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 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 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or beta-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.
Hayden
J M
JM
Department of Medicine, Loma Linda University, Jerry L. Pettis Veterans Administration Medical Center, California 92357, USA.
Strong
D D
DD
Baylink
D J
DJ
Powell
D R
DR
Sampath
T K
TK
Mohan
S
S
D000199:Actins / Q000032:analysis / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D001859:Bone Neoplasms / Q000737:chemistry* / Q000473:pathology; D017931:DNA Primers / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis / Q000235:genetics*; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000032:analysis / Q000235:genetics; D007438:Introns; D012516:Osteosarcoma / Q000737:chemistry* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta; D014407:Tumor Cells, Cultured
D000199:Actins; C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D004273:DNA, Neoplasm; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta