Received for publication October 28, 2004.
Revised December 11, 2004.
Accepted for publication December 14, 2004.

RSK4 and PAK5 are novel candidate genes in diabetic rat kidney and brain

Abstract

The orphan nuclear receptor HNF4 is of pivotal importance for liver development and hepatocellular differentiation and plays an essential role in a regulatory circuitry to control a wide range of metabolic processes. It also targets genes in other organs including pancreas, kidney, intestine and colon, promotes expression of an epithelial phenotype, triggers de novo formation of functional tight junctions and contributes to epithelial cell polarity. Specifically, HNF4 dysfunction leads to metabolic disorders including diabetes. We employed the chromatin immunoprecipitation (ChIP) cloning procedure and a bioinformatoric approach to search for candidate genes associated with impaired liver, pancreas and kidney function. We identified two novel targets regulated by HNF4, which participate in the control, at least in part, in cell cycle regulation and are members of the mitogen activated kinase pathway. In multiple ChIP assays RSK4 and PAK5 were confirmed and in vitro binding of HNF4 was evidenced by electrophoretic mobility shift assays (EMSA) using oligonucleotides, which harbor novel binding sites. We also employed EMSA to probe for binding sites in promoters of HNF1, apolipoprotein B, 1-antitrypsin and angiotensinogen. We further studied RSK4 and PAK5 kinase expression in streptozotocin-induced diabetic rat kidney and brain and observed significant repression of HNF4, RSK4 and PAK5 as determined by quantitative real-time RT-PCR. RSK4 and PAK5 may provide a molecular rational for late stage complications in disease and further studies are warranted to explore these targets for the treatment of diabetic nephro- and neuropathy, frequently seen in patients with HNF4 dysfunction.

Key words: DNA binding sites, Promoter analysis, Regulation of gene expression, Transcription targets