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Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands., Competing Interests: The authors have declared that no competing interests exist.

Hepcidin is the key regulator of systemic iron availability that acts by controlling the degradation of the iron exporter ferroportin. It is expressed mainly in the liver and regulated by iron, inflammation, erythropoiesis and hypoxia. The various agents that control its expression act mainly via the BMP6/SMAD signaling pathway. Among them are exogenous heparins, which are strong hepcidin repressors with a mechanism of action not fully understood but that may involve the competition with the structurally similar endogenous Heparan Sulfates (HS). To verify this hypothesis, we analyzed how the overexpression of heparanase, the HS degrading enzyme, modified hepcidin expression and iron homeostasis in hepatic cell lines and in transgenic mice. The results showed that transient and stable overexpression of heparanase in HepG2 cells caused a reduction of hepcidin expression and of SMAD5 phosphorylation. Interestingly, the clones showed also altered level of TfR1 and ferritin, indices of a modified iron homeostasis. The heparanase transgenic mice showed a low level of liver hepcidin, an increase of serum and liver iron with a decrease in spleen iron content. The hepcidin expression remained surprisingly low even after treatment with the inflammatory LPS. The finding that modification of HS structure mediated by heparanase overexpression affects hepcidin expression and iron homeostasis supports the hypothesis that HS participate in the mechanisms controlling hepcidin expression., Competing Interests: The authors have declared that no competing interests exist.

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-β or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS) can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.