Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

Abstract

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Iron, erythropoietin, and inflammation regulate hepcidin in Bmp2‐deficient mice, but serum iron fails to induce hepcidin in Bmp6‐deficient mice

“…Although chronic dietary iron loading and deficiency modulated BMP2 expression, BMP2 levels were not changed by an isolated increase in serum iron and transferrin saturation in response to oral iron gavage. Although a prior report suggested that liver BMP2 may be modulated by apo‐transferrin administration, we were not able to validate the specificity of BMP2 antibody used in that study using our Bmp2 fl/fl ;Tek‐Cre + mice (data not shown) . A similar lack of modulation by serum iron was also seen for BMP6, at least at the mRNA level, as previously reported .…”

GDF11 contributes to hepatic hepcidin (HAMP) inhibition through SMURF1‐mediated BMP‐SMAD signalling suppression

“…We hypothesized that SMURF1‐mediated HAMP suppression was dependent on ERK1/2 signalling. ERK1/2 signalling results in the phosphorylation of the SMAD linker domain for polyubiquitination by SMURF1, and degradation (Sapkota et al , ), and we have previously demonstrated activation of ERK1/2 signalling in hepatocytes from β‐thalassaemic mice, a mouse model in which HAMP regulation is dominated by erythropoiesis (Chen et al , ). Thus, the evaluation of ERK1/2 signalling demonstrated that ERK1/2 phosphorylation is increased by GDF11 in a dose‐dependent manner (Fig A), but not affected by SMURF1 overexpression (Figure S6C).To further explore whether ERK signalling is required for GDF11‐triggered BMP‐suppression of SMAD signalling, we treated Huh7 cells with GDF11 with or without U0126, a MEK/ERK1/2 inhibitor (Fig B, ).…”

“…Mouse primary hepatocytes were obtained from C57BL/6 (wild‐type; WT) mice by two‐step liver perfusion. In brief, the mouse livers were perfused by collagenase IV (Sigma, St. Louis, MO, USA) and 250 000 cells/well were plated on collagen‐coated 6‐well plates in DMEM supplemented with 5% FBS, and cultured as previously described (Chen et al , ). For experiments using rGDF11, Peprotech [Rocky Hill, NJ, USA] or R&D Systems [Minneapolis, MN, USA]), hepatocytes were incubated with 5% FBS and increasing doses of rGDF11 for 24 h. The specific MEK/ERK1/2 inhibitor, U0126 (Promega, Madison, WI, USA), was applied 2–2·5 h prior to cell harvest, and the proteasome inhibitor MG132 (Sigma) was added 4–6 h before cell harvest.…”

Hematological disorders and pulmonary hypertension

“…Interestingly, in a mouse model of β thalassemia, transferrin treatment normalized labile plasma iron levels and RBC survival, and increased hepcidin expression[62]. In addition, increased hepcidin levels were accompanied by increased BMP2 expression in the liver and concomitant decrease in extracellular-signal related kinase (ERK) activation[63]. …”