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Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in
J M Hayden1, D D Strong, D J Baylink
1Department of Medicine, Loma Linda University, Jerry L. Pettis Veterans Administration Medical Center, California 92357, USA.
Abstract:
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.
Insights
Osteogenic protein-1 (OP-1) increases insulin-like growth factor-binding protein 3 (IGFBP-3) expression in human bone cells by enhancing nuclear transcript production. This process requires new protein synthesis and accelerates IGFBP-3 mRNA degradation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Osteogenic protein-1 (OP-1) is crucial for skeletal cell function.
- The insulin-like growth factor (IGF) system plays a key role in bone metabolism.
- Understanding OP-1's modulation of the IGF system is vital for skeletal research.
Purpose of the Study:
- To elucidate the molecular mechanisms by which OP-1 affects the IGF system in human skeletal cells.
- To investigate the time-course effects of OP-1 on IGFBP-3 gene expression in SaOS-2 cells.
Main Methods:
- Time-course analysis of IGFBP-3 mRNA expression in SaOS-2 cells treated with OP-1.
- Assessment of mRNA decay rates for IGFBP-3, IGFBP-4, and beta-actin.
- Measurement of nuclear and cytoplasmic IGFBP-3 transcript levels.
- De novo protein synthesis inhibition experiments.
- Transient transfection assays to analyze the IGFBP-3 promoter region.
Main Results:
- OP-1 significantly increased IGFBP-3 mRNA levels in a time-dependent manner.
- OP-1 accelerated the degradation of IGFBP-3 mRNA without affecting IGFBP-4 or beta-actin.
- OP-1 enhanced IGFBP-3 nuclear transcript abundance, indicating increased transcription.
- The induction of IGFBP-3 required de novo protein synthesis.
- OP-1 did not stimulate activity in the proximal 5'-flanking region of the IGFBP-3 gene.
Conclusions:
- OP-1 stimulates IGFBP-3 expression in human osteoblastic cells.
- The primary mechanism involves promoting IGFBP-3 nuclear transcript production.
- This process necessitates de novo protein synthesis and involves targeted degradation of steady-state IGFBP-3 mRNA.
- Regulatory elements influencing OP-1's effect may lie outside the characterized 5'-flanking region or within gene introns/exons.