CARTILAGE MATRIX GLA PROTEIN, A NEW MARKER OF CHONDROCYTE MATURATION. EFFECTS OF bFGF AND 17betaESTRADIOL.

C Stheneur. MF Dumontier, G Karsenty*. MT Corvol.

INSERM. Paris, France. *Baylor College. Houston. USA.

OBJECTIVE: Our purpose was to study the expression of a new cartilage matrix protein, the Matrix Gla Protein (MGP), during the maturation process of growth plate (GP) cartilage cells. MGP is expressed in cartilage but not in bone during embryonic and post natal development. Its function is still not known. Recent data using MGP-deficient mice suggest that it may inhibit cartilage calcification. The present work aimed to study the in vivo mRNA content of MGP in rabbit cartilage at different ages and in vitro at different stages of chondrocyte maturation (proliferation, hypertrophy, apoptosis and calcification). In addition the effects of growth factors and hormones, known to be involved into GP cartilage maturation during skeletal growth were also studied on chondrocyte MGP mRNA expression.

METHODS: Total RNA was extracted from articular (AR) and GP cartilage from rabbits before and at the age of puberty and studied for MGP mRNA content by Northern blot. At both ages, GP chondrocytes were cultured and analyzed for MGP mRNA expression and cartilage markers (collagen types II and X, proteoglycans (PG)) after incubation with or without bFGF. GH, IGF 1 or 17Bestradiol (E2). In order to characterize a possible transcriptional effect of the effectors on chondrocyte MGP expression, three chimeric plasmids (pMGP) which contained the mouse MGP 5' flanking regions of the gene linked to the Luciferase gene were used in transient DNA transfection of GP cells.

RESULTS: In vivo, MGP mRNA content was three times higher in GP than in AR cartilage of the same animal before puberty, and six fold more elevated at puberty. In vitro, MGP transcripts were nearly undetectable in quiescent chondrocytes, high during the proliferation phase induced by 10 ng/ml bFGF, and at the limit of detection in non dividing hypertrophic chondrocytes. The al II procollagen transcripts were expressed in all culture conditions, while the al X procollagen was only observed in hypertrophic cells. The addition of bFGF ( 1 to 50 ng/ml) induced the proliferation of the cells and decreased the production of chondrocyte proteoglycans while E2 (10^-11M to 10^-8M) did not change the cell number and stimulated chondrocyte PG. MGP mRNA content was up regulated by bFGF in a dose and time dependent manners, with a 3 times higher maximum effect before puberty than at puberty. By contrast, MGP mRNA content was stimulated with low doses (10^-10M) of E2 and not modified with high doses (10^-8M) E2. Finaly, bFGF caused a dose dependent increase in pMGP Luciferase activity, while such an effect was not observed with E2.

CONCLUSIONS: These data show for the first time that MGP gene is a marker of proliferative GP chondrocytes. Basic FGF. which is an inhibitor of chondrocyte maturation, has a transcriptional stimulating effect on the MGP gene expression. E2 probably acts at a post transcriptional level and has a biphasic effect depending on the concentration used. Further studies are needed to characterize MGP function.