Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency

Abstract

Haem-regulated eIF2alpha kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.

Fig 1

Erythroid differentiation of FL in iron deficiency and HRI deficiency. (A) FACS analysis of the erythroid differentiation of E14·5 FL cells. (B) Blood smears of E14·5 embryos. Arrows indicate globin inclusions in Ko-Fe reticulocytes. (C) Cell cycle and apoptosis in E14·5 FL cells. Results are presented as mean ± SD (n = 6).

Fig 2

Expression of Gata1 and Zfpm1 in FL under iron and HRI deficiencies. (A) Expression of Gata1 mRNA. (B) Expression of Zfpm1 mRNA. (C) Expression of Gata1 and Zfpm1 proteins. The results of mRNA expression are presented in mean ± SD (n = 4). The intensities of the autoradiograms in the Western blots were quantified by Alpha EaseFC software. Expression of proteins was normalized with eIF2α. Expression of proteins in Wt+Fe is defined as 1. The normalized values are shown above the autoradiograms.

Fig 3

Expression of Gata1 and Zfpm1 in sorted cell populations of FL under iron and HRI deficiencies. (A) Expression of Gata1 and Zfpm1 mRNAs in three sorted populations. Results are presented in mean ± SD (n = 4). (B) Expression of GATA1 and Zfpm1 proteins in sorted cells. Quantification of Western blots was as described in the legend of Fig 2.

Fig 4

Upregulation of Eif2ak1 during erythroid differentiation. (A) Expression of Eif2ak1 mRNA in three sorted populations. (B) Western blot analyses of HRI, eIF2αP, and eIF2α. Expression of proteins in P1 + P2 of Wt is defined as 1. Quantification of Western blots was as described in the legend of Fig 2.