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M. Ladli et al.
Figure S5). GSK621 provoked 32% cell death at days 5-7 and 70% at days 8-9 (Figure 4B). GSK621 dramatically reduced the proliferation of cells with a more drastic impact on the most mature erythroblasts (Figure 4C).
To gain further insight into the inhibition of mature ery- throblast proliferation by GSK621, the cell cycle was ana- lyzed by quantification of DNA content with propidium iodide. At days 8 and 9, GSK921 induced a reduction in the number of cells in the G2/M phase of the cell cycle and an increase in cells in the early S phase, demonstrating block- age in the S phase (Figure 4D). The protein level of AMPK substrates involved in the cell cycle, P5316 and a target gene of P53, P21 was determined. GSK621-mediated cell cycle arrest was not due to the phosphorylation and consequent stabilization of P53 since there was no variation in their expression, which is in agreement with the absence of defects in G1/S transition (Online Supplementary Figure S6).
We then studied in more detail whether GSK621-mediat- ed AMPK activation could affect erythroblast differentia- tion. In this set of experiments, at days 3-4 cells were imma- ture and did not synthesize hemoglobin, while at days 8-9, more than 80% of the cells were hemoglobinized (Figure 5A). In the presence of GSK621, at days 8-9, the percentage of cells that synthesized hemoglobin was very low.
To decipher more precisely the stage at which the GSK621-mediated activation of AMPK induced cell death, erythroblasts were analyzed for GPA and annexin V by flow cytometry (Figure 5B). GSK621 did not affect imma- ture GPAlow cells at days 3-4, but induced massive death of GPAhigh erythroblasts at days 5-7 (46% annexin V-positive cells with GSK621 versus 16% in control cells) and at days 8-9 (75% versus 16%, respectively). Indeed, after AMPK activation at days 5-7, only 3.5% of erythroblasts were GPAhigh, in contrast to 46.4% in control cells. Furthermore, at day 7 only 7.5% of GSK621-treated cells were band 3high compared to 42% of control cells. At day 9, in the control conditions, erythroid cells continued to differentiate, which is in contrast to immature GSK621-treated cells. Furthermore, morphological studies after staining with May-Grünwald-Giemsa confirmed the blockage in matura- tion. Indeed, in vehicle-treated cultures, at day 9, the popu- lation was mainly constituted of polychromatic erythro- blasts, and at day 14, orthochromatic erythroblasts and reticulocytes, whereas in the GSK621-treated culture, at days 9 and 14, cells were very immature with large nuclei and uncondensed chromatin; no mature cells were detected at day 14 (Figure 5C). The same results (decreased prolifer- ation and survival, differentiation blockage) were obtained with another direct activator, compound 991 (Online Supplementary Figure S7). Overall, our results show that acti- vation of AMPK by direct activators induced a blockage in the cell cycle, proliferation arrest and death of mature ery- throblasts after the basophilic stage.
To reinforce our data, we took advantage of the fact that erythroid progenitors and early precursors can proliferate with delayed differentiation in response to erythropoietin, stem cell factor and dexamethasone.17 We maintained the cells for the indicated number of days in culture medium with vehicle, GSK621, dexamethasone + vehicle or dexam- ethasone + GSK621 (Figure 6A). As expected, the presence of dexamethasone delayed erythroid differentiation, as demonstrated by the absence of a GPAhigh population after 7 days and even 9 days of culture. After 7 days, GSK621 induced cell death, as previously described (Figures 4B and 5B), with 54.6% of cells being positive for annexin V and
35.3% being stained by trypan blue; however, immature erythroblasts treated with dexamethasone + GSK621 were resistant to GSK621-induced cell death. Indeed, only 23.4% of cells were annexin V-positive and 16.6% were stained by trypan blue (Figure 6A,B).
To confirm that the activation of AMPK in mature ery- throblasts provoked cell death, GSK621 was added for 24 and 48 h on day 9 when the erythroblastic population already contained 35% of mature GPAhigh cells (Figure 6C). The GPA/annexin V staining clearly demonstrated that GSK621 induced massive death in mature GPAhigh cells within less than 48 h. With GSK621, 52% of the total cells were positive for annexin V and 43% were stained by try- pan blue versus 16% and 10%, respectively, of the control cells.
Overall, our results demonstrated that the activation of AMPK was deleterious for mature GPAhigh cells, specifically in contrast to immature erythroblasts (from the progenitor stage to the basophilic stage), which were not affected.
AMPK activation induced autophagy and apoptotic death of mature erythroblasts
In erythroblasts, AMPK activation leads to ULK1 phos- phorylation at S555 (Figure 4A), which is well known to be important for the induction of autophagy in several types of cells.18 In GSK621-treated erythroblasts, LC3B-II accumula- tion was clearly detected by immunoblotting (Figure 7A, left panel). The induction of autophagy was confirmed by the use of chloroquine, which blocks the degradation of autophagosomes.19 Indeed, in addition to GSK621, chloro- quine treatment further increased LC3B-II, showing that the activation of AMPK by GSK621 in mature erythroblasts induced autophagy (Figure 7A, right panel).
We, therefore, wondered whether GSK621 provoked cas- pase-dependent apoptotic cell death and treated cells with a pan-caspase inhibitor Q-VD-OPh (QVD) in addition to GSK621. When caspase activity was blocked by QVD for 48 h (as demonstrated by the anti-cleaved-caspase 3 immunoblot), mature erythroblasts were protected from GSK621-induced cell death, showing that AMPK activation induced caspase-dependent apoptotic cell death (Figure 7B).
Discussion
Ampk a1-/-, Ampk β1-/- and Ampk γ1-/- mice develop hemolytic anemia, and the plasma membrane of their red blood cells shows elasticity defects.5–8 The membrane com- position evolves continuously throughout erythropoiesis and during red blood cell maturation; the defects due to the absence of Ampk are most likely initiated during erythro- poiesis. We, therefore, studied the role of AMPK during human erythropoiesis.
As in murine red blood cells,5 a1 is the only catalytic sub- unit expressed in erythroblasts, a2 is not detected and we showed that the heterotrimer a1/β1/γ1 is predominant from erythroid progenitors to orthochromatic erythro- blasts.
During the earliest stages of terminal differentiation, from progenitors to the basophilic stage, AMPK is activated and then its activation is drastically reduced to the reticulocyte stage. Several kinases and phosphatases regulate AMPK activation.1,20 AMPK is activated by phosphorylation at T172 by three upstream kinases: LKB1, which seems to be constitutively active, CaMKK2 which is activated by an
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haematologica | 2019; 104(5)