Abstract
The insulin-like growth factor-binding proteins (IGFBPs) are a family of six proteins that modulate the biological activity of IGF-I and IGF-II and determine their bioavailability to tissues. One of the IGFBPs, IGFBP-1, is distinctive in the dynamic response of its levels in human plasma to metabolic changes. Parallel changes occur in IGFBP-1 mRNA and IGFBP-1 transcription in rat liver. Using the well differentiated H4-II-E rat hepatoma cell line as a model system, we demonstrated previously that IGFBP-1 transcription is positively regulated by dexamethasone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regulation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 microM cycloheximide for 1.5 h did not prevent the induction of IGFBP-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assays) by dexamethasone. By contrast, cycloheximide treatment abolished the decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreased IGFBP-1 transcription in the absence of cycloheximide (> 50% inhibition in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcription. Similar results were observed with a second protein synthesis inhibitor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGFBP-1 transcription. These results suggest that although insulin decreases IGFBP-1 gene transcription in the presence of protein synthesis inhibitors, IGFBP-1 mRNA levels are maintained because of stabilization of the mRNA. Stabilization was demonstrated directly in actinomycin D-treated cells, where the t1/2 of IGFBP-1 mRNA increased from approximately 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive labile proteins contribute to the maintenance of basal IGFBP-1 promoter activity and the rapid turnover of IGFBP-1 mRNA, which determine the dynamic regulation of IGFBP-1 gene expression.
Highlights
(IGFBPs) are a family of six proteins that modulate polypeptides chemically related to insulin thatare widely the biological activity of IGF-I and IGF-I1 and deter- expressed in fetalandadult mammalian tissues (1).They mine their bioavailability to tissues
We examine the effect of cycloheximide on the induction of IGF-binding proteins (IGFBPs)-1 gene expression by dexamethasone and its inhibitiobny insulin inH4-11-Ecells to determine whether de m u 0 protein synthesis is required for the regulapended in 10 mM Tris-HCI, pH 7.9, containing 10 mM NaCl, 5 mM
Since PEPCK gene transcription was inhibited by insulin alone or in combination with cycloheximide (35),these results suggest that cycloheximide stabilizes PEPCK mRNA as idt oes IGFBP-1 mRNA
Summary
Materials-Porcine insulin was obtained from Lilly, dissolved in 10 mM acetic acid (5 mg/ml), and stored at -20 "C. Dexamethasone (Sigma) was dissolved in ethanol to a stock concentration of 1mM. Bovine serum albumin (BSA; radioimmunoassay grade), cycloheximide, anisomycin, and actinomycin D were purchased from Sigma. Linearized DNA (10 pg)from plasmid pGem-7Zf(-) (Promega, Madison, WI) or filters without DNA were used as targets for background hybridization. Quaatitation of Hybridization Results-Hybridized radioactivity in the Northern blot and transcription run-on experiments were quantitated by @-scanningusing a computer-driven radioactivity scanner (AMBIS Scanning System, Automated Microbiology Systems Inc., San Diego, CA). Results are expressed as counts hybridized, following subtraction of background radioactivity hybridized to an equivalent area of the Northern bIot or nuclear transcript radioactivity hybridized to filters containing eitherno DNA orplasmid DNA in the same incubation. Hormonal Treatment of Cells-For experiments, cells were plated in 100-mm culture dishes and grown to confluence in the presence of
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