Characterization of a putative insulin-responsive element and its binding protein(s) in rat angiotensinogen gene promoter: Regulation by glucose and insulin

We previously demonstrated that high glucose activates angiotensinogen (ANG ) expression and that insulin inhibits this activation. The present studies aim to investigate whether insulin regulates ANG gene expression in kidney proximal tubular cells at the transcription level via interaction of the p utative insulin-response element (IRE) with its binding protein(s) in the 5 ' -flanking region of the ANG gene. Fusion genes containing various length s of the 5 ' -flanking region of the rat ANG gene fused to a human GH (hGH) gene as reporter were constructed and transiently introduced into rat immo rtalized renal proximal tubular cells (IRPTCs). The expression of the fusio n genes was monitored by the amount of immunoreactive hGH secreted into the medium as assayed by a specific RIA for hGH. Insulin inhibited the express ion of pOGH (rANG N-1498/+ 18), pOGH (rANG N-1120/+18) and pOGH (rANG N-882 /+18) but not pOGH (rANG N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-68 8/+18) and pOGH (rANG N-53/+18) in high-glucose (i.e. 25 mM) medium. Site-d irected mutagenesis of nucleotides N-874 to N-867 (5 ' CCC GCC CT 3 ') in t he 5 ' -flanking region of the rat ANG gene abolished the response to insul in. Insulin also inhibited the expression of the fusion gene containing the DNA fragment ANG N-882 to N-855 inserted upstream of the ANG gene promoter (N-53/+18), but had no effect on a mutant of N-882 to N-855. Gel mobility shift assays revealed that the labeled putative rat ANG-IRE motif (N-878 to N-864, 5 ' CCT TCC CGC CCT TCA 3 ') was bound to the nuclear proteins of I RPTCs. This binding was displaced by unlabeled ANG IRE and IRE of human gly ceraldehyde phosphate dehydrogenase but not by mutants of ANG-IRE and IRE o f the rat glucagon gene. Southwestern blotting analysis revealed that the l abeled putative ANG-IRE motif bound to a major nuclear protein with an appa rent molecular mass of 48 kDa. Finally, high glucose levels enhanced 48-kDa nuclear protein expression and induced an additional 70-kDa nuclear protei n expression in IRPTCs, as revealed by Southwestern blotting. Insulin inhib ited both 48- and 70-kDa nuclear proteins expression induced by high glucos e levels. Its inhibitory effect was reversed by the presence of PD98059 tan inhibitor of mitogen-activated protein kinase, MAPK) but not by wortmannin tan inhibitor of phosphatidylinositol 3-kinase). These studies demonstrate that insulin action on ANG gene expression is at the transcriptional level . The molecular mechanism (s) Of insulin action is mediated, at least in pa rt, via interaction of the functional IRE with unidentified 48- and 70- kDa nuclear proteins in the rat ANG gene and is MAPK dependent.