CELL-SPECIFIC REGULATION OF IRS-1 GENE-EXPRESSION - ROLE OF E-BOX ANDC EBP BINDING-SITE IN HEPG2 CELLS AND CHO CELLS/

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signal s downstream. We have previously shown that the levels of IRS-1 mRNA v aried in different tissues. To elucidate the molecular mechanisms of t he tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the ch loramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions res ponsible for positive or negative regulation in each cell line mere id entified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further ana lyzed. Within this region a fragment from -1645 to -1605 bp upregulate d the IRS-1 promoter only in HepG2 cells, whereas a fragment from -160 5 to -1585 bp downregulated only in CHO cells. In the gel mobility shi ft assay, several nuclear proteins that bind to these fragments were d etected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identi fied in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 g ene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells an d the two nuclear proteins that bind to the C/EBP binding site regulat e it negatively in CHO cells.