193, P = 0.307), which was in fact not changed. By multivariate analysis, Tf sat was the only independent predictor of Hamp mRNA levels (R2 = 0.23, β = 0.444, and P < 0.001). These data provide further evidence that Tf sat independently regulates hepatic Hamp mRNA expression. Next, we investigated the role of the BMP6-SMAD signaling pathway in hepcidin induction by acute and chronic iron administration. Similar to prior studies,17, 18 chronic iron treatment significantly increased hepatic Bmp6 mRNA levels in comparison to the baseline group,

with a temporal progressive increase similar to LIC (compare Figs. 4A and 1C). Although one prior study suggested that the small intestine may also be a source of BMP6 in response to iron,29 we did not see any effects of chronic iron treatment on Bmp6 mRNA expression VX-809 molecular weight in either the duodenum (Supporting Fig. 2), in buy ABT-888 agreement with other studies,19, 30 or the spleen, another key iron homeostasis organ (Supporting Fig. 3).

The Spearman’s rho test confirmed a strong correlation between hepatic Bmp6 mRNA levels and LIC (r = 0.902, P < 0.001), and multivariate regression analysis demonstrated that LIC was the only factor associated with hepatic Bmp6 mRNA levels, independent of Tf sat, serum iron, hemoglobin, and mean cellular hemoglobin concentration (R2 = 0.846, β = 1.032, P < 0.001). These data suggest that LIC may have a role in hepatic Bmp6 induction by iron. In contrast, hepatic Bmp6 mRNA was not significantly increased by acute iron administration

(Fig. 4B, black bars), where Tf sat increased but LIC did not change. The mock groups (Fig. the 4B, gray bars) did show a small but significantly lower Bmp6 expression at 1 and 24 hours after gavage in comparison to the baseline group, and at 1 and 4 hours after gavage in comparison to the corresponding iron timepoints; however, the overall trend of both iron and mock groups were similar, possibly reflecting an effect from the gavage itself or circadian fluctuations of hepatic Bmp6 mRNA. Importantly, we did not find any correlation between Tf sat and hepatic Bmp6 mRNA levels (r = 0.237, P = 0.068). Additionally, we did not see a corresponding decrease in hepatic Smad1/5/8 phosphorylation or Bmp6-Smad target gene expression by mock gavage (see Figs. 5B, 6C,D, gray bars), suggesting that this small decrease in Bmp6 expression in the mock group was not functionally relevant. We also did not find significant increases in duodenal or splenic Bmp6 mRNA in response to acute iron administration (Supporting Figs. 2, 3). Together, these data suggest that Tf sat does not induce hepcidin expression by stimulating Bmp6 mRNA expression. Next, we analyzed the intracellular signaling mediators and targets of BMP6 signaling (P-Smad1/5/8 protein, Id1 mRNA, and Smad7 mRNA) in the liver after both chronic and acute iron administration. In the chronic iron administration setting, hepatic P-Smad1/5/8 protein (Fig. 5A), Id1 mRNA (Fig. 6A), and Smad7 mRNA (Fig.