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miR-451 inhibits Cab39 for stress erythropoiesis
7D and E). Together, these results demonstrate that increased apoptosis of miR-144/451−/− erythroblasts is p53- dependent.
Discussion
Although the biological functions of miR-144/451 have been studied extensively, few studies have been per- formed in animal models. Moreover, less is known about the miR-144/451-regulated molecular pathways underly- ing the phenotypes observed after miR-144/451 depletion. Our results provide further explanation, in addition to that of elevated oxidative stress caused by aberrant 14-3-3ζ accumulation and consequent FoxO3 sequestration, for the defective erythropoiesis and hemolytic anemia seen in miR-144/451−/− mice.13,15 Upon various erythropoietic stresses, miR-144/451 depletion up-regulated expression of the miR-451 target Cab39 with consequent activation of
the AMPK/mTOR pathway, leading to increased ery- throid apoptosis. Manipulating both AMPK and mTOR activities altered the apoptotic rate in miR-144/451−/− ery- throblasts. In contrast, reduced miR-451 levels in glioma cells as an adaptation to metabolic stress derepress Cab39 to activate the LKB1/AMPK/mTOR pathway and enhance survival.21 Thus, miR-451 may employ a common path- way to regulate stress responses in different cell types, but the net effects are context-dependent. During erythro- poiesis, mTOR activity is relatively high, and the pathway appears to exert a positive effect on precursor expansion and protection against erythropoietic stress,32 consistent with the current study.
Contrasting effects of AMPK activity on apoptosis have been observed during various cellular stresses.25 In some cases, AMPK functions to balance cellular redox state and promote survival during metabolic or genotoxic stress.33,34 In other cases, AMPK activation during stress causes increased apoptosis.35,36 In this study, we showed that in
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Cab39 shRNA
D
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Figure 6. Attenuation of Cab39/AMPK/mTOR signaling rescues erythroid apoptosis after miR- 144/451 depletion. (A) shRNA suppression of Cab39 in miR-144/451−/− erythroblasts as shown by Western blot. (B) Apoptosis was measured by flow cytometry, using Annexin V as an indicator. Cab39 shRNA-expressing nucleated erythroblasts were indicated as Ter119+Hoechst+ cells. (C) The apoptotic index in nucleated erythroid cells after knockdown of Cab39. Luciferase (luc) shRNA was used as a neg- ative knockdown control. **P<0.01 (t-test). N=5 fetal liver (FL) erythroblasts of each genotype were used. (D) shRNA suppression of AMPKα in miR-144/451−/− erythroblasts, as shown by Western blot. (E) The percentage of apoptotic nucleated erythroblasts after knockdown of AMPKα. **P<0.01 (t-test). N=4 FLs of each genotype were used. (F) shRNA suppression of TSC2 in miR-144/451−/− erythroblasts, as shown by Western blot. (G) The apoptotic rates of nucleated erythroblasts after knockdown of TSC2. **P<0.01 (t-test). N=4 FLs of each genotype were used.
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