Inhibition of Hamp1 Expression by TMPRSS6, GDF15, PIAS4 and SMAD6 Utilize Several Target Sites in the Hamp1 Promoter

Hepcidin, the key regulator of iron homeostasis, is up-regulated by iron excess, bone morphogenetic proteins (BMPs) and inflammatory cytokines and down-regulated by hypoxia and anemia. Known positive regulators at the level of transcription factors include SMAD1/4, STAT3 and CEBPα. In this study, we focused on negative regulators of hepcidin regulation:

• Matriptase II/TMPRSS6 (Transmembrane serine protease 6, a recently identified negative regulator in which disruption leads to anemia in mice as well in humans);

• Protein inhibitor of activated STATs no. 4 (PIAS4);

• Growth and differentiation factor 15 (GDF15, a potential erythroid negative regulator); and

• SMAD6 (Mothers against decapentaplegic homolog 6, an inhibitory SMAD blocking the SMAD/BMP pathway).

All tested inhibitors significantly decreased expression of the luciferase reporter under the control of 2.5 Kb murine Hamp1 promoter with GDF15 and PIAS4 Hamp1 specific since none of the inhibitors were able to reduce expression of the luciferase reporter under the control of the murine Hamp2 promoter. Inhibition of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter by SMAD6, unlike TMPRSS6, PIAS4 and GDF15, did not require liver specific transcription factors since the inhibition could also be observed in transfected HEK293T cells. GDF15, PIAS4, TMPRSS6 and SMAD6 all reduced basal level expression of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter as well as the total level of reporter expression induced by IL-6 and BMP-4. Nevertheless, GDF15 did not affect responsiveness (fold induction) to IL-6 and BMP-4. PIAS4 and TMPRSS6 inhibited responsiveness to IL-6 but had little effect on responsiveness to BMP-4. In contrast, SMAD6 did not affect responsiveness to IL-6 but reduced responsiveness to BMP-4. Deletion of the −140 bp −260 bp region of the murine Hamp1 or double deletion of the BMP-RE1 and BMP-RE2 motifs severely reduced the ability of all inhibitors to reduce reporter expression. Deletion of the STAT site abrogated PIAS4 inhibition while deletion of either BMP-RE1 or BMP-RE2 motifs alone partially reduced inhibition by TMPRSS6 and SMAD6. We conclude that there are several independent pathways that inhibit hepcidin expression.