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Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.
Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.