RSK4 and PAK5 Are Novel Candidate Genes in Diabetic Rat Kidney and Brain

Abstract

The orphan hepatic nuclear factor (HNF) 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. In particular, HNF4␣ 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 HNF4␣, which participate in the control, at least in part, in cell-cycle regulation and are members of the mitogenactivated kinase pathway. In multiple ChIP assays, ribosomal S6 kinase 4 (RSK4) and p21-activated kinase 5 (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 used 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 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 HNF4␣ dysfunction.

HNF4α and the Ca-Channel TRPC1 Are Novel Disease Candidate Genes in Diabetic Nephropathy

“…Furthermore, protocols using streptozotocin (STZ) to cause ␤-cell toxicity and to result in diabetes are well known. Thus, administration of STZ to rats caused diabetic nephropathy and neuropathy (21,22), and we observed significant reduction of HNF4␣ transcript level in kidney and liver as reported previously (17). Likewise, TRPC1 gene expression in kidney and liver of STZ-administered rats was repressed, as was HNF4␣ itself (Table 3).…”

“…In addition, we used the ChIP cloning protocol to search for novel disease candidate genes targeted by HNF4␣ after formaldehyde crosslinking of nucleo-protein complexes. Notably, highly differentiated human epithelial Caco-2 cell cultures have been used successfully to study HNF4␣ function (17). Initially we screened immunoprecipitated DNA for enriched promoter sequences of HNF1␣ to ensure specificity of the ChIP assay (5,6).…”

“…Nuclear extracts from rat liver and kidney were prepared as described by Gorski et al (15), whereas nuclear extracts from Caco-2 cells were isolated by the method of Dignam et al (16) with minor modifications, as detailed previously (17). Details for Western blot analysis and electrophoretic mobility shift assays have been published previously by Niehof and Borlak (17). The antibody directed against HNF4␣ (sc-8987x) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany).…”

“…ChIP experiments were carried out with Caco-2 cells. All ChIP procedures and cloning and sequence analyses were carried out as described previously (17). Sequences were identified and annotated by database searches (GenBank Version Build 36.2, maintained by NCBI).…”

“…doi.org/10.2337/db007-1065]). Super shift experiments were done with an HNF4␣-specific antibody (sc-6556x; Santa Cruz Biotechnology), details previously published (17). ChIP, cloning, and sequence analysis.…”