CYCLOHEXIMIDE STABILIZES INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-1(IGFBP-1) MESSENGER-RNA AND INHIBITS IGFBP-1 TRANSCRIPTION IN H4-II-ERAT HEPATOMA-CELLS

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 huma n 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 prev iously that IGFBP-1 transcription is positively regulated by dexametha sone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regul ation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 muM cycloheximide for 1.5 h did not prevent the induction of IGFB P-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assay s) by dexamethasone. By contrast, cycloheximide treatment abolished th e decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreas ed IGFBP-1 transcription in the absence of cycloheximide (>50% inhibit ion in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcriptio n. Similar results were observed with a second protein synthesis inhib itor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGF BP-1 transcription. These results suggest that although insulin decrea ses IGFBP-1 gene transcription in the presence of protein synthesis in hibitors, 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 approxima tely 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive l abile 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.