Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes

Abstract

Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced.

Use of FGF-2 and FGF-18 to direct bone marrow stromal stem cells to chondrogenic and osteogenic lineages

“…In the present study, FGF-18 also upregulated MEF2C , a key osteogenic regulatory transcription factor, and osteogenic genes such as ALPL , COL1A1 and BGLAP . Osteocalcin (BGLAP) is a highly abundant bone protein secreted by osteoblasts, FGF-2 also stimulates production of BGLAP by chondrocytes [47]. The Gla domain of BGLAP interacts with calcium and hydroxyapatite and promotes bone mineralization and represents a useful early marker of bone formation.…”

Sp proteins and Runx2 mediate regulation of matrix gla protein (MGP) expression by parathyroid hormone

“…Further, we showed that PTH induction of MGP expression involves both PKA and ERK/MAPK signaling pathways, and that the 748 bp murine Mgp promoter region is sufficient for transcriptional activation by PTH in MC3T3-E1 cell [Suttamanatwong et al, 2007]. Several consensus transcription factor-binding sites are present in this region of the murine Mgp promoter including AP-1, AP-2 and runt domain binding sequences [Stheneur et al, 2003]. The purpose of this study was to identify specific DNA elements in the Mgp promoter necessary for regulation by PTH and to characterize the transcription factors that elicit this regulation.…”

Activation of the peroxisome proliferator–activated receptor α pathway potentiates interleukin‐1 receptor antagonist production in cytokine‐treated chondrocytes

“…Rabbit chondrocytes were cultured in Ham's F-12 containing 10% FCS as described above, trypsinized, and plated in 6-well plates. Cells were then transfected at 70% confluence by calcium phosphate precipitation as described (13). Briefly, cells were incubated for 4 hours with the precipitate obtained with 0.02M CaCl 2 in HEPES buffer (pH 7.10) and 2 g DNA/well, by adding either 1 g of the chimeric ps1680/sIL1Ra promoter-luciferase construct with 0.5 g of ␤-galactosidase expression vector plus 0.5 g of an empty pGL3 basic vector, or 1 g of ps1680/sIL-1Ra promoterluciferase construct plus 0.5 g of a PPAR␣ expression vector with 0.5 g of ␤-galactosidase expression vector.…”