TGFβ Signaling Regulates Hepcidin Gene Expression

Abstract Abstract 992 Introduction: Hepcidin regulates iron metabolism by reducing duodenal iron absorption and iron release from macrophages and hepatocytes. In inflammatory states, including infection, neoplasia, and heart failure, cytokines induce hepcidin synthesis leading to the development of anemia of inflammation. The regulation of hepcidin gene expression by bone morphogenetic proteins (BMPs), members of the TGFβ family of growth factors, has been extensively investigated. In contrast, less is known about the regulation of hepcidin gene expression by other stimuli, including TGFβ itself. Although TGFβ expression is increased in inflammatory states, the role of TGFβ in the induction of hepcidin gene expression is controversial. To further elucidate the role TGFβ in iron metabolism, we investigated the regulation of hepcidin gene expression in the hepatoma cell line, HepG2. Methods: HepG2 cells were incubated with TGFβ (0.1, 0.5, 1, 2.5, and 5 ng/ml) for varying durations. RNA was extracted for measurement of levels of mRNAs encoding hepcidin, PAI-1 (a TGFβ-target gene), and Id-1 (a BMP-target gene). Cellular proteins were extracted to measure levels of phosphorylated TGFβ-responsive SMADs (using antibodies directed against phosphorylated SMAD2 or SMAD3) and levels of phosphorylated BMP-responsive SMADs (using antibodies directed to phosphorylated SMADs 1 and 5, SMAD1/5). The mechanisms by which TGFβ regulates hepcidin were investigated by pretreating cells with cycloheximide, an inhibitor of protein synthesis (50 μg/mL); Noggin (250 ng/mL) or LDN-193189 (100 nM), inhibitors of BMP signaling; or SB-431542 (5 μM), an inhibitor of the TGFβ type 1 receptor, Alk5. In additional experiments, HepG2 cells were transfected with an siRNA directed against Alk5, 72 hours before exposure to TGFβ. Results: In HepG2 cells, TGFβ induced hepcidin gene expression in a time- and dose-dependent manner: hepcidin mRNA levels were maximal at 2 hours after stimulation with TGFβ (1 ng/ml) and declined thereafter. Incubation of HepG2 cells increased PAI-1 and Id-1 mRNA levels, although increased PAI-1 mRNA levels persisted for at least 8 hours whereas Id-1 mRNA levels peaked at 2 hours. Cycloheximide did not block the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, or Id-1. TGFβ induced phosphorylation of SMADs 2 and 3, as well as SMAD1/5. Pretreatment of HepG2 cells with LDN-193189 (at concentrations that inhibit all four BMP type I receptors, as well as Alk1 which is a target of both BMPs and TGFβ) did not block the ability of TGFβ to induce hepcidin or Id-1 gene expression or phosphorylation of SMADs 2, 3, or 1/5. Pretreatment with Noggin gave similar results. Inhibition of Alk5 with SB-421542 blocked the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, and Id-1, as well as phosphorylation of SMADs 2, 3, or 1/5. TGFβ-stimulated hepcidin gene expression was inhibited by siRNA-mediated knockdown of Alk5. Conclusion: In HepG2 cells, TGFβ induces hepcidin gene expression via a mechanism which requires Alk5. Although, in addition to phosphorylation of SMADs 2 and 3, TGFβ induces phosphorylation of BMP-responsive SMADs, the failure of cycloheximide to inhibit the induction of hepcidin gene expression by TGFβ suggests that synthesis of BMPs is not required. Moreover, the inability of LDN-193189 to inhibit TGFβ-stimulated hepcidin gene expression suggests against a role for activation of Alk1 by TGFβ. Taken together our findings suggest that TGFβ stimulates hepcidin gene expression via a mechanism that requires Alk5 and may be mediated by signaling either via SMADs 2 and 3 or SMAD1/5. Targeting the regulation of hepcidin gene expression by TGFβ may offer a novel therapeutic approach to the anemia of inflammation. Disclosures: No relevant conflicts of interest to declare.