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Role of AMPK in human erythropoiesis
increase in cytosolic Ca2+, and possibly TAK1, which is acti- vated by cytokines. The phosphatases PP1, PP2A and PP2C dephosphorylate T172 and the kinases GSK3, PKA, PKB, and PKC inhibit AMPK activation; they may contribute to the reduced AMPK activation in mature erythroblasts. Our data from the quantitative mass spectrometry analysis of human erythropoiesis11 did not allow us to quantify these kinases and phosphatases because of their absence, their very weak expression or the scarcity of peptides generated. Nevertheless, our western blot studies showed the expres- sion of LKB1 along erythroid differentiation and suggested that LKB1 could be the upstream activating kinase for AMPKa1. Further studies are needed to understand the kinetics of AMPK activation and its upstream regulators during erythroid terminal differentiation.
Our results in human erythroblasts show that knock- down of the expression of the a1 subunit by shRNA induces a decrease in cell proliferation and does not inhibit cell survival or erythroid maturation. Red blood cells from Ampk a1-/- mice have defects in membrane elasticity leading to hemolytic anemia. The absence of the a1 subunit in human erythroblasts induces important changes in the expression of membrane proteins and could potentially affect the expression of membrane proteins involved later in erythrocyte membrane elasticity. As we previously showed, phosphorylation of adducin at Ser724 is increased in red blood cells from Ampk a1-/- and Ampk γ1-/- mice. Our data demonstrate that this modification is also present ear- lier in human erythroblasts. Interestingly, in sickle cell dis- ease, the reduction of red blood cell deformability is associ-
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Figure 7. GSK621-mediated AMPK activation leads to autophagy and apoptosis. (A) GSK621-mediated AMPK activation induces autophagy. Erythroid cells were incubated in the absence (vehicle) or presence of 20 mM GSK621 from day 0 to the indicated days of culture (left panel). Chloroquine (10 mM) was added for 4 h before harvesting the cells (right panel). LC3BII was detected by western blot experiments using specific antibodies. Anti-β actin or anti-HSC70 antibodies were used as loading controls. (B) GSK621 provokes the caspase-dependent apoptosis of mature day 8 erythroblasts. Mature erythroblasts were incubated in the absence (vehicle) or presence of 20 mM GSK621 or 20 μM GSK621 and 10 mM QVD for 48 h; cells were labeled with anti-GPA and for annexin V binding and analyzed by flow cytometry. A representative experiment and the percentage of annexin V-positive cells from three experiments are shown. Results are expressed as the mean ± SD. *P<0.05, **P<0.01. Efficiency of QVD to block caspase activity was determined by western blot using an anti-caspase 3 antibody that specifically detects the cleaved isoform of caspase 3. β-actin was used as a loading control. d: day; CQ: chloroquine; GPA: glycophorin A; QVD: Q-VD-OPh.
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