SYNTHESIS AND PHOSPHORYLATION OF OSTEOPONTIN BY AVIAN EPIPHYSEAL GROWTH-PLATE CHONDROCYTES AS AFFECTED BY DIFFERENTIATION

The regulation of synthesis and phosphorylation of osteopontin in rela tion to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture, Osteopontin gene expression was evalua ted in the tibia growth-plate of 3-week-old chickens by in situ hybrid ization, The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated, Within the hypertrophic region, a poorly labeled area sepa rated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation, In culture, prolif erative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activi ty, Chondrocytes not treated with ascorbic acid, exhibited lower level s of osteopontin mRNA than the treated cells, The phorbol ester TPA-an activator of protein kinase C-and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression, Chondrocytes secreted low lev els of phosphorylated osteopontin to the medium, EGF treatment resulte d in the appearance of phosphorylated osteopontin in the medium, witho ut affecting the synthesis of other proteins, FGF and TGF(beta), but n ot IGF-I or IGF-II, also caused phosphorylation of osteopontin, Ascorb ic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further, Parathyroid ho rmone caused a dose-dependent attenuation of osteopontin phosphorylati on and inhibited the EGF-dependent osteopontin phosphorylation, The re sults suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms, The response to the various controlling agents varies with the state of differentiation, Both processes-the synthesis and phosphorylation of osteopontin-are un der the control of local growth factors which are involved in bone gro wth and calcification.