DIFFERENTIAL-EFFECTS OF RETINOIC ACID AND GROWTH-FACTORS ON OSTEOBLASTIC MARKERS AND CD10 NEP ACTIVITY IN STROMAL-DERIVED OSTEOBLASTS/

The effects of retinoic acid (RA) on the expression of osteoblastic-re lated cell markers was examined. A marrow stromal osteogenic cell line , MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen alpha(2) (1), osteonectin, osteopontin, biglycan, and alk aline phosphatase (ALK-P). Gene expression was unchanged in response t o RA triggering for 24 hr. Furthermore, cell growth and enzymatic acti vities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. The se parameters were examined in MBA-15 and clonal populations represent ing different stages of differentiation. The cell's growth rate was un changed, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines exam ined while CD10/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CD10/NEP activit y at the beginning of the culture period, reaching basal level with ti me. This activity was greatly increased over control level in MBA-15.6 , a mature stage of osteoblasts. Furthermore, the response of cell lin es to various growth factors was tested subsequent to priming the cult ures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measure d when cells were exposed to transforming growth factor beta (TGF beta ). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that ha d remained unchanged following priming of the cell with RA. Insulin-li ke growth factor I (IGF-I) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cel ls. Cellular response to bone-seeking hormone, parathyroid hormone (PT H), and prostaglandin E(2) (PGE(2)) was monitored by activation of int racellular cAMP. Treatment with RA caused a dramatic decrease in MBA-1 5.6 cell responses to PTH and PGE(2), but no significant effects could be observed in other clonal lines. (C) 1994 Wiley-Liss, Inc.