Regulation of Insulin-like Growth Factor Binding Protein-5 mRNA Abundance in Rat Intestinal Smooth Muscle

• Published in: Biochemical and biophysical research communications 2000-08, Vol.275 (2), p.422-427
• Main Authors: Hou, Y.T., Xin, X.P., Li, L., Zimmerman, E.M.
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; 5' Untranslated Regions; Animals; Base Sequence; Cell Nucleus - metabolism; Cells, Cultured; Cloning, Molecular; Colon - cytology; Colon - metabolism; cycloheximide; Cycloheximide - pharmacology; cytoplasmic extract; DNA Primers; IGF-I; IGFBP-5; Insulin-Like Growth Factor Binding Protein 5 - genetics; Insulin-Like Growth Factor I - pharmacology; insulin-like growth factor-binding protein 5; mRNA binding proteins; Muscle, Smooth - metabolism; protein synthesis inhibitor; Protein Synthesis Inhibitors - pharmacology; puromycin; Rats; Rats, Inbred Lew; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-Binding Proteins - genetics
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.3283

IGF-I increases abundance of IGFBP-5 mRNA in rat intestinal smooth muscle cells (RISM), and IGFBP-5 protein in RISM conditioned media. The translational blocker, cycloheximide, decreased the abundance of IGFBP-5 mRNA to undetectable 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. Anisomycin, emetine, and puromycin abolished IGFBP-5 mRNA as seen with cycloheximide. Cycloheximide had a dose- and time-dependent effect on IGFBP-5 mRNA. IGF-I increased IGFBP-5 nuclear transcripts by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that IGF-I acts at least partially by increasing IGFBP-5 mRNA transcription. Protein synthesis inhibitors did not affect IGFBP-5 nuclear transcripts, therefore, they affect only mature mRNA. The IGFBP-5 mRNA 3′ and 5′ UTRs were cloned and their sequences searched for adenosine-uridine rich elements (AUREs), elements shown to regulate RNA stability. RNA mobility gel shift assay showed two protein activities that bind to nt 922 to 2076 of the 3′ UTR, a region that contains an AURE. One protein activity (BA2) was decreased in cytoplasmic extracts from cycloheximide-treated RISM. These data demonstrate that IGFBP-5 mRNA integrity is dependent on protein synthesis. The 3′ UTR of IGFBP-5 contains elements shown to bind proteins important for RNA stability regulation. This region binds RISM cytoplasmic proteins, and may mediate the dramatic effect of cycloheximide on IGFBP-5 abundance. RNA–protein interactions may be important to IGFBP-5 mRNA stability and ultimately, to IGFBP-5 actions.