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Role of AMPK in human erythropoiesis
Cell proliferation
Cell proliferation was determined by trypan blue exclusion dye.
Statistics
Results are expressed as means ± standard deviation (SD). A Student t test was used to determine statistical significance. P val- ues <0.05 were considered statistically significant.
Results
AMPK a1 activation is tightly regulated during human erythroid differentiation
Because AMPK occurs as a heterotrimeric complex con- taining catalytic a subunits and regulatory β and γ subunits, we aimed to identify which isoforms are expressed in human erythroblasts and to study their variation during human erythroid differentiation. Human primary erythroid progenitors were maintained for up to 12 days in culture and were able to differentiate from the pro-erythroblastic stage (day 2) to the reticulocyte stage (day 12) (Figure 1A and Online Supplementary Figure S1 for cell morphology). During maturation, erythroblasts progressively acquired cell surface-specific markers such as GPA (from the pro-ery- throblastic stage), band 3 (from the basophilic-erythroblas- tic stage) and decreased expression of a4 integrin at the orthochromatic erythroblast stage. They also started to syn- thesize hemoglobin from the basophilic stage. Western blot analysis demonstrated that the a1 catalytic subunit was expressed while the a2 isoform was not detectable and that the expression of a1 was constant along erythroid differen- tiation (Figure 1B). The regulatory subunits, β1 and γ1, were expressed throughout erythroid differentiation. We previ- ously determined the copy number of individual proteins for each stage of erythroid differentiation by an absolute quantitative proteomics analysis.11 We confirmed the expression of a1, β1 and γ1, while a2, β2 and γ2 isoforms were not detectable by mass spectrometry analysis (Online Supplementary Figure S2). Overall, our results suggest that a1/β1/γ1 is the heterotrimeric complex that is predomi- nantly present in human erythroblasts.
Despite the global constant expression of AMPK, the acti- vation of this protein might be modulated during differen- tiation. We, therefore, studied AMPK activation by detec- tion of phosphorylation of the a1 catalytic subunit at Thr172 and phosphorylation of one of its substrates, ACC, at Ser79 (Figure 1C). From day 2 to day 6, the phosphoryla- tion of AMPK and ACC was clearly detectable, but con- comitantly decreased from day 8 to the end of differentia- tion. LKB1 was expressed throughout erythroid differentia- tion. These data showed the biphasic pattern of activation of AMPK during erythroid differentiation with clear activa- tion in immature erythroblasts from the pro-erythroblast stage until day 6 when the cells were GPAmed/band 3low (basophilic erythroblasts) and reduced activation in mature GPAhigh/band 3med erythroblasts from day 8 to day 12, corre- sponding to stages from polychromatic erythroblasts to reticulocytes.
The absence of AMPK induces decreased proliferation and alterations in the expression of membrane proteins of human erythroblasts
To decipher the role of AMPK in erythroblasts, we inhib- ited AMPK expression by a specific shRNA targeting the a1
catalytic subunit. Cells were infected by a lentivirus coding for either shAMPKa1 or shControl (shCtrl) at day 1 and then at day 4 after CD36 cell sorting. The decrease in a1 AMPK expression resulted in an expected decrease of the phosphorylation of AMPK and its substrates ACC and ULK1 (Figure 2A). No compensatory expression of the a2 catalytic subunit was observed in response to inhibition of the a1 isoform (Online Supplementary Figure S3). The inhibi- tion of AMPK a1 expression did not significantly modify the differentiation of the cells. Indeed at days 6, 8 and 10 of culture, the cell population was mainly composed of basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts, respectively, and this pattern was not different when AMPKa1 was knocked down (Figure 2B). The morphology of the shCtrl and shAMPKa1 cells was very similar (Online Supplementary Figure S4). The percentages of hemoglobinized cells estimated at the indi- cated stages of differentiation were identical between the shCtrl and shAMPKa1 cells (Figure 2C). shAMPKa1 ery- throblasts showed a reduced ability to proliferate compared to shCtrl erythroblasts (Figure 2D). The inhibition of AMPKa1 expression did not significantly affect the viability of the cells measured by the trypan blue exclusion assay (Figure 2E) or by annexin V flow cytometry analysis (data not shown).
Because red cells from Ampka1-/- and Ampkγ1-/- mice are highly resistant to osmotic stress and poorly deformable, the expression of membrane proteins involved in these processes was studied (Figure 3A). In shAMPKa1 polychro- matic and orthochromatic erythroblasts, western blot experiments showed that the phosphorylation of adducin on Ser726 was increased while the expression of spectrins and ankyrin was not affected (Figure 3A). Western blots demonstrated that the global expression of band 3 was sig- nificantly decreased in the shAMPKa1 cells while its expression at the cell surface, measured by FACS, was abnormally increased (Figure 3B). FACS analyses also showed a decrease of the cell surface expression of GPA at each stage of differentiation for the shAMPKa1 cells in comparison to ShCtrl (39.9% at day 6/basophilic erythrob- lasts, 50% at day 8/polychromatlic erythroblasts and 58.8% at day 10/orthochromatic erythroblast versus 100% for shCtrl) (Figure 3C). Overall, the decrease in AMPK expression induced major abnormalities in the expression of the membrane proteins band 3 and GPA and, as in murine Ampk knockout mice, led to enhanced phosphory- lation of adducin. Furthermore, AMPKα1 knockdown pro- voked a decrease in cell proliferation without affecting cell viability and erythroblast maturation.
Proliferation and survival of mature GPAhigh erythroblasts are specifically and drastically diminished by GSK621-mediated AMPK activation
To further investigate the role of AMPK in erythroid cells, the activation of AMPK was enhanced by GSK621, a direct, potent, novel activator of AMPK.12-15 In primary erythro- blasts, the activation of AMPK by GSK621 was dose-depen- dent, with increased phosphorylation of T172 AMPKa, and also gradual stimulation of the phosphorylation of the sub- strates of AMPK, ACC at Ser79 and ULK1 at Ser 555, from 5 to 20 mM (Figure 4A). The latter dose was then used in the experiments. GSK621 was added to the medium from day 0 after CD36 cell sorting until the indicated days. GSK621 induced the phosphorylation of AMPK and its substrates at each stage of erythroid maturation (Online Supplementary
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