Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway.

Erythropoietin (Epo) stimulation of its receptor's downstream signaling pathways and optimum function of GATA-1 transcription factor are both essential for normal erythroid cell development. Epo-receptor (EpoR) signaling and GATA-1 regulate proliferation, survival, differentiation, and maturation of erythroid cells. Whether any signal that is generated by EpoR targets GATA-1 or affects GATA-1 transcriptional activity is not known. Here, we demonstrate that stimulation of EpoR results in phosphorylation of GATA-1 at serine 310 (S310) in primary fetal liver erythroid progenitors and in cultured erythroid cells. We show that phosphorylation of GATA-1 is important for Epo-induced maturation of fetal liver erythroid progenitor cells. The PI3-kinase/AKT signaling pathway is identified as a mediator of Epo-induced phosphorylation of GATA-1. AKT serine threonine kinase phosphorylates GATA-1S310 in vitro and in erythroid cells and enhances GATA-1 transcriptional activity. These data demonstrate that EpoR signaling phosphorylates GATA-1 and modulates its activity via the PI3-kinase/AKT signaling pathway.

AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism.

Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that overexpression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6, formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 overexpression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any fatty acids into phospholipids (PLs) or triacylglycerides (TAGs) was unknown. Here we show that Acsl6 overexpression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, oleic acid (OA) and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 overexpression. Additionally, the level of total cellular PLs was increased in Acsl6 overexpressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 overexpression increased the incorporation of [(14)C]-labeled OA, AA, or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of fatty acids into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its overexpression increases DHA and AA internalization primarily during the first 24 h of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth.

Acyl-CoA synthetase 2 overexpression enhances fatty acid internalization and neurite outgrowth.

During neurodevelopment neurons increase phospholipid synthesis to generate additional plasma membrane that makes up the growing neurites. Compared with most cell types, neurons contain a high percentage of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and docosahexaenoic acid (DHA). By utilizing PC12 cell lines as a model neuronal cell line, we examined the internalization rate of AA, DHA, and non-essential oleic acid (OA), as well as their effects on neurite outgrowth. When wild type cells were differentiated, the rate of AA and DHA internalization increased 50% more than the rate of OA internalization. When media were supplemented with AA or DHA, the average neurite length was increased by approximately 40%, but supplementation with the same amount of OA had no effect. We also increased the levels of acyl-CoA synthetase-1 (ACS1) and ACS2 proteins to determine whether they contribute to PUFA internalization or neurite outgrowth. Overexpression of ACS1 increased the rate of OA internalization by 55%, and AA and DHA uptake was increased by 25%, but there was no significant change in neurite outgrowth. In ACS2-overexpressing cells, in contrast, the rate of OA internalization increased by 90%, AA by 115%, and DHA by 70%. The average aggregate neurite length in ACS2-overexpressing cells was increased by approximately 40% when the media were supplemented with PUFAs, but there was no change with OA supplementation. Taken together, these results support the hypotheses that ACSs are rate-limiting for fatty acid internalization and that ACS2 enhances neurite outgrowth by promoting PUFA internalization.

Cytokines and BCR-ABL mediate suppression of TRAIL-induced apoptosis through inhibition of forkhead FOXO3a transcription factor.

Cytokine-provided survival signals are known to suppress apoptosis through inhibition of mitochondrial pathways that involve Bcl-2 family members. Here we show that in hematopoietic cells, cytokines also regulate death receptor-mediated pathways. We demonstrate that hematopoietic cytokines such as IL-3 and erythropoietin in normal cells, as well as BCR-ABL oncoprotein in transformed cells, inhibit transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using small interfering RNAs, we show that the inhibition of TRAIL function is sufficient to partially rescue cytokine-deprived cells from apoptosis. Finally, we demonstrate that cytokine and BCR-ABL suppression of TRAIL transcription is mediated through phosphorylation and inhibition of the forkhead FOXO3a transcription factor. BCR-ABL-induced inhibition of TRAIL transcription in hematopoietic cells may provide a novel mechanism for tumorigenicity in chronic myeloid leukemia.