Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary Cultured Human Erythroid Precursor Cells From Apoptosis

Abstract

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34+ cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 μmol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V–binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

Quiescent CD34+ early erythroid progenitors are resistant to several erythropoietic ‘inhibitory’ cytokines; role of FLIP

“…However, despite the fact that IFNs efficiently activated the STAT-5 protein in erythroid cells, they induced apoptosis, which indicates that activation of STAT proteins alone is probably not sufficient to protect erythroid cells from undergoing apoptosis. Similarly, in our previous study, we demonstrated that the PI-3K-AKT axis, which plays an important antiapoptotic role in early erythroid cells (Haseyama et al, 1999;Myklebust et al, 2002;Bouscary et al, 2003), was activated in both CD34 + cells and early erythroblasts by KL. Furthermore, we noticed that this potential prosurvival signalling by KL was not negatively affected at the time of KL stimulation by any of the inflammatory cytokines used in our studies.…”

Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Akt1, Bad, and caspase‐mediated pathways

“…Chong et al Erythropoietin and neuronal apoptotic pathways 1115 during these injury paradigms. In regards to EPO, we demonstrate that it independently phosphorylates and increases the activation of Akt1 in neurons similar to its capacity to activate Akt1 in erythroid progenitors (Haseyama et al, 1999;Uddin et al, 2000). EPO appears to be dependent upon the activation of Akt1 to prevent neuronal injury, since prevention of Akt1 phosphorylation with inhibitors of PI-3K activity significantly reduces the ability of EPO to protect neurons.…”

Biological significance of MAPK, AKT and JAK‐STAT protein activation by various erythropoietic factors in normal human early erythroid cells

“…Interestingly, although both KL and EpO inhibited apoptosis in human BFU‐E‐derived cells (Fig 3), only KL strongly stimulated AKT phosphorylation (Fig 5 ). To explain this discrepancy, we investigated whether EpO activates The JAK‐STAT pathway, which was recently reported to play, similar to the PI‐3K‐AKT axis, an important role in inhibiting apoptosis in human haematopoietic cells (Haseyama et al , 1999). To address this issue, we examined both the activation of JAK‐2 (Fig 6A) as well tyrosine phosphorylation of STAT‐5 (Fig 6B and C).…”