Abstract
Highly related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK). Gel shift studies reveal that oligonucleotide probes containing an IRS from the IGFBP-1 or PEPCK gene form a similar complex with hepatic nuclear proteins. Unlabeled competitors containing the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit the formation of this complex. Antibody against C/EBPbeta (but not other C/EBP proteins) supershifts this complex, and Western blotting of affinity purified proteins confirms that C/EBPbeta is present in this complex. Studies with affinity purified and recombinant protein indicate that C/EBPbeta does not interact directly with the IRS, but that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS reveal that the ability to interact with factors required for the formation of this complex correlates well with the ability of insulin to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene constructs with a C/EBP-binding site (but not an HNF-3/forkhead site or cAMP response element) maintains the effect of insulin on promoter activity. Together, these findings indicate that a nucleoprotein complex containing C/EBPbeta interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression.
Highlights
Related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK)
These findings indicate that a nucleoprotein complex containing CAAT/enhancer-binding proteins (C/EBPs) interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression
Initial studies revealed that a 32P-labeled double-stranded oligonucleotide probe containing the IGFBP-1 insulin response element (BP1) forms multiple nucleoprotein complexes with nuclear extracts prepared from rat H4IIE hepatoma cells (Fig. 1A, left panel)
Summary
Materials and Expression Vectors—-Antibodies against C/EBP␣ (sc61), C/EBP (sc-150), CEBP␦ (sc-151), ATF-1 (sc-270, which recognizes CREB 1 p43 and CREM-1), ATF-3 (sc-188), ATF-4/CREB-2 (sc200), c-Jun (sc-44, which recognizes with JunB and JunD p39), c-Fos (sc-253, which recognizes FosB, Fra-1, and Fra-2), Rb p110 (sc-50-G), NF p50 (sc-7178, which recognizes NF p105), NF p60 (sc-7151), YY1 (sc-281), FKHR (sc-9809), FKHRL1 (sc-9813), and the glucocorticoid receptor (sc-1003) were obtained from Santa Cruz Biotechnology. Gel shift conditions reported to optimize the binding of recombinant C/EBP to a probe containing the PEPCK IRS and flanking sequence [29] were used as noted (Fig. 6A). Binding activity in every other fraction was measured by gel shift assay using the ⌬IRS. oligo as a probe. Proteins were eluted with a 0.1–1 M KCl gradient in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol and binding activity was monitored by gel shift assay. Gel shift assays were performed with either the ⌬IRS. (round 1 and 3) or the ⌬IRS. oligo (round 2 of affinity chromatography) labeled as a probe, thereby ensuring that the purified proteins would interact both with the affinity matrix and with two different oligonucleotide probes containing an IRS. Transfected cells were refed with serum-free medium with/ without 100 nM human insulin 18 h prior to lysis and analysis of luciferase activity [6]
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