Coordinate regulation of liver ferroportin degradation and de novo synthesis 1 determines serum iron levels in mice

The iron hormone hepcidin is transcriptionally activated by iron or inflammation via distinct, partially overlapping pathways. We addressed how iron affects inflammatory hepcidin levels and the ensuing hypoferremic response. Dietary iron overload did not mitigate hepcidin induction in LPS-treated wt mice but prevented effective inflammatory hypoferremia. Likewise, LPS modestly decreased serum iron in hepcidin-deficient Hjv-/- mice, model of hemochromatosis. Synthetic hepcidin triggered hypoferremia only in control but not iron-loaded wt animals. Furthermore, it dramatically decreased hepatic and splenic ferroportin in Hjv-/- mice on standard or iron-deficient diet, but only triggered hypoferremia in the latter. Mechanistically, iron induced liver ferroportin mRNA translation, thereby antagonizing hepcidin-mediated hypoferremia. Conversely, iron depletion suppressed de novo ferroportin synthesis in Hjv-/- livers, allowing exogenous hepcidin to cause hypoferremia. Consequently, prolonged LPS treatment eliminating ferroportin mRNA permitted hepcidin-mediated hypoferremia in iron-loaded mice. Thus, liver ferroportin mRNA translation is critical determinant of serum iron and finetunes hepcidin-dependent functional outcomes. Our data indicate a crosstalk between hepcidin/ferroportin and IRE/IRP systems. Moreover, they suggest that hepcidin supplementation therapy is more efficient combined with iron depletion.

genetic iron overload models and appeared to decrease in Hjv-/-mice on IDD (Fig. 2D). LIC was 94 substantially reduced in Hjv-/-mice in response to IDD, but also compared to wt mice on HID 95 (Fig. 2E). The quantitative LIC data are corroborated histologically by Perls staining (Fig. 2M). 96 Dietary iron loading increased splenic iron in wt mice and confirmed that Hjv-/-mice fail to 97 retain iron in splenic macrophages (Fig. 2N). As expected, hepcidin and Hamp mRNA were 98 induced in HID-fed wt mice and were low in Hjv-/-mice on SD, and further suppressed to 99 undetectable levels following IDD intake ( Fig. 2F-G). 100 LPS reduced serum iron and transferrin saturation in hyperferremic wt mice on HID and 101 Hjv-/-mice on SD or IDD, but not below baseline of control wt mice on SD, the only animals 102 that developed a robust hypoferremic response ( Fig. 2A-B). The LPS treatment was associated 2C-E). Notably, LPS-treated wt mice on HID and Hjv-/-mice on IDD exhibited dramatic 106 6 differences in Hamp mRNA but similar blunted hypoferremic response to the acute 107 inflammatory stimulus. Thus, the profound hepcidin induction in iron-loaded wt mice cannot 108 reduce serum iron below that of iron-depleted Hjv-/-mice with negligible hepcidin. 109 The LPS treatment strongly suppressed liver Fpn(+IRE) (ferroportin IRE isoform)

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To assess the potential role of hepcidin, we first analyzed ferroportin in the liver, an 116 organ that contributes to iron sequestration during inflammation. Immunohistochemical staining 117 of liver sections revealed strong ferroportin expression in Kupffer cells, predominantly in 118 periportal areas, under all experimental conditions (Figs. 3A and S1). Hepatocellular staining is 119 also evident in the iron overload models, mostly in periportal hepatocytes (Fig. S1), and in line 120 with recent data (12). LPS triggered a shift of ferroportin in Kupffer cells from elongated 121 dendritic branches to round intracellular compartments. LPS did not affect the intensity or 122 distribution of Kupffer cell ferroportin in Hjv-/-mice (Fig. S1), in agreement with previous 123 findings (9). 124 We further analyzed ferroportin in liver homogenates by Western blotting. Levels of 125 biochemically detectable liver ferroportin differed substantially between wt and Hjv-/-mice. 126 Thus, they were relatively low in the former and highly induced in the latter (Fig. 3B), 127 independently of iron load. The differences were more dramatic compared to those observed by 128 7 immunohistochemistry (Figs. 3A and S1). Conceivably, the strong ferroportin signal in Western 129 blots from Hjv-/-liver homogenates reflects high ferroportin expression in hepatocytes, the 130 predominant cell population. Yet, hepatocellular ferroportin is less visible by 131 immunohistochemistry because the signal is substantially weaker compared to that in Kupffer 132 cells. Interestingly, the LPS treatment profoundly suppressed total liver ferroportin in Hjv-/-133 mice on SD but not IDD, while it modestly affected it in wt mice (Fig. 3B). These data are 134 consistent with negative regulation of ferroportin by residual LPS-induced hepcidin in Hjv-/-135 mice on SD, which could explain the small drop in serum iron and transferrin saturation under 136 these acute inflammatory conditions, as reported (9). However, liver ferroportin remained 137 detectable and apparently functional, as it did not allow significant iron sequestration and 138 hypoferremia. Notably, a lack of robust hypoferremic response is also evident in LPS-treated wt 139 mice on HID, despite maximal hepcidin and minimal liver ferroportin levels.

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Western blotting confirmed that total liver ferroportin is highly induced in Hjv-/-mice 183 (Fig. 5B). Again, the signal intensity can be attributed to protein expressed in hepatocytes. The  Taken together, our data suggest that synthetic hepcidin overcomes endogenous hepcidin 193 deficiency in Hjv-/-mice. However, it only triggers hypoferremia in these animals following 194 relative iron depletion. On the other hand, in iron-loaded wt mice with already high endogenous 195 hepcidin, excess synthetic hepcidin fails to promote hypoferremia. 196 Iron-dependent regulation of ferroportin mRNA translation in the liver. We hypothesized 197 that the functional outcomes of exogenous hepcidin may not merely depend on its capacity to 198 degrade tissue ferroportin, but also on iron-dependent ferroportin regeneration via de novo 199 synthesis. We addressed this in the liver, which can export iron to plasma from ferroportin-  Livers of wt mice on HID had significantly (p<0.01) higher iron content compared to Hjv-/-mice 215 on IDD. Iron redox speciation analysis by CE-ICP-MS revealed a profound increase in Fe 2+ /Fe 3+ 216 ratios in livers of Hjv-/-mice on SD, which was corrected by dietary iron depletion (Fig. 6C).

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Nevertheless, there was no difference in Fe 2+ /Fe 3+ ratios among livers of wt mice on SD or HID, 218 and Hjv-/-mice on IDD. We conclude that an increase in total iron content, rather than excessive 219 accumulation of redox active Fe 2+ drives Fpn(+IRE) (and Fth1) mRNA translation in the liver.

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Effective hypoferremic response to hepcidin in mouse models of iron overload following 221 prolonged LPS treatment that eliminates ferroportin mRNA. We reasoned that complete   We used a ~200-fold excess of synthetic hepcidin to directly assess its capacity to divert 258 iron traffic in iron-loaded mice. Hepcidin injection caused hypoferremia in control wt mice on 259 SD and significantly reduced serum iron in wt mice on HID and Hjv-/-mice on SD, but not 260 below baseline (Fig. 4). Thus, synthetic hepcidin failed to cause hypoferremia in iron overload 261 models with either high or low endogenous hepcidin. Importantly, synthetic hepcidin promoted 262 13 robust hypoferremia in relatively iron-depleted Hjv-/-mice on IDD, with undetectable 263 endogenous hepcidin. It should be noted that synthetic hepcidin had similar effects on tissue 264 ferroportin among wt or Hjv-/-mice, regardless of iron diet (Fig. 5). It reduced intensity of the 265 ferroportin signal in Kupffer cells and splenic macrophages of wt mice without significantly 266 affecting biochemically detectable total protein levels. In addition, it dramatically reduced total 267 ferroportin in the liver and spleen of Hjv-/-mice. However, in all experimental settings there was 268 residual tissue ferroportin, which appears to be functionally significant. 269 We reasoned that at steady-state, tissue ferroportin may consist of fractions of newly 270 synthesized protein, and protein that is en route to hepcidin-mediated degradation. Conceivably, 271 the former may exhibit more robust iron export activity, at least before its iron channel gets Hjv-/-mice, in line with the restoration of hepcidin-mediated hypoferremic response (Fig. 4). and D), it is implausible that NTBI uptake by Zip14 and/or DMT1 substantially contributes to 306 inflammatory hypoferremia. Lcn2 is an acute phase protein that can sequester intracellular iron 307 bound to catecholate siderophores (29), and is more likely to transport iron to tissues during infection. In any case, synthetic hepcidin did not affect expression of iron transporters (Fig. S4).

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This excludes the possibility for a synergistic effect on LPS-induced tissue iron uptake that could 310 promote effective hypoferremia in the iron overload models.

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In conclusion, our data reveal a crosstalk between the hepcidin pathway and the IRE/IRP