Sotatercept, An Activin Receptor-2a Ligand Trap, Modulates Hepcidin Levels In Primary Human Hepatocytes

Sotatercept, a recombinant human fusion protein containing the extracellular domain of ActRIIA binds to and inhibits activin and other members of the TGFß superfamily to induce a rapid increase in red cell number and hemoglobin. We sought to investigate if RAP-011, the murine ortholog of sotatercept, might regulate iron availability, which is important for erythropoiesis, through modulating hepcidin levels. Hepcidin is a circulating peptide hormone that negatively regulates iron transport into the plasma membrane by binding the iron transporter, ferroportin, and causing degradation of the complex. Hepcidin is primarily secreted by hepatocytes and also by hematopoietic cells, macrophages, kidney, heart, pancreas, and adipose cells (Gantz, T., Blood, 2003). The hepcidin promoter can be activated by both SMAD-4 and STAT-3, downstream targets of BMP-6 and IL-6 respectively (Andriopoulos Jr, B., Nature Genetics, 2009). BMP-6 binds its receptor, triggering downstream SMAD-1/5/8 signaling leading to SMAD-4 activation and initiation of HAMP (the gene encoding hepcidin) transcription. SMAD-4 signaling is also implicated in STAT-3 activation of HAMP transcription downstream of inflammatory signals such as IL-6 (Wang, R.H., Cell Metabolism, 2005).

In the current study, we evaluated the effect of RAP-011 on hepcidin expression and regulation in primary human hepatocytes. Consistent with its role as an activin receptor ligand trap, RAP-011 reduced baseline p-SMAD 1/5/8 expression and subsequently decreased HAMP expression. Next, we asked whether RAP-011 could also decrease HAMP expression under conditions of exogenous BMP-6 stimulation. When hepatocytes were treated with 50 ng BMP-6, an approximately 50-fold induction of HAMP was observed; interestingly, RAP-011 treatment decreased this induction by half. Similarly, treatment of the hepatocytes with the inflammatory cytokine IL-6 led to nearly a 7-fold induction of HAMP which was reduced to baseline levels following the addition of RAP-011. Hepcidin protein levels, although much more variable, mirrored that of HAMP expression following BMP-6 and combined BMP-6 + RAP-011 treatment. Protein changes following IL-6 stimulation were less apparent; this may be due to the fact that the peak of IL-6-stimulated HAMP expression occurred earlier than the 48 hours post-treatment at which the protein was assayed. Our data demonstrate that RAP-011 can modulate SMAD signaling and HAMP expression in primary human hepatocytes and suggests that RAP-011 may allow for increased iron availability for erythropoiesis. Importantly, RAP-011 was able to rescue IL-6-induced HAMP expression, suggesting that the drug might be able to restore iron availability in antinflammatory environment. Anemia of inflammation is associated with diseases such as renal, Castleman’s, and Rheumatoid arthritis. Hepcidin has been shown to play a role in restricting iron and promoting anemia in these pathologies (Steinbicker, A.U., Blood, 2011). In summary, RAP-011, through a yet unknown mechanism, may help to regulate iron availability by regulating hepcidin in pathological situations such as inflammation, iron restriction, or stress erythropoiesis.