Hepatic peptide hormone hepcidin, a key regulator of iron metabolism, is induced by inflammatory cytokine interleukin-6 (IL-6) in the pathogenesis of anemia of inflammation or microbial infections. Small heterodimer partner-interacting leucine zipper protein (SMILE)/CREBZF is a transcriptional corepressor of nuclear receptors that control hepatic glucose and lipid metabolism. Here, we examined the role of SMILE in regulating iron metabolism by inflammatory signals. Overexpression of SMILE significantly decreased activation of the Janus kinase 2-signal transducer and activator of transcription 3 (STAT3)-mediated hepcidin production and secretion that is triggered by the IL-6 signal in human and mouse hepatocytes. Moreover, SMILE co-localized and physically interacted with STAT3 in the nucleus in the presence of IL-6, which significantly suppressed binding of STAT3 to the hepcidin gene promoter. Interestingly, epigallocatechin-3-gallate (EGCG), a major component of green tea, induced SMILE expression through forkhead box protein O1 (FoxO1), as demonstrated in FoxO1 knockout primary hepatocytes. In addition, EGCG inhibited IL-6-induced hepcidin expression, which was reversed by SMILE knockdown. Finally, EGCG significantly suppressed lipopolysaccharide-induced hepcidin secretion and hypoferremia through induction of SMILE expression in mice. These results reveal a previously unrecognized role of EGCG-inducible SMILE in the IL-6-dependent transcriptional regulation of iron metabolism.

Hepcidin, a major regulator of systemic iron homeostasis, is mainly induced in hepatocytes by activating bone morphogenetic protein 6 (BMP-6) signaling in response to changes in the iron status. Small heterodimer partner-interacting leucine zipper protein (SMILE), a polyphenol-inducible transcriptional co-repressor, regulates hepatic gluconeogenesis and lipogenesis. Here, we examine the epigallocatechin-3-gallate (EGCG) effect on BMP-6-mediated SMAD1/5/8 transactivation of the hepcidin gene. EGCG treatment significantly decreased BMP-6-induced hepcidin gene expression and secretion in hepatocytes, which, in turn, abated ferroportin degradation. SMILE overexpression significantly decreased BMP receptor-induced hepcidin promoter activity. SMILE overexpression also significantly suppressed BMP-6-mediated induction of hepcidin mRNA and its secretion in HepG2 and AML12 cells. EGCG treatment inhibited BMP-6-mediated hepcidin gene expression and secretion, which were significantly reversed by SMILE knockdown in hepatocytes. Interestingly, SMILE physically interacted with SMAD1 in the nucleus and significantly blocked DNA binding of the SMAD complex to the BMP-response element on the hepcidin gene promoter. Taken together, these findings suggest that SMILE is a novel transcriptional repressor of BMP-6-mediated hepcidin gene expression, thus contributing to the control of iron homeostasis.

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2′-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol.