EFFECTS OF TRANSFORMING GROWTH-FACTOR-BETA ON BONE NODULE FORMATION AND EXPRESSION OF BONE MORPHOGENETIC PROTEIN-2, OSTEOCALCIN, OSTEOPONTIN, ALKALINE-PHOSPHATASE, AND TYPE-1 COLLAGEN MESSENGER-RNA IN LONG-TERM CULTURES OF FETAL-RAT CALVARIAL OSTEOBLASTS

Transforming growth factor beta (TGF-beta) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-beta is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes prolif eration of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-beta(1) on first subcultu res of fetal rat osteoblasts obtained from calvarial bones and culture d from confluence with ascorbic acid and beta-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear b y day 8. Similar to experiments described by Antosz et al., TGF-beta a dded to confluent cultures inhibited the formation of bone nodules. Bo th the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-beta(1). TGF-beta also i mpaired the expression of genes associated with bone formation, includ ing type I collagen, alkaline phosphatase, osteopontin, and osteocalci n. TGF-beta also inhibited the expression of mRNA for the bone morphog enetic protein 2 (BMP-2). These results, showing suppression of marker s representative of osteoblast differentiation, suggest that the effec ts of TGF-beta to stimulate bone formation in vivo are not likely a re sult of effects on differentiated mineralizing osteoblasts but, as sug gested by previous studies, more likely are caused by effects on osteo blast precursors. These results also suggest that endogenous BMP-2 exp ression in fetal rat calvaria cells is important for bone cell differe ntiation.