in press Cancedda, R., Capasso, O., Castagnola, P., Descalzi-Cancedda, F., and Quarto, N. (1985) J. Cell Biochem. 2 8 , 7-14 Castagnola, P., Moro, G., Descalzi-Cancedda, F., and Cancedda, R. (1986) J. Cell Biol. 1 0 2 , 2310-2317 Castagnola, P., Torella, G., and Cancedda, R. (1987) Dew. Biol. 1 2 3 , 332-337 Castagnola, P., Dozin, B., Moro, G., and Cancedda, R. (1988) J. Cell Biol. 106,461-467 Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 1 6 2 , 156159 Craig, A. M., Nemir, M., Mukherjee, B. B., Chambers, A.F., and 157, Denhardt, D. T. (1988) Biochem.Biophys.Res.Commun. 166-173 Descalzi Cancedda, F., Manduca, P., Tacchetti, C., Fossa, P., Quarto, R., and Cancedda, R. (1988) J . Cell Biol. 1 0 7 , 2455-2463 Descalzi Cancedda, F., Dozin, B., Rossi, F., Molina, F., Cancedda, R., Negri, A,, and Ronchi, S. (1990) J. Biol. Chem. 2 6 5 , 19060-19064 Doty, S. B., and Schofield, B. H. (1976) Progr. Histochem. Cytochem. 8 , 1-38 Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) Nucleic Acids Res. 16,6127-6145 Fisher, L. W., Hawkins, G. R., Tuross, N., and Termine, J. D. (1986) J . Biol. Chem. 262,9702-9708 Franzen, A., and Heingard, D. (1985) Biochem. J. 2 3 2 , 715-724 Franzen, A., Oldberg, A., and Solursh, M. (1989) Matrix 9 , 261-265 Gerstenfeld, L. C., Gotoh, Y., McKee, M. D., Nanci, A., Landis, W. J., and Glimcher, M. J. (1990) Anat. Rec. 2 2 8 , 93-103 Gibson, G. J., Beaumont, B. W., and Flint, M. H.(1984) J . Cell Biol. 99,208-216

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