Ablation of Hepatocyte Smad1, Smad5, and Smad8 Causes Severe Tissue Iron Loading and Liver Fibrosis in Mice

Abstract

A failure of iron to appropriately regulate liver hepcidin production is central to the pathogenesis of hereditary hemochromatosis. SMAD1/5 transcription factors, activated by bone morphogenetic protein (BMP) signaling, are major regulators of hepcidin production in response to iron; however, the role of SMAD8 and the contribution of SMADs to hepcidin production by other systemic cues remain uncertain. Here, we generated hepatocyte Smad8 single (Smad8^fl/fl ;Alb-Cre⁺ ), Smad1/5/8 triple (Smad158;Alb-Cre⁺ ), and littermate Smad1/5 double (Smad15;Alb-Cre⁺ ) knockout mice to investigate the role of SMAD8 in hepcidin and iron homeostasis regulation and liver injury. We found that Smad8;Alb-Cre⁺ mice exhibited no iron phenotype, whereas Smad158;Alb-Cre⁺ mice had greater iron overload than Smad15;Alb-Cre⁺ mice. In contrast to the sexual dimorphism reported for wild-type mice and other hemochromatosis models, hepcidin deficiency and extrahepatic iron loading were similarly severe in Smad15;Alb-Cre⁺ and Smad158;Alb-Cre⁺ female compared with male mice. Moreover, epidermal growth factor (EGF) failed to suppress hepcidin in Smad15;Alb-Cre⁺ hepatocytes. Conversely, hepcidin was still increased by lipopolysaccharide in Smad158;Alb-Cre⁺ mice, although lower basal hepcidin resulted in lower maximal hepcidin. Finally, unlike most mouse hemochromatosis models, Smad158;Alb-Cre⁺ developed liver injury and fibrosis at 8 weeks. Liver injury and fibrosis were prevented in Smad158;Alb-Cre⁺ mice by a low-iron diet and were minimal in iron-loaded Cre^- mice. Conclusion: Hepatocyte Smad1/5/8 knockout mice are a model of hemochromatosis that encompasses liver injury and fibrosis seen in human disease. These mice reveal the redundant but critical role of SMAD8 in hepcidin and iron homeostasis regulation, establish a requirement for SMAD1/5/8 in hepcidin regulation by testosterone and EGF but not inflammation, and suggest a pathogenic role for both iron loading and SMAD1/5/8 deficiency in liver injury and fibrosis.