Functional but abnormal adult erythropoiesis in the absence of the stem cell leukemia gene

Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.     

Hematopoiesis: how does it happen?

Hematopoiesis entails the generation of stem cells, the proliferation and maintenance of multipotential progenitors, and lineage commitment and maturation. During the past year, critical components of these steps have been defined. Notable are gene-targeting experiments in mice in which one or more hematopoietic lineages have been shown to be ablated upon inactivation of several nuclear regulatory proteins (GATA-2, Tal-1/SCL, Rbtn2/LMO2, PU.1, Ikaros, E2A, and Pax-5), and experiments that establish GATA-1 as a factor capable of programming at least three lineages (erythroid, thrombocytic, and eosinophilic) from a transformed avian progenitor cell.     

A non-apoptotic role for Fas/FasL in erythropoiesis

Issues remain to be elucidated in the developmental regulation of erythropoiesis. In particular the role of Fas, a member of the tumor necrosis factor family of receptors despite much work remains unclear. During erythropoiesis, Fas is expressed at low levels on erythroblasts. For most cell types, Fas to FasL interaction causes apoptotic cell death via caspase activation. Here, we show that in humans, early erythroid progenitors are refractory to apoptosis triggered through Fas. Further during early human erythropoiesis, Fas triggered caspase activation provides a positive stimulus for erythroid maturation, and does not alter cellular proliferation or trigger apoptosis.     

A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress

The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members--Atg4C and Atg4D--contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (ΔN63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, where it undergoes further processing to a ~42 kDa mitochondrial form. Interestingly, caspase cleavage is not needed for Atg4D mitochondrial import, because ~42 kDa mitochondrial Atg4D is observed in cells treated with caspase inhibitors and in cells expressing caspase-resistant Atg4D (DEVA(63)). Using HeLa cell lines stably expressing ΔN63 Atg4D, we showed that mitochondrial Atg4D sensitizes cells to cell death in the presence of the mitochondrial uncoupler, CCCP, and that mitochondrial cristae are less extensive in these cells. We further showed that the organization of mitochondrial cristae is altered during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ΔN63 Atg4D, and that these cells have elevated levels of mitochondrial reactive oxygen species (ROS) during late stages of erythropoiesis. Together these data suggest that the import of Atg4D during cellular stress and differentiation may play important roles in the regulation of mitochondrial physiology, ROS, mitophagy and cell viability.