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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 13, Issue of May 5, pp. 9645-9652,1993 Printed in U. S. A. 0 1993 by The American Society for Biochemistry and Molecular Biolom, Inc.

Expression and Role of c-myc in Chondrocytes Undergoing Endochondral Ossification*

(Received for publication, September 25, 1992, and in revised form, January 28, 1993)

Masahiro IwamotoS, Kimitoshi YagamiS, Phyllis Lu Vallet, Bjorn R. Olsens, Christos J. Petropoulosll , Donald L. Ewert**, and Maurizio PacificiS From the $Department of Anatomy-Histology, University of Pennsylvania Dental School, Phihdelphia, Pennsylvania 19104- 6003, the $Department of Anatomy and Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, the (IABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Developmental Center, Frederick, Maryland 21 702, and **The Wistar Institute, Philadelphia, Pennsylvania 19104

To analyze the relationship between c-myc gene expression and chondrocyte proliferation and maturation during endochondral ossification, Day 18-19 chick embryo sterna were pulse-labeled with [3H]thymidine, and serial sections were processed for autoradiography and in situ hybridization. Proliferating chondrocytes, located in four distinct areas of the de- veloping sternum, all contained high levels of c-myc transcripts, whereas postmitotic chondrocytes (such as hypertrophic chondrocytes) contained undetectable amounts. These findings were confirmed by Northern blot analysis and by the observation that antisense c- myc oligomer treatment inhibited proliferation in cultured chondrocytes. Constitutive overexpression of c- myc by retroviral vectors in immature chondrocyte cultures (c-myc cultures) maintained the cells in a proliferative state and blocked their maturation into hypertrophic chondrocytes. The lack of maturation in the c-myc cultures was corroborated by analysis of type X collagen gene regulation. Control immature cultures contained strong repressor activity for the type X collagen gene promoter, as revealed by transfection assays; repressor activity was lost upon maturation and activation of type X collagen synthesis. In the c-myc cultures, however, repressor activity persisted. Thus, c-myc participates in the normal changes in proliferation accompanying chondrocyte maturation in vivo and in culture. The decreases in c-myc expression and cell proliferation appear to be required for completion of maturation.

During endochondral ossification, chondrocytes undergo a complex process of maturation that involves changes in cell proliferation as well as changes in size, shape, and gene expression. In the avian growth plate, the immature chondrocytes are rapidly proliferating cells characterized by a small size, irregular shape, and low synthesis of extracellular matrix components (Stocum et al., 1979; Howlett, 1979). As the rate of cell proliferation decreases, the chondrocytes first become flat and then reacquire a round cell shape; these changes are accompanied by a marked increase in the production of characteristic extracellular matrix molecules, which include collagen types I1 and IX and aggrecan. The round postmitotic

AR 39705 (to M. P.), AR 36819 and AR 36820 (to B. R. O.), and CA * This work was supported by National Institutes of Health Grants

39000 and CA 53058 (to D. L. E.) and by an investigator award from the Arthritis Foundation (to P. L.). 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

cells finally develop into mature hypertrophic chondrocytes that produce large quantities of type X collagen, matrix vesi- cles, and alkaline phosphatase (Ali, 1976; Capasso et al., 1984; Schmid and Linsenmayer, 1985; Oshima et al., 1989).

The i n vivo mechanisms that regulate chondrocyte proliferation during maturation in the growth plate have not been identified. c-myc is a normal endogenous nuclear protein thought to be closely involved in the regulation of proliferation in a variety of cell types (Kelly et al., 1984; Sejersen et al., 1985). c-myc gene expression is rapidly induced following stimulation of cell proliferation by growth factors or serum in cultured cells; this is followed by a marked decrease in expression as the cells re-enter a quiescent state (Kelly et al., 1984; Bravo et al., 1985; Dean et al., 1986). Inhibition of proliferation in cultured articular chondrocytes by oxygen free radicals is accompanied by decreased c-myc gene expression (Vincent et al., 1991). In cells undergoing differentiation, such as skeletal muscle or kidney epithelial progenitor cells, c-myc gene expression is high during the early stages of cytodifferentia- tion when the progenitor cells are mitotically active. As the cells terminally differentiate and become postmitotic, c-myc gene expression decreases significantly (Sejersen et al., 1985; Endo and Nadal-Ginard, 1986).

The possible participation and role of c-myc in chondrocyte maturation during endochondral ossification have not been studied previously. If c-myc were to be involved in regulating mitotic activity in chondrocytes i n vivo, one would expect to find high c-myc gene expression in chondrocytes in the proliferative zone of growth plate, but very low expression in quiescent chondrocytes in the prehypertrophic and hypertrophic zones. In addition, suppression of c-myc gene expression in immature chondrocytes in culture should inhibit proliferation, whereas overexpression should maintain the cells in a proliferative state and prevent maturation. The latter prediction is sustained by the finding that overexpression of v-myc, the viral counterpart of c-myc, does promote chondrocyte proliferation and blocks maturation (AlemEt et al., 1985; Gionti et al., 1985; Horton et al., 1988; Quarto et al., 1992). Finally, if c-myc-overexpressing chondrocytes remain proliferative and immature for many cell generations, they should retain molecular mechanisms that prevent the expression of maturation-related genes, such as the type X collagen gene. In this study, we have tested these predictions and have obtained strong evidence in their support.

MATERIALS AND METHODS

In Situ Hybridizations and Autoradiography-Sterna were dis- sected from Day 1&19 chick embryos and immediately frozen in liquid nitrogen. Frozen longitudinal 8-pm-thick sections were thaw- mounted on 3-aminopropyltriethoxysilane-coated slides and quickly dried. Sections were fixed for 5 min at room temperature with 3%

9645

9646 e-myc and Chondrocyte Development

paraformaldehyde in diethyl pyrocarbonate-treated 0.1 M phosphate- buffered saline containing 5 mM MgC1’. After rinsing three times with phosphate-buffered saline, sections were prehybridized by incu- bation with 50% formamide, 4 X standard saline phosphate EDTA, 1 X Denhardt’s solution, 500 pg/ml salmon sperm DNA, 10% dextran sulfate, and 0.1% SDS for 1 h at 37 “C. Sections were then incubated with fresh hybridization solution containing 300 ng/ml digoxigenin- 11-dUTP-labeled 25-mer antisense c-myc oligomer (see below) for 16-18 h at 37 “C in a humid chamber. After hybridization, slides were sequentially rinsed with 2, 1, and 0.1 X SSPE in 50% formamide for 30 min each at 37 “C. Slides were incubated for 30 min with a 1:500 dilution of alkaline phosphatase-conjugated anti-digoxigenin Fab fragments in blocking solution (Boehringer Mannheim); after rinsing, enzymatic activity was detected by the nitro blue tetrazolium/5- bromo-4-chloro-3-indolyl phosphate reaction (Boehringer Mann- heim).

The sequence of the c-myc oligomer was selected by computer analysis of DNA data banks and corresponded to nucleotides +265 to +290 of chicken c-myc exon 2 (5”CGACCAGCTGGAGATGGT- GACGGAG-3’) (Watson et al., 1983). Sense and antisense oligomers were labeled with digoxigenin-11-dUTP in the presence of dATP by the 3”tailing procedure (Boehringer Mannheim). Specificity of hybridization was further established by Northern blot analysis; the antisense (but not sense) oligomer hybridized to the major 2.2-kb’ c- myc mRNA present in proliferating chondrocytes and absent in postmitotic hypertrophic chondrocytes (see below).

For autoradiography, freshly isolated sterna were incubated for 3 h in serum-free Dulbecco’s modified Eagle’s medium containing 50 pCi/ml [3H]thymidine (80 Ci/mmol). Tissue was rinsed and quickly frozen in liquid nitrogen. Longitudinal frozen sections were prepared as described above and were processed for autoradiography using Kodak NTB-2 emulsion as described (Pacifici et al., 1983); companion sections were processed for in situ hybridization with c-myc oligomers as described above.

Northern and Dot Blot Analyses-Total cellular RNA was isolated from cartilage by the method of Smale and Sasse (1992) with minor modifications. The peripheral and core regions of Day 18-19 chick embryo upper sterna were surgically separated under a dissecting microscope and kept at 4 “C. As shown under “Results,” two upper sternal peripheral regions were identified in the course of this study; the region used to isolate RNA was the one surrounding the core region and in contact with the ribs. Cartilage fragments (0.1-0.2 g, wet weight) were quickly homogenized in 2 ml of 4 M guanidine isothiocyanate, 0.1 M Tris-HC1, pH 7.5, and 1% 2-mercaptoethanol. The homogenate was mixed with 100 pl of 10% sodium lauryl sarcos- ine and centrifuged for 5 min in a microcentrifuge. The supernatant (2 ml) was layered over an equal volume of cesium trifluoroacetate with a density of 1.6 g/ml and containing 1 mM EDTA. Samples were centrifuged at 147,000 X g for 20 h at 18 “C. After removal of the supernatant, the pelleted material was dissolved in 200 p1 of diethyl pyrocarbonate-treated water; extracted once with phenol/chloroform/ isoamyl alcohol (25:24:1); mixed with 20 pl of 3 M sodium acetate, pH 4.8; and precipitated with 2 volumes of ethanol. After centrifugation, the RNA was resuspended in diethyl pyrocarbonate-treated water.

RNA was isolated from cultured chondrocytes (see below) by the guanidine isothiocyanate method (Chomczynski and Sacchi, 1987) as detailed previously (Pacifici et al., 1991).

RNA samples from tissue or cultured cells were denatured by glyoxalation, electrophoresed on agarose gels, transferred to Hybond- N membranes (Amersham Corp.) by capillary blotting, and hybridized to 32P-labeled riboprobes. Blots were washed at high stringency and exposed to Kodak films at -70 “C for various lengths of time. When necessary, blots were dehybridized at 90 “C in 0.05 X standard saline phosphate EDTA for 5 min and hybridized to a different probe. A final rehybridization was carried out using an 18 S ribosomal RNA probe to control for variations in RNA concentrations, gel loading, and transfer efficiency. The plasmids used were the chick type X collagen cDNA pDLrlO (Leboy et al., 19881, a 197-bp subclone of pYN3116 (Ninomiya et al., 1986) and the thick full-length c-myc cDNA MlOC.

Dot blots were performed using a Schleicher & Schuell apparatus according to the manufacturer’s instructions as described previously (Ewert et al., 1990).

Short-term Cell Cultures-Immature and hypertrophic chondrocytes were enzymatically isolated from the caudal one-third portion

The abbreviations used are: kb, kilobase(s); bp, base pair(s); BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; CAT, chloramphenicol acetyltransferase.

of sterna (Pacifici et al., 1991) and zones 2 and 3 of the proximal tibial growth plate (Stocum et al., 1979) of Day 18-19 chick embryos, respectively. Cells were grown in standard monolayer cultures for 5- 7 days without subculturing (passage 0) and then analyzed. The medium used was Dulbecco’s modified high glucose Eagle’s medium containing 10% defined fetal calf serum (HyClone Laboratories) and 50 units/ml penicillin and streptomycin (complete medium) (Pacifici et al., 1983). After this brief culture period, the sternal cells remained immature as indicated by their small cell size, absence of type X collagen production, and very low levels of alkaline phosphatase activity (Pacifici et al., 1991); in contrast, the tibial cells remained very large in size and were all positive for type X collagen synthesis and rich in alkaline phosphatase activity. To determine the cell doubling times in these cultures, the entire cell population was recovered by enzymatic digestion (see below), and cell number was determined as a function of culture age.

Alkaline phosphatase assays were performed as detailed previously (Pacifici et al., 1991); activity was expressed as nanomoles of para- nitrophenyl phosphate hydrolysis/30 min/106 cells.

Viral Vector Construction-The details of the construction of retroviral vectors containing the chicken c-myc and v-myc genes are described elsewhere.’ Briefly, a 1.6-kb chicken c-myc cDNA clone (M10C-2) was prepared that contains the entire first exon, spans the major open reading frame that initiates in exon 2, and ends within exon 3; the clone lacks a 500-bp region representing much of the 3’- untranslated sequences. A v-myc retrovirus was constructed that differed from c-myc only in the coding regions (exons 2 and 3) by adding exon 1 of c-myc to the 5’-end of the v-myc gene of the MC29 virus. In this way, insert length in the v-myc and c-myc viruses was kept equal. Replication-competent c-myc and v-myc retroviruses were constructed by subcloning the respective myc clones into the replication-competent retrovirus vector RCAS(A) (Hughes et al., 1987). Cells producing high titer retroviruses were generated by transfecting virus-free chick embryo (SPAFAS Inc.) fibroblast cultures with recombinant retrovirus DNA. Culture supernatants containing high titer virus stocks, as determined by end point dilution of infectious units, were used to infect chondrocyte cultures.

Viral Infection and Long-term Cell Cultures-Immature chondrocytes isolated from the caudal one-third portion of Day 18-19 chick embryo sterna as described above were mixed with recombinant virus at 10 focus forming units/cell in 1 ml of medium containing 40 pg/ ml DEAE-dextran and incubated for 1 h at room temperature. After viral adsorption, cells were plated at 1 X 106/100-mm tissue culture dishes in complete medium and grown for 4-7 days. Control cultures were mocked-infected and grown along with the infected cultures. After 4-7 days in primary culture, the dishes contained both floating and attached chondrocytes. The floating cells were first recovered by centrifugation, resuspended in balanced salt solution, and added back to their respective dishes. The entire cell population was treated with 0.1% trypsin and 0.02% EDTA for 15-20 min to detach the adherent cells, recovered by centrifugation, rinsed with medium, and counted. Aliquots were used to determine average cell diameter microscopi- cally; a minimum of 50 cells were analyzed, and their diameters were averaged. Cells were plated into secondary cultures (passage l), allowed to grow for 4-7 days until subconfluent, and passaged again. This subculturing procedure was then repeated many times as speci- fied under “Results.” The total cell numbers determined at each passage were used to calculate the cumulative number of cell generations from the onset of the culture. Cultures received fresh complete medium every other day throughout the periods of time studied; at each feeding, floating cells were recovered by centrifugation and added back to their respective dishes.

Oligonucleotide Treatment-Immature chondrocytes isolated from the caudal region of Day 18-19 chick embryo sterna were plated in complete medium on Vitrogen-coated 24-well plates at 30,000 cells/ well. To further increase cell adhesion, 12 units/ml testicular hyaluronidase (Calbiochem) was added to the medium (Leboy et al., 1989). On the next day, cultures received fresh medium containing the indicated doses of phosphorothioated 15-mer sense, antisense, or mixed sense/antisense c-myc oligomers. Control dishes received vehicle alone. The oligomers correspond to the first 15 bases from the translation start site of chicken c-myc (sense oligomer, 5’- ATGCCGCTCAGCGCC-3’) (Watson et al., 1983); when used as a mixture, the sense and antisense oligomers were mixed in equal amounts 24 h prior to use. Cultures were fed daily with medium containing fresh oligomer and hyaluronidase for the duration of the experiments. To determine cell number, the entire cell population

’ C. J. Petropoulos and S. H. Hughes, manuscript in preparation.

c-myc and Chondrocyte Development 9647

was recovered by enzymatic treatment as described above, and cell number was determined in a hemocytometer.

Immunofluorescence-Cells were detached from the culture dishes by treatment with 0.1% trypsin and 0.02% EDTA for 3 min, rinsed, and allowed to adhere for 10 min to 35-mm dishes that had been precoated with 0.05% polylysine. Cells were fixed with 70% ethanol for 5 min and processed for immunofluorescence as described previously (Pacifici et al., 1983). The antibodies used were a rabbit antiserum to chick type X collagen (Pacifici et al., 1991), the monoclonal antibody 2B1 to chick type I1 collagen (Mayne et al., 1993) (kindly provided by Dr. Richard Mayne, University of Alabama at Birming- ham), and a rabbit antiserum to the viral structural protein P27 (SPAFAS Inc.). The bound antibodies were detected by rhodamine- conjugated secondary antibodies (Cappel) and viewed under epifluo- rescence microscopy.

Construction of Type X Collagen Gene Promoter-CAT Vectors- The chloramphenicol acetyltransferase plasmid used was pBLCAT3, a promoterless vector that contains a polylinker 5’ of the CAT-coding region (Luckow and Schutz, 1987). Type X collagen promoter fragments (see Fig. 8) were cloned into the polylinker region using restriction endonucleases and phosphorylated linkers (New England BioLabs, Inc.). The 640-bp fragment (which contains the transcription start site, 558 bp of 5’-flanking region, and 82 bp of the first untranslated exon) was excised from the chick genomic clone PL 10 (Lu Valle et al., 1988) using restriction endonucleases HindIII and SacI. The addition of Sal1 linkers to the 3’-SacI site allowed the 640- bp fragment to be ligated in the correct orientation into the HindIII and Sal1 sites of the polylinker region of pBLCAT3 (640 CAT vector). The 1740-bp fragment was excised from PL 10 using NsiI and Socl. After addition of SalI linkers, the fragment was ligated into the SalI site of the polylinker of pBLCAT3 and oriented using the internal HindIII site (1740 CAT vector). The 1700-bp fragment (5’ of the 1740-bp fragment) was excised from PL 10 with PstI and NsiI and ligated into the PstI site of the polylinker of the 1740 CAT vector, resulting in the construction of the 3440 CAT vector. The PstI site is located just 5’ of and adjacent to the Sal1 site in which the 1740-bp fragment had been ligated. Since the 3’-NsiI site of the 1700-bp fragment is compatible for ligation with PstI but does not regenerate a PstI site, digestion of the resulting construct with PstI and HindIII (the internal site in the 1740-bp fragment) allowed us to determine the orientation of the 1700-bp fragment. Similar procedures were used to construct the 4840 CAT vector, which contains additional 5’- flanking region of the type X collagen gene. All plasmids were purified by polyethylene glycol precipitation (Lis, 1980), followed by cesium chloride/ethidium bromide equilibrium centrifugation (Radloff et al., 1967).

In control experiments, unrelated DNA fragments up to 3.3 kb in size and ligated 5’ of the 640 CAT construct described above (in pBLCAT3) did not significantly alter CAT activity; this indicates that the size of the insert is not a major factor in determining promoter activity.

Transfections-Transient transfections were performed using the high efficiency calcium phosphate coprecipitation method in the presence of BES (Chen and Okayama, 1987). Each 60-mm dish of cells was cotransfected with 4 pg of the type X collagen promoter- CAT vector constructs and 1 pg of the &galactosidase plasmid pCHllO (Hall et al., 1983) in complete medium for 8-14 h. The medium was then replaced with fresh medium, and cells were incubated for an additional 48 h prior to harvest according to Gorman et al. (1982). Cells were resuspended in 110 p1 of 0.25 M Tris-HC1, pH 7.8 subjected to three cycles of freeze/thaw; and centrifuged for 5 min at 4 “C in a microcentrifuge. Forty 111 of the supernatant were used in a P-galactosidase assay (Herbomel et al., 1984). Aliquots of each sample containing 5 units of &galactosidase activity were incubated with 0.1 pCi of [“C]choramphenicol for 10 min at 37 “C and then subjected to thin-layer chromatography. CAT activity was determined by scintillation counting; values are expressed as percent of CAT activity of cells transfected with the 640 CAT vector. Efficiency of transfection with the various CAT constructs detailed above varied from 1 to 9% in different experiments as determined by P-galactosidase cytochemical staining (Fisher et al., 1988); transfection efficiency was independent of stage of chondrocyte maturation.

Definition-Throughout this study, chondrocytes that produce typical cartilage markers (including aggrecan and collagen types 11 and IX) but do not express mature traits (high alkaline phosphatase activity, type X collagen, hypertrophy, and mineralized matrix) are referred to as “differentiated immature chondrocytes.”

RESULTS

Endogenous c-myc Gene Expression during Chondrocyte Maturation-In the first set of experiments, we analyzed C-

myc gene expression during the changes in cell proliferation that accompany chondrocyte maturation in vivo. As a model system, we studied the Day 18-19 chick embryo sternum, which contains maturing chondrocytes in its upper cephalic portion (Fig. lA, us) and immature chondrocytes in the lower caudal portion (Fig. 1A, Is) (Gibson et al., 1984). T o precisely map cell proliferation in these various tissue areas, freshly isolated sterna were pulse-labeled with 13H]thymidine for 3 h, and longitudinal sections were analyzed by autoradiography. The immature lower one-third portion of the sternum contained relatively few [3H]thymidine-labeled chondrocytes (Fig. IE, Is), whereas the adjacent middle portion contained numerous labeled chondrocytes, many of which displayed a flattened cell morphology (Fig. lD, ms). In the upper cephalic portion, the distribution of proliferating cells was more complex. As expected, the central region of the upper sternum, containing mature hypertrophic cells (Gibson et al., 1984), did not exhibit incorporation of [3H]thymidine (Fig. lC, core); in contrast, two relatively small zones at the periphery of the core region (Fig. 1, A and B, per) contained many labeled cells that were smaller in size than the hypertrophic chondrocytes in the core region.

To determine whether all the above-mentioned areas of chondrocyte proliferation were associated with c-myc gene expression, similar longitudinal sternal sections were processed for in situ hybridization using digoxigenin-labeled 24- mer antisense c-myc oligonucleotides. Clearly, chondrocytes in the most proliferative areas shown above exhibited high levels of c-myc transcripts (Fig. 1, F and H ) ; in contrast, mitotically quiescent chondrocytes, such as hypertrophic chondrocytes in the upper sternal core region and slow proliferating chondrocytes in the caudal region, contained low to undetectable levels of transcripts (Fig. 1, G and I , respectively). Companion longitudinal sections hybridized with sense oligomer produced no detectable hybridization (data not shown).

The in situ hybridization findings were verified by Northern blot analysis. Total RNAs were isolated from the proliferating and quiescent hypertrophic zones of the upper sternum (Fig. L 4 , per and core, respectively) and hybridized to a 32P-labeled c-myc riboprobe. The proliferating chondrocytes contained large amounts of the 2.2-kb c-myc mRNA, whereas the quiescent hypertrophic cells contained -10% as much (Fig. 1J, lanes 1 and 2, respectively). This was the opposite of type X collagen mRNA levels, which were undetectable in proliferating upper sternal chondrocytes, but high in hypertrophic cells (Fig. lK, lanes I and 2, respectively). A similar Northern blot analysis could not be carried out with the middle proliferative zone of the sternum (Fig. 1, D and H ) because this zone was difficult to dissect free of adjacent nonproliferative cells.

To corroborate these in uiuo data, we determined the steady- state c-myc mRNA levels in immature and hypertrophic chondrocytes in culture and the relationship between these levels and the mitotic activity of the cells. Immature caudal sternal chondrocytes and mature hypertrophic tibial chondrocytes from Day 18-19 chick embryos were maintained in primary monolayer culture for 5-7 days. As expected, the immature cultures were composed of cells uniformly small in size, round in shape, engaged in active cell proliferation, and with a doubling time of -24 h. In contrast, the hypertrophic cultures contained cells very large in size, epithelioid to polygonal in shape, and with a doubling time of >I00 h (see Giaretti et al. (1988)). Northern blot analysis revealed that the immature

9648 c-myc and Chondrocyte Development

A

%

us ms Is I I I I

B per C core D E

F G H I - J per core kb "F-

c-myc iW - 2.2

K type x A* - 2.5

1 2

FIG. 1. Analysis of chondrocyte proliferation and c-myc gene expression in sternum. Day 18 chick embryo sternum was pulse- labeled with [3H]thymidine for 3 h; longitudinal sections were then processed for autoradiography to reveal areas of chondrocyte proliferation and for in situ hybridization to detect c-myc gene expression. A, low magnification micrograph of a longitudinal section to illustrate the upper (us), middle (ms), and lower (Is) portions of the sternum. In the upper portion, two peripheral regions (per) and a central region (core) are also indicated. Bar, 1.06 mm. Shown are higher magnification micrographs of portions of a longitudinal section processed for [3H]thymidine autoradiography (B-E) . B and C, upper sternal peripheral and core portions, respectively; D, middle sternal portion; E, lower sternal portion. Also shown are higher magnification micrographs of portions of a section processed for in situ hybridization (F-Z). F and G, upper sternal peripheral and core portions, respectively; H , middle sternal portion; I , lower sternal portion. Bar, 210 pm. Autoradiographs are shown of a Northern blot containing RNAs from upper sternal peripheral ( l a n e I ) and core ( l a n e 2 ) chondrocytes ( J and K ) ; the blot was first hybridized to a 32P-labeled c-myc probe ( J ) and dehybridized and rehybridized to a 32P-labeled type X collagen probe ( K ) .

proliferating chondrocytes contained readily detectable levels of the 2.2-kb c-myc RNA, whereas the slow to nonproliferating hypertrophic chondrocytes contained barely detectable levels (Fig. 2A). The pattern of c-myc gene expression was opposite of that of type X collagen gene expression; the immature proliferating cells contained no detectable type X collagen mRNA, whereas the hypertrophic cells contained large amounts of it (Fig. 2B). Identical results were obtained with mature hypertrophic chondrocytes from the upper sternum (data not shown). Rehybridization of the blot to an 18 S ribosomal probe confirmed that the lanes contained the appropriate amounts of RNA (Fig. 2C).

To establish a more direct correlation between c-myc gene expression and chondrocyte proliferation, we analyzed the effects of antisense myc oligonucleotide treatment on the proliferation of immature lower sternal chondrocyte cultures, an approach previously used to link c-myc gene expression to proliferation in other cell types (Heikkila et al., 1987; Holt et al., 1988). Chondrocyte proliferation was inhibited in a time- and dose-dependent manner by phosphorothioated antisense ( A S ) oligomer treatment (Fig. 3, A and B); this effect was specific and not due to toxic effects since neither sense (S) oligomer nor a mixture of antisense plus sense (AS + S) oligomers affected proliferation significantly (Fig. 3, A and B ) . Lack of toxic effects by the various oligonucleotide treat-

ments was also demonstrated by the finding that incorporation of radiolabeled amino acids into newly synthesized proteins was unchanged on a per cell basis. In fact, proteoglycan synthesis measured by [35S]sulfate incorporation was stimu- lated 30-40% by antisense oligonucleotide treatment (data not shown), in good agreement with the inverse relationship between proliferation and matrix synthesis seen in chondrocytes (Kato and Iwamoto, 1990; Iwamoto et al., 1991; Adams et al., 1991).

c-myc Ouerexpresswn and Chondrocyte Proliferation-The maturation process of chondrocytes and the associated changes in cell proliferation and phenotypic expression can be reproduced in long-term cultures of immature caudal sternal chondrocytes. Initially in these cultures, the cells are small in size, proliferate readily as shown above, and synthesize mainly collagen types I1 and IX and aggrecan. After 3-5 weeks in culture, many of the cells undergo hypertrophy, express the type X collagen gene, and reduce significantly their proliferative activity (Castagnola et al., 1987; Giaretti et al., 1988; Pacifici et al., 1991). We asked whether constitutive overexpression of c-myc controlled by a retroviral promoter would alter the pattern of mitotic activity of caudal sternal chondrocytes undergoing maturation in culture.

Freshly isolated caudal sternal chondrocytes were infected with a retroviral vector encoding chicken c-myc. Parallel


Proliferating Hypertrophic chondrocytes chondrocytes

2 6 20 2 6 20pgRNA

A - c-rnyc

B

-Type X collagen

C

FIG. 2. Northern blot analysis of c-myc and type X collagen gene expression in immature proliferating lower sternal and mature hypertrophic tibial chondrocytes in culture. Different amounts (2, 6, and 20 pg) of total cell RNAs were loaded on each lane; separated by gel electrophoresis; blotted; and sequentially hybridized to "P-labeled c-myc ( A ) , type X collagen ( B ) , and 18 S ribosomal (C) probes.

- 1 3 5

Days in culture

B

Oligomer (pM)

FIG. 3. Effects of phosphorothioated antisense c-myc oligomer on proliferation in cultured lower sternal chondrocytes. A, proliferating chondrocyte cultures were treated with 1 PM antisense (AS), sense (S), or mixed antisense/sense ( A S + S ) oligomers staring on Day 1 of culture and harvested on Days 3 and 5; parallel control cultures (None) received vehicle alone. Note that only the antisense oligomer given by itself inhibits proliferation, as revealed by cell number determinations. B, companion cultures were treated with various doses of antisense, sense, or mixed antisense/ sense oligomers for 4 days, and cell numbers were determined at the end of treatment. Note that the antisense oligomer inhibits proliferation in a dose-dependent manner, whereas the sense or mixed antisense/sense oligomers have minimal effects. *, p < 0.05.

cultures were infected with similar vectors encoding v-myc (for comparison to c-myc) or with c-myc in the reverse orientation (to exclude possible effects of viral structural proteins on the chondrocyte phenotype); additional cultures were mock-infected and served as controls. The various cell populations were grown for several weeks after infection and subcultured every 4-5 days when still subconfluent. At each subculture, the total cell number was determined and used to calculate the cumulative number of cell generations from the start of the culture. During the first two to three subcultures (passages 1-3), all the various cell populations grew a t similar rates and had undergone similar numbers of cell generations (Fig. 4). Starting at the fourth passage (Days 16-18 of culture), both control cultures and those infected with virus encoding c-myc in the reverse orientation (Fig. 4, reverse c-myc) began to decrease their rates of cell growth. In contrast, the cultures overexpressing c-myc or v-myc continued to replicate a t con-

n &

5 * C

C 0 .s 40 a, c a, rn

- c-myc - v-myc - control = 20 reverse c-myc

G n " 0 20 40 60 80 100

Days in culture

FIG. 4. Cell generation number in control and myc cultures. Freshly isolated lower sternal chondrocytes were infected with retroviral vectors encoding c-myc, v-myc, or c-myc in the reverse orientation (reverse c-myc). Companion cells were mock-infected and served as control. Cultures were grown until subconfluent; the entire cell population was then recovered by enzymatic treatment, counted, and subcultured. This procedure was repeated every 4-5 days; the specific day of subculturing is indicated by the position of the symbols along the graph. The cell generation number in each culture was calculated from the cumulative number of cells present at each passage.

Control

c-myc

v-myc

6.4 3.2 1.6 0.8 0.4 0.2

FIG. 5. Dot blot analysis of myc gene expression in control (first row), c-myc (second row), and v-myc (third row) chondrocyte cultures (passage 5). Serial dilutions of RNA samples corresponding to the indicated number of cells were dot-blotted, hybridized to a 32P-labeled c-myc probe, and analyzed by autoradiography.

stant rates during the next 6-8 weeks, reaching a total of 60- 65 cell generations.

To confirm that the infected cells were overexpressing c- myc or v-myc compared to control cells, total RNAs were isolated from infected and uninfected Day 24 cultures (passage 5) (see Fig. 4) and used to determine the steady-state levels of myc RNAs by dot blot analysis and hybridization with a nick-translated 32P-labeled c-myc cDNA probe. The uninfected cultures contained levels of c-myc transcripts barely detectable by this method (Fig. 5, first row); in sharp contrast, cultures infected with c - m y or v-myc retroviral vectors contained very high levels of these transcripts (second and third rows, respectively). Similar high levels of these transcripts were found in c-myc and v-myc cultures at later passages.

c-myc Overexpression and Chondrocyte Maturation-To determine whether the maturation of chondrocytes was inhibited by overexpression of c-myc, we compared the cell diameter, type X collagen gene expression, and alkaline phosphatase activity in passage 5 control cultures and cultures infected with the c-myc retroviral vector (RCAS) (hereafter termed c- myc cultures). The control cultures contained chondrocytes with an average diameter of 23 f 3.1 pm (Fig. 6, A, C, and E ) ; this amounted to an increase in volume of >&fold compared to the volume of chondrocytes in early passage 1 cultures, which averaged 11 f 2.1 pm in diameter (data not shown). As revealed by immunocytochemistry, every control cell was

9650 e-myc and Chondrocyte Development

Control c-myc A Type X Type II Total RNA

- FIG. 6. Phase and immunofluorescence micrographs of con-

trol and c-myc chondrocyte cultures (passage 5) . Cultures were fixed and stained with antibodies to P27 viral protein or type I1 or X collagen. A and B, C and D, and E and F, control cultures stained with antibodies to P27 and collagen types I1 and X, respectively. C and H , I and J , and K and L, c-myc cultures stained with antibodies to P27 and collagen types I1 and X, respectively. Bar, 110 pm.

engaged in active type I1 collagen production, indicating that the cells possessed a normal chondrocyte lineage phenotype (Fig. 6, C-D). In addition, the cultures contained -25-30% of type X collagen-producing cells (Fig. 6, E-F), indicating that they were undergoing maturation.

In comparison to control cultures, the c-myc cultures contained cells significantly smaller in diameter (15 ? 2.3 pm) (Fig. 6G), amounting to 30-35% of the average cell volume displayed by control cells. Immunofluorescence showed that every cell in the c-myc cultures was engaged in active type I1 collagen synthesis (Fig. 6, ZJ), but not type X collagen synthesis (Fig. 6, K-L), indicating that the c-myc cells retained their differentiated phenotype, but had not matured. Immunostaining with an antiserum to viral structural protein P27 revealed that every chondrocyte contained this viral antigen and thus had been infected by the retroviral vector (Fig. 6H); this viral antigen was not detectable in control cells (Fig. 6B). Similar overall results were obtained with v-myc cultures (data not shown). Systematic immunofluorescence analyses of c-myc cultures as old as passage 20 (-80 days in culture) demonstrated that the chondrocytes retained a differentiated phenotype for a t least 50 cell generations (data not shown).

To corroborate the above data, we determined the steady- state levels of type X and I1 collagen RNAs in control and c- myc passage 5 cultures by Northern hybridization. The control cultures contained large amounts of both the 2.5-kb type X collagen mRNA and the 5.2-kb type I1 collagen mRNA (Fig. 7A, lanes 1 and 4, respectively); in comparison, the c- myc (as well as v-myc) cultures containzd no detectable type X collagen mRNA (lanes 2 and 3, respectively) and lower levels of type I1 collagen mRNA (lanes 5 and 6, respectively). Each lane in the Northern blot contained similar amounts of RNAs as indicated by methylene blue staining (lanes 7-9).

The control and myc cultures differed also in the levels of alkaline phosphatase activity, an enzyme associated with chondrocyte maturation and matrix mineralization. The control cultures possessed significant amounts of alkaline phosphatase activity, amounting to -5 units/1O5 cells. In contrast, the c-myc and v-myc cultures contained -10% as much activ-

"i* r

- 28s

- 18s

1 2 3

B 6 ]

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4 5 6 7 8 9

OJ- control c-myc v-myc

FIG. 7. A , Northern blot analysis of type X ( l anes 1-3) and type I1 (lanes 4-6) collagen mRNA levels in control, c-myc, and v-myc passage 5 cultures. Total cell RNAs were separated by gel electrophoresis, blotted, and hybridized sequentially to the appropriate labeled probes. Note that control cultures contain abundant type X and I1 collagen transcripts (lanes 1 and 4, respectively). In contrast, c-myc and v-myc cultures contain no detectable type X collagen RNA (lanes 2 and 3, respectively) and lower levels of type I1 collagen RNA (lanes 5 and 6, respectively) compared to control cultures. Each lane contained similar amounts of RNAs as revealed by methylene blue staining of the membrane (lanes 7-9). B, histogram showing alkaline phosphatase (APase) activity in control, c-myc, and v-myc cultures. Note that control cultures contain -9-fold more alkaline phosphatase activity than either c-myc or v-myc cultures. The asterisk on the y axis indicates nanomoles of para-nitrophenyl phosphate hydrolyzed per 30 min/105 cells.

ity as control cultures (Fig. 7B). c-myc Overexpression and Type X Collagen Gene Promoter

Activity-In a final set of experiments, we analyzed the molecular mechanisms by which c-myc overexpression sup- presses type X collagen gene expression. The design of these experiments was based on recent studies from our laboratories (Lu Valle et al., 1991, 1993) on the regulation of type X collagen gene expression during chondrocyte maturation. We found that multiple negative elements present in the 5'- flanking region of the gene act in an additive manner to restrict type X collagen gene expression to hypertrophic chondrocytes. Briefly, a plasmid containing a 640-bp promoter fragment, including 558 bp upstream of the transcription start site and 82 bp of the first exon fused to the reporter gene CAT (640 CAT construct) (Fig. 8A), elicited high CAT activity after transfection into both passage 0 immature lower sternal chondrocytes and mature hypertrophic upper sternal chondrocytes. When additional upstream sequences (1100-, 1700-, and 1400-bp fragments) were ligated to the 5'-end of the 640 CAT construct to generate 1740 CAT, 3440 CAT, and 4840 CAT constructs, respectively (Fig. 8A), they acted ad- ditively to reduce CAT activity in the immature chondrocytes, but had no significant effect in hypertrophic chondrocytes. Thus, these negative elements and putative repressor proteins appear to prevent type X collagen gene activity in immature


A Exon1 Exon2 5' I I I n &3

1400 I 1700 1100 640

1 4840CAT

I 3440CAT

1740CAT

640CAT CAT gene

B C D E F

H I I

?" - - ""- """"

" _ " 4840 3440 1740 640 4840 3440 1740 660 4840 3440 1740 640

FIG. 8. Analysis of type X collagen promoter activity in control and c-myc cultures. A, schematic representation of the type X collagen gene. Exons 1 and 2 are shown as open boxes. 640 is the 640-bp promoter fragment that contains the transcription start site and 558 bp of 5"flanking sequence. 1100,1700, and 1400 are the DNA fragments located 5' of the 640-bp fragment. The promoter- CAT constructs are depicted below. The thick solid lines represent the portions of the promoter and upstream sequences that are present in the different constructs; the shaded boxes represent the reporter gene CAT. B-E, histograms describing the CAT activity generated by the various CAT constructs in passage 0, 1,2, and 4 control lower sternal chondrocyte cultures, respectively. F, histogram describing the CAT activity of the constructs in passage 5 c-myc cultures. G-I, representative autoradiographs of thin-layer chromatograms to determine (in duplicate) the CAT activity of the different constructs in passage 0 control, passage 2 control, and passage 5 c-myc cultures, respectively. These autoradiographs are from the same series of experiments used to calculate the data shown in B-F.

chondrocytes; loss of such repressors by hypertrophic cells allows activation of the gene. In this study, we asked whether the absence of type X collagen gene expression in c-myc cultures may be due to suppressor mechanisms similar to those operating in control immature chondrocytes.

To approach this question, we first analyzed how the activity of the above four constructs (4840 CAT, 3440 CAT, 1740 CAT, and 640 CAT) changed during maturation of control immature lower sternal chondrocytes over time and passage number in culture. In agreement with observations described in our previous studies (Lu Valle et al., 1991, 1993), the 640 CAT plasmid produced high CAT activity regardless of the age of control cultures (Fig. 8, B-E and G-H). In comparison, the CAT activity directed by 4840 CAT, 3440 CAT, and 1740 CAT plasmids was very low in passage 0 cultures, but progressively increased in passage 1, 2, and 4 control cultures (Fig. 8, B-E and G-H). This indicated that, as the number of mature type X collagen-producing cells increased over time in culture, repressor activity was progressively lost. When the passage 5 c-myc cultures were examined in a similar fashion, the four plasmids produced patterns of CAT activity indistin- guishable from those produced by immature passage 0 cultures (Fig. 8, F and I); these cells only allowed minimal activity of the large 4840 and 3440 CAT constructs.

DISCUSSION

The results of this study show for the first time that endogenous c-myc gene expression changes during chondrocyte maturation in vivo. We show that chondrocytes in every proliferative zone of the sternum contain significant amounts

of c-myc transcripts; in contrast, slow to nonproliferating chondrocytes, such as hypertrophic chondrocytes in the core zone of the upper sternum, contain significantly reduced levels of this transcript. Similarly, we show that actively proliferating chondrocytes in culture express the c-myc gene at high levels, whereas slow proliferating hypertrophic cells exhibit markedly reduced expression. A more direct link between chondrocyte proliferation and endogenous c-myc gene expression is suggested by our finding that antisense c-myc oligomer treatment markedly reduces proliferation of cultured chondrocytes. Thus, the close association between proliferation and endogenous c-myc gene expression and function indicates that, as in other differentiating cell types involving changes in mitotic activity (Sejersen et al., 1985; Endo and Nadal- Ginard, 1986), maturing chondrocytes utilize c-myc-dependent mechanisms to regulate their proliferation during endochondral ossification.

Our results with retroviral expression vectors show that de- regulated constitutive c-myc expression directly or indirectly maintains the chondrocytes in an immature state and pre- vents maturation. The cells remain differentiated, highly proliferative, and small in size and express typical cartilage matrix molecules such as type I1 collagen. However, contrary to control age-matched cells, they fail to enlarge and produce type X collagen and high alkaline phosphatase activity and retain strong repressor activity directed toward the 5"flanking regulatory sequences of the type X collagen gene. The effects of de-regulated c-myc are reminiscent of those observed in chondrocytes continuously treated with basic fibroblast growth factor or parathyroid hormone (Kato and Iwamoto, 1990; Koike et al., 1990) or expressing v-myc (confirmed here) (Alemh et al., 1985; Gionti et al., 1985; Horton et al., 1988; Quarto et al., 1992). In each instance, these exogenous factors also maintain the chondrocytes in an immature proliferative state and interfere with maturation. Clearly, a decrease or cessation of proliferative activity appears to play an important role in the progression and completion of the maturation process of chondrocytes.

The apparent lack of major effects of de-regulated c-myc on the differentiated immature phenotype of chondrocytes is not completely surprising. Other differentiated cell types have been found to contain c-myc transcripts or protein, although usually at levels lower than in their respective progenitor undifferentiated cells (Dotto et al., 1986; Jaffredo et al., 1989; Endo and Nadal-Ginard, 1986; Hirvonen et al., 1990). One effect of de-regulated c-myc on chondrocytes shown here is the partial inhibition of type I1 collagen gene expression. Previous studies have indicated that there is usually an inverse relationship between the rate of chondrocyte proliferation and matrix production both in culture and in vivo, regardless of the stimulus or condition used to modulate proliferation (Kato and Iwamoto, 1990; Iwamoto et al., 1991; Oshima et al., 1989; Stocum et al., 1979; Adams et al., 1991; Quarto et al., 1992). For example, type X collagen gene expression increases when chondrocyte proliferation is decreased by a shift from monolayer to suspension culture (Adams et al., 1991). Thus, the partial decrease in type I1 collagen gene expression in c-myc chondrocyte cultures is likely not a direct effect of de-regulated c-myc gene expression, but rather a secondary effect of rapid continuous cell proliferation. If SO, this would imply that, in immature chondrocytes, c-myc may be a modulator of mitotic activity, but may have little direct effect on the mechanisms regulating their differentiated phenotype.

How then does c-myc interfere with maturation? One possibility already suggested above is that de-regulated c-myc would maintain the immature chondrocytes in a proliferative state, and its inhibition of maturation would be a secondary

9652 c-myc and Chondrocyte Development

or indirect effect of continuous proliferation. A second possibility is suggested by the finding that c-myc associates with another helix-loop-helixhasic leucine zipper-containing factor (Max) to form sequence-specific DNA-binding heterodimer complexes (Blackwood and Eisenman, 1991; Luscher and Eisenman, 1990). It has been suggested that a small number of Max-related proteins may exist and that the resulting myc- Max complexes may regulate specific biological mechanisms (Blackwood and Eisenman, 1991). Thus, it is possible that myc-Max heterodimer(s) could function as repressor(s) by directly binding to regulatory sequences of genes needed for or associated with chondrocyte maturation, such as the repressor sequences present in the type X collagen gene. This possibility would suggest that myc-Max-dependent mechanisms may influence genes associated with chondrocyte maturation, but may have little or no direct effect on genes characteristic of differentiated immature chondrocytes. A DNA sequence for myc-Max binding (Halazonetis and Kandil, 1991) is present in the 5"flanking region of the type X collagen gene promoter in degenerate form3; we are currently testing whether this sequence is functional and may have a direct role in regulating type X collagen gene expression.

If decrease or withdrawal from mitotic activity is needed for chondrocyte maturation, what is the role and biological significance of such mitotic activity to begin with? Tradition- ally, the mitotic activity of chondrocytes in the proliferative zone of the growth plate has been thought to provide additional cells for interstitial growth of cartilaginous models of long bones (Kember, 1983). If so, chondrocyte proliferation would simply serve to expand the cell population, but would have no other role. However, support for additional roles of proliferation comes from studies from this and other laboratories (Castagnola et al., 1987; Pacifici et al., 1990; Adams et al., 1991). For example, Castagnola et al. (1987) maintained chick tibial chondrocytes in cultures containing 20% (high serum cultures) or 4% (low serum cultures) fetal calf serum. As to be expected, they found that the chondrocytes under- went more cell replications in high than low serum cultures. Interestingly, they also found that the low serum cultures produced much less type X collagen than high serum cultures. They concluded that a number of cell divisions may be needed for progression toward maturation and type X collagen gene expression.

In conclusion, the chondrocyte maturation process involves an initial period of rapid cell proliferation, followed by withdrawal from mitotic activity. Both phases appear to have important distinct roles in maturation. By forcing the cells to persist in a proliferative state, de-regulated c-myc expression may prevent the decrease or cessation of mitotic activity needed for completion of the maturation process.

Acknowledgments-We thank Dr. Richard Mayne for the type I1 collagen monoclonal antibody 2B1, Drs. G. Smale and J. Sasse for communicating their RNA isolation procedure from cartilage before publication, J. DuHadaway for technical assistance, and Dr. S. L. Adams for critical reading of the manuscript.

P. Lu Valle, M. Iwamoto, M. Pacifici, and B. R. Olsen, unpub- lished observations.

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