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M. Ladli et al.
ated with increased phosphorylation of adducin at Ser 726.21 Thus, it would be interesting to determine whether AMPK plays a role in this pathology. Our results suggest that expression of band 3 is regulated by AMPKa1. Through in vitro studies, Thali et al. identified band 3 as a potential direct substrate for AMPK.22 An attractive hypoth- esis would be that AMPK induces band 3 phosphorylation resulting in an increase in its global expression but a less efficient expression at the cell surface of erythroblasts.
To activate AMPK specifically, we used direct activators of AMPK because these molecules bind directly to the β subunit and do not affect the AMP/ATP ratio as metformin does. Several recent studies have demonstrated the involve- ment and specificity of GSK621 in activating AMPK.12-15 In hematopoietic cells, GSK621 has been reported to be more potent in primary acute myeloid leukemia cells and cell lines than the direct activator A-769662.12
In the present study, we demonstrated, through the use of direct activators (GSK621 and compound 991), that AMPK activation in mature erythroblasts (GPAhigh) (poly- chromatic to reticulocytes) induced apoptotic cell death, whereas no such effect was observed in similarly treated immature erythroblasts. Furthermore, the fact that GSK621 induced the apoptosis of mature erythroblasts after only 48 h of treatment but did not affect erythrob- lasts that were maintained in an immature state (by dex- amethasone) after 9 days of GSK621 excludes a potential toxic effect due to the accumulation of compounds. We propose that maintaining AMPK activation after the basophilic stage, when AMPK is not normally activated, induces cell cycle arrest followed by the induction of autophagy and caspase-dependent apoptosis. Thus, our
work suggests that AMPK activation during the final stages of erythropoiesis is deleterious.
The present work highlights the role of AMPK in erythro- poiesis and adds further support to the involvement of AMPK in the regulation of hematopoiesis. In hematopoietic stem cells, AMPK deficiency partially phenocopies the mitochondrial defects observed in Lkb1-/- mice without affecting hematopoietic stem cell maintenance,23 Obba et al. recently demonstrated that the activation of AMPK is cru- cial for CSF-1-induced autophagy and human monocyte differentiation into macrophages.24 Our results demonstrate the importance of the finely tuned regulation of AMPK dur- ing adult human erythropoiesis. This observation is of sig- nificant value since deciphering the molecular mechanisms regulating proliferation, survival and differentiation of ery- throblasts is necessary to better understand how erythroid progenitors and precursors can physiologically give rise to red blood cells. The use of direct AMPK activators is being considered as a therapeutic treatment in several chronic metabolic diseases. Phase I and II trials investigating the use of the activators PXL770 (clinical trial NCT03395470) and compound 0304 (betagenon.se) are in progress in patients with non-alcoholic hepatic steatosis or type 2 diabetes. These activators could induce the apoptosis of mature ery- throblasts in the bone marrow so it will be necessary to analyze hematologic parameters to prevent potential ane- mia.
Acknowledgments
ML was funded by the Ministère de l’Enseignement Supérieur et de la Recherche and the Labex GRex. This work was supported by the Laboratory of Excellence Labex GRex.
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