Page 62 - 2019_05-HaematologicaMondo-web

M. Ladli et al.
a major sensor of energy status that maintains cellular energy homeostasis but also exerts non-metabolic func- tions such as the maintenance of cell survival, cell polarity and regulation of the cell cycle.3,4
Erythropoiesis is a tightly regulated process that permits the production of around two million red cells each second throughout a human life, while the total cell number has to be kept within a narrow margin. This extremely dynamic process is also very flexible, since it must increase rapidly in response to blood loss and hypoxia. Furthermore, main- taining homeostasis is crucial and an imbalance in erythro- poiesis can lead to the development of erythroid patholo- gies such as polycythemias and anemia.
We and other groups have previously demonstrated that AMPK plays a crucial role in the integrity and sur- vival of red blood cells. We showed that mice that are globally deficient in the catalytic subunit, Ampka1 but not in those lacking the isoform Ampka2, as well as those globally deficient in the regulatory subunits Ampkβ1 and Ampkγ1, develop regenerative hemolytic anemia caused by increased sequestration of abnormal erythrocytes. Ampka1-/-, Ampkβ1-/- and Ampkγ1-/- mice develop splenomegaly and iron accumulation due to a compensa- tory response through extramedullary erythropoiesis in the spleen and enhanced erythrophagocytosis. The life- span of erythrocytes from Ampka1-/- and Ampkγ1-/- mice was shorter than that of wild-type littermates. Moreover, Ampka1-/- and Ampkγ1-/- erythrocytes were highly resist- ant to osmotic stress and poorly deformable in response to increasing shear stress, which is consistent with a loss of membrane elasticity.5-8
The defects in Ampk-deficient erythrocytes suggested that alterations might occur early during terminal ery- throid maturation but no data were available on the importance of AMPK in human erythropoiesis. We, therefore, decided to investigate whether AMPK could be implicated in regulating the proliferation, survival and dif- ferentiation of human erythroid precursors.
In the present study, we analyzed the expression and activation of AMPK along human erythroid differentia- tion. Our experiments show that AMPK is highly activat- ed in immature erythroblasts and weakly active in mature erythroblasts. We studied the impact of knocking down AMPK and of AMPK activation by direct activators. In erythroblasts, the knockdown of the AMPK α1 catalytic subunit expression by short hairpin (sh) RNA induced a decrease in cell proliferation and alterations in the expres- sion or phosphorylation of membrane proteins whereas no defect in hemoglobin synthesis or erythroid matura- tion was observed. The activation of AMPK is necessary in immature erythroblasts but maintaining the activation in mature erythroblasts is deleterious, demonstrating that AMPK activation has to be tightly regulated during human terminal erythroid differentiation.
Methods
Materials
adducin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Actin (A5441) antibodies, dexamethasone and chloroquine were purchased from Sigma- Aldrich (Lyon, France). Anti-ankyrin antibodies were obtained from Neuromab (Davis, CA, USA) (#75-380) and anti β-spectrin antibodies from Abcam (Cambridge, UK). Compound GSK-621 was purchased from Selleckchem (Houston, TX, USA) and 991 (5-[[6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol-2- yl]oxy]-2-methyl-benzoic acid) was synthesized by Spirochem (Basel, Switzerland).
Cell lines and cell culture
CD34+ cells were obtained from human donors who gave informed consent in accordance with the Declaration of Helsinki and the study was approved by the French ministry of higher education and research review board. Granulocyte colony-stim- ulating factor-mobilized CD34+ cells were purified from periph- eral blood after cytapheresis. CD34+ cells were isolated by posi- tive selection using an immunomagnetic procedure (MACS CD34 isolation kit; Miltenyi Biotech (Paris, France). CD34+ cells were cultured in 5% CO2 at 37°C for 7 days in IMDM medium (Life Technologies, Waltham MA, USA) containing 1% gluta- mine, 15% BIT 9500 (Stem Cell Technologies), 100 ng/mL stem cell factor, 10 ng/mL interleukin-6 and 10 ng/mL interleukin-3 (Miltenyi Biotech). After 7 days of culture, CD36+ cells corre- sponding to a highly purified population of human erythroid progenitors were obtained by positive selection on CD36 immunomagnetic beads (CD36 unlabeled antibodies purchased from Beckman Coulter, Villepinte, France) coupled to anti- mouse IgG1 microbeads purchased from Miltenyi Biotech) . CD36+ cells were then cultured with 2 U/mL erythropoietin, 100 ng/mL stem cell factor and 10 ng/mL interleukin-3 for up to 14 days for erythroid differentiation. GSK621 or compound 991 was added from day 0 after CD36+ selection; cells were counted daily and diluted to a final concentration of 0.8 x106 cells/mL by the addition of fresh medium containing the indicated concen- tration of AMPK activator. Because of interindividual variability, the kinetics of erythroid differentiation varies between different human samples. Thus, the days of culture corresponding to the same stage of differentiation have been grouped.
AMPK a1 and a2 antibodies were obtained from Graham Hardie (University of Dundee, UK);9,10 antibodies against the AMPK β1 and γ1 isoforms, phospho-Thr 172 AMPK, phospho- Ser 79 ACC, phospho-Ser 555 ULK1, LKB1, LC3B and cleaved caspase 3 were from Cell Signaling Technology (Danvers, USA) and anti-HSC70, anti-a spectrin, anti-band 3 and anti-P-Ser 726
Flow cytometry
Cells were labeled as previously described.11 Briefly, PC7-con- jugated anti-glycophorin A (GPA), APC-conjugated anti-cd49d (α4 integrin) or an appropriate isotype control were purchased from Beckman Coulter; anti-BRIC6 (anti-band 3) was from the NHSBT International Blood Group Reference Laboratory (Bristol, UK). FITC-conjugated annexin V was used to measure the percentage of cell apoptosis.
Lentiviral constructs, lentiviral production and cell infection
Lentiviral constructs for control and AMPKa1 shRNA [(SHC002 and SHCLNG-NM006251 (TRCN00000000859), respectively)] were purchased from Sigma (Lyon, France). To obtain recombinant lentiviruses, 293T cells were transiently transfected by calcium phosphate precipitation with three differ- ent plasmids: pCMV-G (VSVG envelope coding sequence), pCMV-gag-pol and a recombinant pLKO.1 vector encoding either a control or AMPKa1 shRNA. Supernatants containing infectious lentiviral particles were concentrated by ultracentrifu- gation. Infections of human erythroblasts were performed at day 1 and at day 4 after CD36 cell sorting and culture in the pres- ence of interleukin-3, stem cell factor and erythropoietin, as described above.
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haematologica | 2019; 104(5)