Expression of HNF4alpha variants in pancreatic islets and Ins-1 beta cells

Background: Hepatocyte nuclear factor (HNF4alpha) 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 beta cell behaviour.

Methods: Expression of HNF4alpha 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 HNF4alpha9, which lacks both AF-1 and the F domain. Islets also expressed the HNF4alpha P1 promoter variants HNF4alpha1/alpha2, and Hep3B cells expressed HNF4alpha3. When ectopically expressed in COS-7 cells, HNF4alpha1, alpha3, alpha7, and alpha9 each stimulated an HNF4alpha-dependent promoter. Variants containing exon 1B (HNF4alpha4 - alpha6) were not detected. Lack of canonical splicing signals and species conservation argues against exon 1B usage.

Conclusions: This is the first report of HNF4alpha9 expression in any tissue. Our findings extend our understanding of HNF4alpha gene transcription and function. This knowledge may be useful in efforts to recover or establish regulated insulin secretion.