OSTEOGENIC PROTEIN-1 STIMULATES PRODUCTION OF INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-3 NUCLEAR TRANSCRIPTS IN HUMAN OSTEOSARCOMA CELLS

To begin delineating molecular mechanisms by which osteogenic protein- 1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP -1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 o steosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-depen dent manner (from 3-36 h; treatment X time interaction, P < 0.001). Th e stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by trans cript stabilization; actually, OP-1 treatment selectively increased th e decay of mRNA for IGFBP-3 (T-1/2 = 5 h vs. 24 h for OP-1 and control s), but not for IGFBP-4 or beta-actin. Conversely, OP-1 acutely increa sed IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1-24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 exp eriments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protei n synthesis. Transient transfection experiments were undertaken to iso late putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP -3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activit y in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclu sion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblast ic cells by a mechanism that largely promotes the production of IGFBP- 3 nuclear transcripts, a process that requires de novo protein synthes is, and overrides an OP-1-induced targeted degradation of IGFBP-3 stea dy-state mRNA.