Bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in a mouse model of iron deficiency anemia.

Iron deficiency is a potent stimulator of fibroblast growth factor 23 (FGF23), a hormonal regulator of phosphate and vitamin D metabolism, that is classically thought to be produced by bone-embedded osteocytes. Here, we show that iron-deficient transmembrane serine protease 6 knockout (Tmprss6-/-) mice exhibit elevated circulating FGF23 and Fgf23 messenger RNA (mRNA) upregulation in the bone marrow (BM) but not the cortical bone. To clarify sites of Fgf23 promoter activity in Tmprss6-/- mice, we introduced a heterozygous enhanced green fluorescent protein (eGFP) reporter allele at the endogenous Fgf23 locus. Heterozygous Fgf23 disruption did not alter the severity of systemic iron deficiency or anemia in the Tmprss6-/- mice. Tmprss6-/-Fgf23+/eGFP mice showed green fluorescence in the vascular regions of BM sections and showed a subset of BM endothelial cells that were GFPbright by flow cytometry. Mining of transcriptomic data sets from mice with normal iron balance revealed higher Fgf23 mRNA in BM sinusoidal endothelial cells (BM-SECs) than that in other BM endothelial cell populations. Anti-GFP immunohistochemistry of fixed BM sections from Tmprss6-/-Fgf23+/eGFP mice revealed GFP expression in BM-SECs, which was more intense than in nonanemic controls. In addition, in mice with intact Tmprss6 alleles, Fgf23-eGFP reporter expression increased in BM-SECs following large-volume phlebotomy and also following erythropoietin treatment both ex vivo and in vivo. Collectively, our results identified BM-SECs as a novel site for Fgf23 upregulation in both acute and chronic anemia. Given the elevated serum erythropoietin in both anemic models, our findings raise the possibility that erythropoietin may act directly on BM-SECs to promote FGF23 production during anemia.

NCOA4 is regulated by HIF and mediates mobilization of murine hepatic iron stores after blood loss.

The mechanisms by which phlebotomy promotes the mobilization of hepatic iron stores are not well understood. NCOA4 (nuclear receptor coactivator 4) is a widely expressed intracellular protein previously shown to mediate the autophagic degradation of ferritin. Here, we investigate a local requirement for NCOA4 in the regulation of hepatic iron stores and examine mechanisms of NCOA4 regulation. Hepatocyte-targeted Ncoa4 knockdown in nonphlebotomized mice had only modest effects on hepatic ferritin subunit levels and nonheme iron concentration. After phlebotomy, mice with hepatocyte-targeted Ncoa4 knockdown exhibited anemia and hypoferremia similar to control mice with intact Ncoa4 regulation but showed a markedly impaired ability to lower hepatic ferritin subunit levels and hepatic nonheme iron concentration. This impaired hepatic response was observed even when dietary iron was limited. In both human and murine hepatoma cell lines, treatment with chemicals that stabilize hypoxia inducible factor (HIF), including desferrioxamine, cobalt chloride, and dimethyloxalylglycine, raised NCOA4 messenger RNA. This NCOA4 messenger RNA induction occurred within 3 hours, preceded a rise in NCOA4 protein, and was attenuated in the setting of dual HIF-1α and HIF-2α knockdown. In summary, we show for the first time that NCOA4 plays a local role in facilitating iron mobilization from the liver after blood loss and that HIF regulates NCOA4 expression in cells of hepatic origin. Because the prolyl hydroxylases that regulate HIF stability are oxygen- and iron-dependent enzymes, our findings suggest a novel mechanism by which hypoxia and iron deficiency may modulate NCOA4 expression to impact iron homeostasis.

Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice.

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.

A novel splicing mutation of the alpha-spectrin gene in the original hereditary pyropoikilocytosis kindred.

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia due to abnormalities of the red blood cell (RBC) membrane skeleton. In the original HPP kindred, there is compound heterozygosity for an allele encoding a structural variant of alpha-spectrin (L207P) and an alpha-spectrin allele associated with a defect in alpha-spectrin production. To identify the molecular defect in the production-defective allele, reticulocyte alpha-spectrin cDNA from one of the original HPP patients was analyzed. Transcripts from the production-defective, non-L207P allele demonstrated a pattern of abnormal splicing between exons 22 and 23, resulting in insertion of intronic fragments with an in-frame premature termination codon. A G to A substitution at position +5 of the donor consensus splice site of IVS 22 was identified in the inserts. Following gene transfer into tissue culture cells, there was complete absence of normally spliced alpha-spectrin gene transcripts derived from a minigene containing the IVS 22 +5 mutation.