Page 88 - 2021_02-Haematologica-web

R. Agoro et al.
could have provided evidence for a possible mechanism for the effect of cFGF23 on iron absorption during hypo- ferremia.
Our study also demonstrates that disruption of FGF23 signaling prevents LPS-induced hypoferremia by decreas- ing hepcidin (Figure 5D and E) and abrogating iron seques- tration in liver and spleen (Figure 6). Suppression of hep- cidin by cFGF23 in LPS-treated mice appears to be inde- pendent of the IL6/STAT3 pathway (Online Supplementary Figure S3). However, cFGF23 significantly decreases induc- tion of Bmp6 transcription by LPS (Online Supplementary Figure S5), suggesting that cFGF23 may be affecting the BMP6/SMAD pathway, an important transcriptional path- way for hepcidin regulation. In support of this, our data show that splenic expression of erythroferrone (Erfe), a negative regulator of hepcidin, is decreased in mice treated with LPS, whereas mice treated with the cFGF23 under LPS conditions have significantly higher Erfe expression (Online Supplementary Figure S6). Erfe has been shown to suppress BMP/SMAD signaling in vivo and in vitro, and to inhibit hepcidin induction by BMP6.73 Thus, our data sug- gest that cFGF23 decreases hepcidin induction by LPS by increasing Erfe, and possibly suppressing the BMP6/SMAD pathway.
We recently described that inhibition of FGF23 signaling using 10 mg/kg of the C-tail FGF23 rescues anemia and iron deficiency in a CKD mouse model by stimulating erythro- poiesis, increasing serum iron and ferritin levels, and atten- uating chronic inflammation.29 In this experimental condi- tion, where we observed highly elevated FGF23 levels and absolute iron deficiency characterized by a decrease in circu- lating iron29 and iron stores (Online Supplementary Figure S7), the C-tail FGF23 significantly reduced circulating FGF23 lev- els and improved the impaired iron distribution by elevating circulating iron and ferritin levels29 and increasing liver iron content (Online Supplementary Figure S7). In the present study, we show that even by decreasing the concentration
of the C-tail FGF23 to 1 mg/kg, Epo is still induced at the steady state and after LPS (Figure 7D), and this concentration of the C-tail FGF23 is also sufficient to alleviate LPS-induced functional iron deficiency (Figure 6). However, this concen- tration is not sufficient to attenuate hepatic IL-6 and IL-1α induction (Figure 5A and C), but it did significantly decrease TNF-α expression (Figure 5B). This is consistent with other studies showing that recombinant FGF23 and increased pro- duction of endogenous FGF23, such as in Hyp mice, stimu- late TNF-α expression in macrophages.46 Thus, inhibition of FGF23 signaling represents a therapeutic target against absolute and functional iron deficiency pathologies.
Taken together, our results show for the first time that FGF23 signaling inhibition alleviates LPS-induced acute hypoferremia.
Disclosures
No conflicts of interests to disclose.
Contributions
RA developed and directed the project, performed research and data analysis, and wrote the manuscript; MYP and CLH per- formed research, data and statistical analyses; SJ and AG per- formed research; GC purified and crystallized the C-tail FGF23 and analyzed its crystal structure; MM designed, refined, ana- lyzed, and interpreted the crystal structure of the C-tail FGF23; DS developed and directed the project, performed data analysis, supervised the study, and wrote the manuscript.
Acknowledgments
The authors would like to thank Anna Montagna and Elena Healy for technical assistance.
Funding
This work was supported by funds from the US Department of Defense (W81XWH-16-1-0598) to DS, and the NIH (NIDCR) to MM (DE 13686).
References
1. Gozzelino R, Arosio P. Iron homeostasis in health and disease. Int J Mol Sci. 2016;17(1).
2. Layoun A, Huang H, Calve A, Santos MM. Toll-like receptor signal adaptor protein MyD88 is required for sustained endotox- in-induced acute hypoferremic response in mice. Am J Pathol. 2012;180(6):2340-2350.
3. Schmidt PJ. Regulation of iron metabolism by hepcidin under conditions of inflamma- tion. J Biol Chem. 2015;290(31):18975- 18983.
4. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood. 2003;102(3):783-788.
5.Kemna E, Pickkers P, Nemeth E, van der Hoeven H, Swinkels D. Time-course analy- sis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS. Blood. 2005;106(5):1864-1866.
6. Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a puta- tive mediator of anemia of inflammation, is a type II acute-phase protein. Blood. 2003;101(7):2461-2463.
7.Nemeth E, Tuttle MS, Powelson J, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306(5704):
2090-2093.
8.Ganz T, Nemeth E. Iron homeostasis in
host defence and inflammation. Nat Rev
Immunol. 2015;15(8):500-510.
9. Weiss G. Iron metabolism in the anemia of
chronic disease. Biochim Biophys Acta.
2009;1790(7):682-693.
10. Babitt JL, Lin HY. Mechanisms of anemia in
CKD. J Am Soc Nephrol. 2012;23(10):1631-
1634.
11. Wolf M. Update on fibroblast growth fac-
tor 23 in chronic kidney disease. Kidney
Int. 2012;82(7):737-747.
12. David V, Martin A, Isakova T, Spaulding C,
Qi L, Ramirez V, et al. Inflammation and functional iron deficiency regulate fibrob- last growth factor 23 production. Kidney Int. 2016;89(1):135-146.
13. Farrow EG, Yu X, Summers LJ, et al. Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) pheno- type in fibroblast growth factor-23 (Fgf23) knock-in mice. Proc Natl Acad Sci U S A. 2011;108(46):E1146-55.
14. White KE, Carn G, Lorenz-Depiereux B, Benet-Pages A, Strom TM, Econs MJ. Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int. 2001;60(6):2079-2086.
15.Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF23 as a
causative factor of tumor-induced osteo- malacia. Proc Natl Acad Sci U S A. 2001; 98(11):6500-6505.
16. Perwad F, Zhang MY, Tenenhouse HS, Portale AA. Fibroblast growth factor 23 impairs phosphorus and vitamin D metab- olism in vivo and suppresses 25-hydroxyvi- tamin D-1alpha-hydroxylase expression in vitro. Am J Physiol Renal Physiol. 2007;293(5):F1577-1583.
17.
Shimada T, Hasegawa H, Yamazaki Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19(3):429-435.
18.Sitara D, Kim S, Razzaque MS, et al. Genetic evidence of serum phosphate- independent functions of FGF-23 on bone. PLoS Genet. 2008;4(8):e1000154.
19. Sitara D, Razzaque MS, Hesse M, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol. 2004;23(7):421-432.
20. Sitara D, Razzaque MS, St-Arnaud R, et al. Genetic ablation of vitamin D activation pathway reverses biochemical and skeletal anomalies in Fgf-23-null animals. Am J Pathol. 2006;169(6):2161-2170.
21. Eicher EM, Southard JL, Scriver CR, Glorieux FH. Hypophosphatemia: mouse
402
haematologica | 2021; 106(2)