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

The orphan hepatic nuclear factor (HNF) HNF4alpha 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. In particular, HNF4alpha dysfunction leads to metabolic disorders, including diabetes. We used the chromatin immunoprecipitation (ChIP) cloning procedure and a bioinformatic approach to search for candidate genes associated with impaired liver, pancreas, and kidney function. We identified two novel targets regulated by HNF4alpha, 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, ribosomal S6 kinase 4 (RSK4) and p21-activated kinase 5 (PAK5) were confirmed, and in vitro binding of HNF4alpha was evidenced by electrophoretic mobility shift assays (EMSA) using oligonucleotides, which harbor novel binding sites. We also used EMSA to probe for binding sites in promoters of HNF1alpha, apolipoprotein B, alpha1-antitrypsin, and angiotensinogen. We further studied RSK4 and PAK5 kinase expression in streptozotocin-induced diabetic rat kidney and brain and observed significant repression of HNF4alpha, RSK4, and PAK5 as determined by quantitative real-time reverse transcriptase-polymerase chain reaction. RSK4 and PAK5 may provide a molecular rationale 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 HNF4alpha dysfunction.