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Osteogenic Protein-1 Stimulates Production of Insulin-Like Growth Factor Binding Protein-3 Nuclear Transcripts in Human Osteosarcoma Cells¹

Departments of Biochemistry (D.D.S., D.J.B., S.M.), Medicine (J.M.H., D.D.S., D.J.B, S.M.), Microbiology and Molecular Genetics (D.D.S.), and Physiology (S.M.), Loma Linda University and Mineral Metabolism Unit (J.M.H., D.D.S., D.J.B., S.M.), Jerry L. Pettis Veterans Administration Medical Center, Loma Linda California 92357; Baylor College of Medicine (D.R.P.), Houston, Texas 77054; and Creative Biomolecules (T.K.S.), Hopkinton, Massachusetts 01748

Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Mineral Metabolism (151), Pettis VA Medical Center, 11201 Benton Street, Loma Linda, California 92357.

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 osteosarcoma 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-dependent manner (from 3–36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T_1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or ß-actin. Conversely, OP-1 acutely increased 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 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate 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 activity 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 conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

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