Hypoxia Induces Hemoglobin Production in Human Erythroleukemia Cells by Increasing the Secretion of Transforming Growth Factor -β1 and the Expression of Mitoferrin.

Hypoxia induces erythropoiesis by stimulating erythropoietin secretion from kidney in vivo. Hypoxia also stimulates hemoglobin production in cultured murine erythroleukemia cells, but its mechanism is still unknown. We have found that hypoxia induces hemoglobin production in human erythroleukemia cell line, YN-1, and have analyzed the mRNA expression profile of YN-1 cells under hypoxic conditions (1% O₂, 5% CO₂ and 94% N₂) using DNA microarrays, and compared it with that of normoxic conditions (5% CO₂ and 95% room air). For DNA microarray analysis, total RNA was prepared from YN-1 cells, which were incubated under hypoxic or normoxic conditions for 6 hours. We thus found the increased mRNA expression for several genes involved in erythroid differentiation, including transforming growth factor β1 (TGF-β1) and mitoferrin (mitochondrial iron importer, previously known as SLC25A37). Among these candidates, we have focused on TGF-β1, since TGF-β1 is able to induce erythroid differentiation of several erythroleukemia cell lines, including YN-1 cells and K562 cells. We initially confirmed the increased mRNA expression of TGF-β1 under hypoxic conditions by Northern blotting analysis in YN-1 cells and YN-1-0A cells, which is a daughter cell line of YN-1 cells and is maintained in serum-free medium. Likewise, the hypoxic induction of mitoferrin mRNA in YN-1 cells was confirmed by Real Time PCR analysis. Then, using ELISA system, we measured the TGF-β1 concentration in the culture medium of YN-1-0A cells, and have found that hypoxia stimulates TGF-β1 secretion from the cells. Notably, the hypoxia-mediated increases in hemoglobin production and γ-globin mRNA expression were significantly suppressed in YN-1-0A cells, when TGF-β1 activity in the culture medium was neutralized with anti-TGF-β1 antibody. These results suggest that hypoxia stimulates TGF-β1 secretion from YN-1 cells and YN-1-0A cells, which in turn induces the terminal differentiation of erythroid cells in an autocrine manner. Interestingly, in contrast to the hemoglobin production, addition of TGF-β1 into the culture medium did not influence mitoferrin expression, suggesting that hypoxia induces mitoferrin expression by a mechanism independent of TGF-β1. Since several putative hypoxia responsive elements are present in the promoter region of the human mitoferrin gene, hypoxia inducible factor 1 (HIF-1) may be involved in inducible expression of mitoferrin under hypoxic conditions. The hypoxia-mediated induction of mitoferrin expression is important for effective supply of iron for heme biosynthesis in erythroid cells, as several missense mutations of the zebrafish mitoferrin gene are associated with hypochromic anemia and erythroid maturation arrest (Shaw G.C. et al., Nature, vol.440, p96–100, 2006). Taken together, we suggest that hypoxia stimulates hemoglobin production in erythroid cells by coordinately increasing the iron supply into mitochondria and the γ-globin expression, which may be achieved in part by the induction of mitoferrin and the enhanced secretion of TGF-β1, respectively.