Page 94 - 2019_09-HaematologicaMondo-web

J. Liu et al.
idly increased hepatic hepcidin mRNA at 6 h, and the peak was observed at 24 h for compound 93 and at 48 h for compounds 156 and 165 with >2-fold increases (P<0.05) (Figure 2B). Thereafter, hepcidin declined and returned to the baseline level at 96 h, suggesting that these com- pounds could be dosed every 3-4 days for long-term administration. Accordingly, serum hepcidin was increased at 6 h after administration of compounds 93, 156 and 165, and the peak was detected at 24 h for com- pounds 93 and 165 and at 48 h for compound 156 (P<0.05) (Figure 2C). In another experiment, mice were treated with the compounds at a dose of 30 mg/kg body weight every 3 days for a total of 8 or 12 days. In agreement with the previous observations, treatment with compounds 93, 156 and 165 led to persistent stimulation of hepcidin expression over the 12-day period, especially for com- pounds 93 and 165 with more than 3-fold induction of hepatic hepcidin mRNA 12 days after administration (P<0.05) (Figure 2B). In parallel to the mRNA changes, similar increases of serum hepcidin relative to the levels in untreated mice were demonstrated (P<0.05) (Figure 2C). Hepcidin induction resulted in reduced serum iron and increased splenic iron content (P<0.05) (Figure 2D, E). Iron staining of spleen sections confirmed the increase of splenic iron content, particularly in macrophages (in blue, indicated by arrows), compared to the content in untreat- ed mice (Figure 2F). In addition, these compounds were evaluated in mice at a lower dose, 10 mg/kg body weight. As shown in Online Supplementary Figure S5, similar results were observed for these three compounds in modulating hepcidin expression and body iron redistribution. With respect to time of peak and duration of effect. these com- pounds were still active at the lower doses, but less potent than at the higher doses.
To screen for potential toxicities of these compounds, liver, spleen, kidney, lung, heart and bone marrow speci- mens were subjected to histological analysis. No toxic changes were observed after 24 h and 12 days in any of these organs from mice challenged by compounds 93, 156 and 165 at a dose of 30 mg/kg body weight (Online Supplementary Figure S6). No impairment of spontaneous activities (e.g., feeding or movement) was observed. Moreover, no significant changes were found in serum aspartate aminotransferase, alanine aminotransferase or lactate dehydrogenase (Online Supplementary Figure S7A-C).
Inflammation increases hepcidin expression,23 in large part through interleukin-6, so we explored the possibility that our compounds acted by increasing inflammatory mediators. As shown in Online Supplementary Figure S7D, serum interleukin-6 was not detectable in serum before or after compound administration for 24 h and 12 days. Bacterial lipopolysaccharide, at a dose of 5 mg/kg body weight, was used as a positive control stimulant of inter- leukin-6. Furthermore, hepatic inflammation was deter- mined by measuring serum amyloid A1 (SAA1), a down- stream target of interleukin-6, and a very sensitive marker of inflammation. Consistently, Saa1 mRNA levels were not significantly altered in livers from mice treated with these compounds (Online Supplementary Figure S7E), and neither was another inflammatory marker, serum tumor necrosis factor-a (Online Supplementary Figure S7F). In an analysis of the complete blood count, no increase of white blood cells was observed in peripheral blood 24 h follow- ing administration of a single dose or multiple doses for 12 days (Online Supplementary Figure S8). Collectively, our
findings ruled out that compounds 93, 156 and 165 affect hepcidin through inflammatory mechanisms.
Compounds 93, 156 and 165 target SMAD1/5/8 signaling to promote hepcidin expression
Iron/BMP6–SMAD1/5/8 signaling controls hepcidin expression depending on iron status24,25 and we therefore examined whether our compounds modulated this key pathway. As shown in Figure 3A, the level of phosphory- lated SMAD1/5/8 (P-SMAD1/5/8) was increased in livers of mice treated with the three compounds, with com- pound 156 causing the greatest increase in P-SMAD1/5/8. To interpret this finding, the upstream regulator of P- SMAD1/5/8, transmembrane protease serine 6 (TMPRSS6), and its downstream target genes, inhibitor of DNA binding 1 (ID1) and SMAD family member 7 (SMAD7), were examined by qRT-PCR. The mRNA levels of Tmprss6 were suppressed by more than 50% in liver specimens from mice treated with the three compounds, relative to the levels in untreated mice (P<0.05) (Figure 3B). In contrast, Id1 and Smad7 were induced by approxi- mately 1.5 to 2.5 fold in livers from treated mice, relative to the levels in the untreated controls (P<0.05) (Figure 3B). Transferrin receptor 2 (TFR2), HFE, hemojuvelin, TMPRSS6 and bone morphogenetic protein (BMP) recep- tors interact to activate hepcidin expression by upregulat- ing SMAD1/5/8 signaling. The BMP/BMP receptor inter- action enhances SAMD1/5/8 phosphorylation and TMPRSS6 downregulates hepcidin expression by cleaving hemojuvelin and other proteins in the complex.26,27 Consistent with the mRNA changes, the protein levels of TMPRSS6 were reduced in treated mice relative to those in untreated controls (Figure 3A). Additionally, phospho- rylated ERK1/2 (P-ERK1/2) was recently found to repress hepcidin expression by suppressing SMAD1/5/8 phospho- rylation.28 Here, the protein value of P-ERK1/2 was also diminished upon treatment with the compounds (Figure 3A). These results collectively suggest that compounds 93, 156 and 165 increased hepcidin expression by suppressing TMPRSS6 and ERK1/2 and thereby decreasing their inhibitory effects on SMAD1/5/8 phosphorylation.
We next investigated these effects using hepatocytic cell lines, murine Hepa 1-6. Consistent with the in vivo results, compounds 93, 156 and 165 greatly induced hepcidin expression in Hepa 1-6 cells (P<0.05) (Figure 3C) and increased the expression levels of its downstream targets, Id1 and Smad7 (P<0.05) (Figure 3D). Mechanistically, the compounds suppressed ERK1/2 phosphorylation and decreased TMPRSS6 concentration (Figure 3E). Of note, these three compounds differentially suppressed P- ERK1/2 versus TMPRSS6 protein concentrations, suggest- ing that they may differ in their mechanisms of effect on SMAD1/5/8 signaling (Figure 3E).
Compounds 93, 156 and 165 target erythroid regulators to strengthen hepcidin
Given that erythropoietin and erythropoiesis factors, growth differentiation factor 15 (GDF15), twisted gastru- lation BMP signaling modulator 1 (TWSG1) and erythro- ferrone (ERFE), are also involved in regulating hepcidin expression,29-32 we examined these regulators. As shown in Online Supplementary Figure S9, serum erythropoietin level was unchanged in mice after treatment with the com- pounds for 24 h and 48 h. However, the expression levels of Erfe, Gdf15 and Twsg1 were significantly repressed in
1772
haematologica | 2019; 104(9)