Iron enhances hepatic fibrogenesis through TGF-β activation

Introduction Although the accumulation of hepatic iron occurs during chronic liver injury, its role in fibrogenesis remains poorly understood. To date, studies show that excess iron in hepatocytes promotes formation of reactive oxygen species, which then activate hepatic stellate cells (HSC) to secrete collagen matrix. It is unclear however, whether iron directly affects HSC function. Aim Our aims were to investigate the effects of exogenous iron on core fibrogenic and iron-related genes and proteins in HSCs. Method The GRX murine HSC cell line was treated with the physiologically relevant form of transferrin-bound iron, holotransferrin (0.005, 0.05, 0.5, 2 and 5 g/L) for 24 h, with or without the iron chelator deferoxamine (10 μM). Expression of fibrogenic markers (α-SMA, TGF-β and Serpine-1) were analyzed by qRT-PCR, while ferritin was measured by ELISA. Levels of transferrin receptor (TfR1), TGF-β receptors and p-Smad 2 and 4 were analyzed by western blot, and secreted collagen was measured using the Sircol assay. Results HSCs express the iron-uptake and iron-exporter proteins TfR1 and ferroportin, respectively. Treatment with holotransferrin upregulated the expression of TfR1 by 1.8-fold (p<0.05), led to the accumulation of storage iron (ferritin) by up to 2-fold (p<0.01), and activated HSCs: α-SMA mRNA increased by up to 2-fold (p<0.03), TGF-β mRNA was elevated by 1.6-fold (p=0.05) and Serpine-1 mRNA was raised by 2.5-fold (p<0.05). These were accompanied by a 2-fold increase in secreted collagen (p<0.03), and activation of the TGF-β pathway: increased protein expression of TGF-β R1, TGF-b RII, and p-Smad 2 and 4 (p<0.05). Conversely, the addition of deferoxamine significantly lowered ferritin levels by 30% (p<0.03), repressed fibrogenic genes, α-SMA (0.2-fold; p<0.03), and TGF-β (0.4-fold; <0.03), and reduced collagen secretion by up to 60% (p<0.01). Deferoxamine also inhibited TGF-β signaling, with decreased levels of TGF-β RII and p-Smad 2. Conclusion HSCs express the iron transport proteins and are regulated by levels of exogenous iron. Excess iron activates HSCs and the TGF-β pathway, while iron depletion using deferoxamine attenuates the fibrogenic response, suggesting that iron depletion could be a useful adjunctive therapy in the treatment of individuals with advanced liver disease.