MASL1 Is a Critical Modifier of Hepcidin Expression Via Smad1/5/8 Phosphorylation in the Bone Morphogenetic Protein (BMP) Signaling Pathway

Iron is essential for hemoglobin synthesis during terminal erythropoiesis. Hepcidin is the main regulator of iron homeostasis and is repressed by erythropoiesis. Although several candidates have been proposed to act as hepcidin inhibitors and erythroid regulators such as growth differentiation factor 15 (GDF15), twisted gastrulation BMP signaling modulator 1 (TWSG1) or erythroferrone (ERFE), their role in hepcidin repression during erythropoiesis is still unclear. We previously demonstrated that malignant fibrous histiocytoma-amplified sequence 1 (MASL1) is important for terminal erythropoiesis. In addition, down-regulation of MASL1 expression in macrophages strongly enhances IL-6 production following LPS or poly IC stimulation. Therefore, we hypothesized that MASL1 could directly (or indirectly) influence hepcidin expression. We found that endogenous MASL1 expression was significantly decreased in CD34⁺ cells treated with IL-6 compared with EPO-treated CD34⁺ cells at day 3 (0.35±0.05 fold vs 2.34±0.13 fold, P=0.002) and day 7 (1.03±0.75 fold vs 205.31±10.83 fold, P=0.001) of differentiation. In contrast, endogenous hepcidin expression was markedly increased in CD34⁺ cells treated with IL-6 compared with EPO-treated CD34⁺ cells. Interestingly, an increased hepcidin expression was detected in MASL1-knockdown CD34⁺ cells at day 3 of EPO-induced differentiation when compared with mock (47.47±23.49 fold vs 2.10±2.46 fold, P=0.029) or control lentiviral vector (47.47±23.49 fold vs 2.54±1.29 fold, P=0.029). Of note, ERFE, GDF15 and TWSG1 expression were decreased in MASL1-knockdown CD34⁺ cells at day 3 of EPO-induced differentiation. In addition, endogenous MASL1 expression is down-regulation after LPS treatment in PMA-induced THP1 cells but IL-6 is enhanced in MASL1-knockdown-PMA-induced THP1 cells after LPS treatment. In human hepatic cells (Huh-7), we found a significant decrease in hepcidin expression in MASL1-overexpressed Huh-7 cells after BMP2 (5.85±0.32 fold vs 9.98±0.97 fold, P=0.028) or BMP6 (10.07±1.35 fold vs 20.3±0.75 fold, P=0.007) stimulation for 24 hrs. Moreover, up-regulation of MASL1 enhanced the phosphorylation of Erk1/2 proteins while inhibited the phosphorylation of Smad1/5/8 proteins in Huh-7 cells after BMP2 or BMP6 stimulation for 1 hr that consequently affect in down-regulation of hepcidin expression. Strikingly, MASL1 overexpressed-Huh7 cells showed moderately decreased nuclear localization of phospho-Smad1/5/8 after BMP2 or BMP6 treatment. Taken together, these data demonstrate that MASL1 is a critical modifier of hepcidin expression potentially via additional mechanisms related to erythropoiesis and body iron homeostasis. Further clarification of these pathways may be the useful in developing novel treatment of anemias or iron disorders.