Regulation of insulin-like growth factor binding protein-5 mRNA abundance in rat intestinal smooth muscle

IGF-I increases abundance of IGFBP-5 mRNA in rat intestinal smooth muscle c ells (RISM), and IGFBP-5 protein in RISM conditioned media. The translation al blocker, cycloheximide, decreased the abundance of IGFBP-5 mRNA to undet ectable levels, suggesting that IGFBP-5 mRNA integrity is linked to protein synthesis. We studied the mechanism of IGF-I's effect on IGFBP-5 mRNA, and the role of cytoplasmic proteins in modulating IGFBP-5 mRNA abundance. Ani somycin, emetine, and puromycin abolished IGFBP-5 mRNA as seen with cyclohe ximide. Cycloheximide had a dose- and time-dependent effect on IGFBP-5 mRNA . IGF-I increased IGFBP-5 nuclear transcripts by reverse transcription-poly merase chain reaction (RT-PCR), suggesting that IGF-I acts at least partial ly by increasing IGFBP-5 mRNA transcription. Protein synthesis inhibitors d id not affect IGFBP-5 nuclear transcripts, therefore, they affect only matu re mRNA. The IGFBP-5 mRNA 3' and 5' UTRs were cloned and their sequences se arched for adenosine-uridine rich elements (AUREs), elements shown to regul ate RNA stability. RNA mobility gel shift assay showed two protein activiti es that bind to nt 922 to 2076 of the 3' UTR, a region that contains an AUR E. One protein activity (BA2) was decreased in cytoplasmic extracts from cy cloheximide-treated RISM. These data demonstrate that IGFBP-5 mRNA integrit y is dependent on protein synthesis. The 3' UTR of IGFBP-5 contains element s shown to bind proteins important for RNA stability regulation. This regio n binds RISM cytoplasmic proteins, and may mediate the dramatic effect of c ycloheximide on IGFBP-5 abundance. RNA-protein interactions may be importan t to IGFBP-5 mRNA stability and ultimately, to IGFBP-5 actions. (C) 2000 Ac ademic Press.