Expression of HNF4α variants in pancreatic islets and Ins-1 β cells

Background Hepatocyte nuclear factor (HNF4 alpha) is a nuclear receptor essential for endodermal differentiation and cell functions in the adult pancreas, liver, and other tissues. Mutations in the HNF4A gene cause MODY1. Up to nine protein variants arise from two developmentally regulated promoters. Because some variants lack the N-terminal activation function 1 (AF-1) and/or C-terminal inhibitory F domain, defining their tissue-specific regulation and function is important for understanding pancreatic cell behaviour. Methods Expression of HNF4 alpha. variants in islets, rat Ins-1 insulinoma cells, and human Hep3B hepatocellular carcinoma cells was assessed using a long-range reverse transcription-polymerase chain reaction (RT-PCR) strategy capable of recognizing each combination of mRNA termini. Protein expression was verified by immuno-blotting with terminus-specific antibodies and DNA-binding assays. Results Mouse islets and both cell lines express HNF4 alpha 9, which lacks both AF-1 and the F domain. Islets also expressed the HNF4 alpha PI promoter variants HNF4 alpha 1/alpha 2, and Hep3B cells expressed HNF4 alpha 3. When ectopically expressed in COS-7 cells, HNF4 alpha 1, alpha 3, alpha 7, and alpha 9 each stimulated an HNF4 alpha-dependent promoter. Variants containing exon 1B (HNF4 alpha 4 - alpha 6) were not detected. Lack of canonical splicing signals and species conservation argues against exon 1B usage. Conclusions This is the first report of HNF4 alpha 9 expression in any tissue. Our findings extend our understanding of HNF4 alpha gene transcription and function. This knowledge may be useful in efforts to recover or establish regulated insulin secretion. Copyright (c) 2008 John Wiley & Sons, Ltd.