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Regulation of insulin-like growth factor binding protein-5 mRNA abundance in rat intestinal smooth muscle
1Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-0589, USA.
Abstract:
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.
Insights
Insulin-like growth factor I (IGF-I) enhances insulin-like growth factor-binding protein 5 (IGFBP-5) mRNA stability in rat intestinal cells. Protein synthesis is crucial for maintaining IGFBP-5 mRNA levels, involving interactions with cytoplasmic proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Insulin-like growth factor I (IGF-I) influences cellular processes, including gene expression.
- Insulin-like growth factor-binding protein 5 (IGFBP-5) plays a role in regulating IGF-I bioavailability and action.
- The regulation of IGFBP-5 mRNA stability in intestinal smooth muscle cells is not fully understood.
Purpose of the Study:
- To investigate the mechanism by which IGF-I affects IGFBP-5 mRNA abundance in rat intestinal smooth muscle cells (RISM).
- To determine the role of protein synthesis and cytoplasmic proteins in modulating IGFBP-5 mRNA levels.
- To identify regulatory elements within the IGFBP-5 mRNA that contribute to its stability.
Main Methods:
- Treatment of RISM cells with IGF-I and protein synthesis inhibitors (cycloheximide, anisomycin, emetine, puromycin).
- Quantification of IGFBP-5 mRNA and nuclear transcripts using reverse transcription-polymerase chain reaction (RT-PCR).
- Cloning and sequence analysis of IGFBP-5 mRNA 3' and 5' untranslated regions (UTRs) for adenosine-uridine rich elements (AUREs).
- RNA mobility gel shift assays to identify protein binding to the 3' UTR.
Main Results:
- IGF-I increased IGFBP-5 mRNA and protein abundance in RISM cells.
- Protein synthesis inhibitors abolished IGFBP-5 mRNA, indicating dependence on ongoing protein synthesis.
- IGF-I increased IGFBP-5 nuclear transcripts, suggesting transcriptional regulation.
- The 3' UTR of IGFBP-5 contains an AURE that binds cytoplasmic proteins, with one activity (BA2) reduced by cycloheximide treatment.
- These protein-RNA interactions are implicated in regulating IGFBP-5 mRNA stability.
Conclusions:
- IGFBP-5 mRNA integrity and abundance in RISM cells are dependent on active protein synthesis.
- IGF-I likely enhances IGFBP-5 mRNA stability through mechanisms involving cytoplasmic protein interactions with the 3' UTR.
- RNA-protein interactions mediated by the 3' UTR are critical for regulating IGFBP-5 mRNA stability and subsequent cellular actions.
