Insulin-controlled C/EBPα expression determines the impact of TGF-β on HNF4α transcription in hepatocytes

Background and Aims TGF-β has been thought as an HNF4α inhibitor through SMAD complex that initiates hepatocyte epithelial-to-mesenchymal transition. However, we found that SMAD complex is required for HNF4α transcription in hepatocytes. This study scrutinizes regulatory mechanisms of TGF-β signaling in HNF4α transcription.

Methods HNF4α expression was examined in 98 HBV-infected patient samples and primary human and mouse hepatocytes. The effects of TGF-β on HNF4α expression was measured by qPCR and immunoblotting. ChIP assays were used to investigate the factors, which regulates HNF4α transcription.

Results Both SMAD2/3 and C/EBPα were required for constitutively HNF4α expression in hepatocytes. Knockdown of either SMAD2/3 or C/EBPα reduced RNA polymerase II binding to the HNF4A core promoter and decreased HNF4α expression. TGF-β incubation increased HNF4α transcription in hepatocytes at 2h, however decreased its expression at 24h. In contrast to upregulating HNF4α transcription, SMAD2/3 bound to the CEBPA promoter and repressed C/EBPα transcription. Given that the half-life of C/EBPα protein was only 3h, long-term TGF-β incubation led to C/EBPα exhaustion that inhibited HNF4α expression. Impressively, SMAD2/3 binding to the CEBPA promoter was inhibited by insulin administration. Consistent with in vitro observation, 67 patients with hepatic HNF4α immune positivity expressed both p-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either p-SMAD2 or C/EBPα expression. In 18 patients lacking both HNF4α and C/EBPα expression, GLUT2 expression in hepatocytes was undetectable, indicating insulin resistance in these patients.

Conclusions Insulin-controlled C/EBPα expression is crucial to determine the effect of TGF-β on hepatic HNF4α expression.

 

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Publikationsverlauf

Artikel online veröffentlicht:
26. Januar 2022