Abstract
Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both in vitro and in vivo. Specifically, enzymatic removal of heparan sulfate from primary human hepatocytes, CRISPR/Cas9 manipulation of heparan sulfate biosynthesis in human hepatoma cells, or pharmacological manipulation of heparan sulfate–protein interactions using sodium chlorate or surfen dramatically reduced baseline and BMP6/SMAD1/5/8-dependent hepcidin expression. Moreover inactivation of the heparan sulfate biosynthetic gene N-deacetylase and N-sulfotransferase 1 (Ndst1) in murine hepatocytes (Ndst1f/fAlbCre+) reduced hepatic hepcidin expression and caused a redistribution of systemic iron, leading to iron accumulation in the liver and serum of mice. Manipulation of heparan sulfate had a similar effect on IL6-dependent hepcidin expression in vitro and suppressed IL6-mediated iron redistribution induced by lipopolysaccharide in vivo. These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes.
Highlights
Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis
Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear
These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes
Summary
To examine if hepcidin expression depends on HS in primary human hepatocytes, we obtained cells through the Liver Tissue Cell Distribution System (see “Experimental Procedures”). To obtain additional evidence that HS regulates hepcidin expression, we silenced EXOSTOSIN 1 (EXT1) and EXT2 in human hepatocarcinoma cells with siRNAs. EXT1 and EXT2 encode components of the co-polymerase that alternately transfer glucuronic acid and GlcNAc units during HS chain elongation (Fig. 1A). Simultaneous silencing of EXT1 and EXT2 caused a dramatic reduction of HAMP mRNA expression in naive cells and after stimulation with BMP6 (Fig. 1D). Control experiments using siRNA to GFP showed no impact on expression of EXT1, EXT2, HAMP, and ID1, or phosphorylation of SMAD5 (Fig. 1, C–G). Together, these studies demonstrate that HS strongly regulates hepcidin mRNA expression in human hepatocytes
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