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X. Fang et al.
cant change in apoptosis of WT erythroblasts occurred
after shRNA suppression of Cab39 (Figure 6B and C). Similarly, shRNA inhibition of AMPKα and TSC2 inhibit- ed apoptosis of miR-144/451−/− erythroblasts but not WT controls (Figure 6D-G).
To further examine the effects of AMPK/mTOR signal- ing during WT and miR-144/451−/− FL erythropoiesis, we used drugs to manipulate the pathway. Consistent with the results of shRNA studies, the AMPK inhibitor Compound C (CC) reduced apoptosis significantly in miR- 144/451−/−, but not WT erythroblasts (Online Supplementary Figure S4A and B). Conversely, inhibiting mTOR activity with rapamycin or activating AMPK with AICAR induced apoptosis in WT erythroblasts (Online Supplementary Figure S4C-F). Overall, our results with shRNAs and pharmaco- logical inhibitors indicate that miR-451 facilitates fetal ery- throblast survival by inhibiting expression of Cab39, resulting in suppression of LKB1 and AMPK and activation of the downstream mTOR pathway.
The increased apoptosis of miR-144/451−/− erythroblasts is p53-dependent
Depending on cell context, mTOR can regulate p53 pos- itively or negatively to alter rates of apoptosis.28-30 Thus, we investigated p53 levels and effector functions in miR- 144/451−/− erythroblasts that exhibit reduced mTOR activ- ity. p53 level was increased in miR-144/451−/− E14.5 FL ery- throblasts (Figure 7A). To investigate the functional impli- cations of this finding, we crossed miR-144/451−/− mice with “p53 knock-in (KI)” mice, in which both alleles of the normal p53 gene are replaced by a cDNA encoding a 4- hydroxytamoxifen (4-OHT)-dependent form of the fusion protein, p53-estrogen receptor.16,31 There is a lack of endogenous p53 activity in the p53 KI mice unless 4-OHT is applied. In the absence of 4-OHT, loss of p53 function rescued the deficient erythroblast numbers in E14.5 FL from miR-144/451−/− mice (Figure 7B and C). Moreover, apoptosis of E14.5 miR-144/451−/− FL erythroblasts was sig- nificantly reduced in the absence of p53 activity (Figure
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the
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Figure 5. Activation of the Cab39/AMPK/mTOR pathway in miR-144/451−/− erythroblasts. (A) Schematic illustration of the major
signaling components in Cab39/AMPK/mTOR molecular path- way. Cab39 expression leads to increased LKB1 activity in the Cab39/LKB1/STRAD protein com- plex, which activates its substrate AMPK. AMPK activation is vital for modulation of the mTOR signaling cascade and for potential stabiliza- tion of p53 activity, which subse- quently governs the fate of the target cells, including their apoptosis, sur- vival, and/or proliferation. (B) Western blot analysis of the expres- sion of Cab39, p-AMPKα, p-Raptor, p- TSC2, p-p70S6K, p-S6, and p-eIF4B, along with their non-phosphorylated counterparts, in fetal liver (FL) ery- throblasts grown in maturation medi- um for 24 hours, when all the cells were nucleated erythroblasts. 14-3- 3ζ was used as a positive control, and actin was used as a sample load- ing control. Quantitative analyses of the protein intensity are shown in (C- I) as follows: (C) Cab39, (D) p-
AMPKα/AMPKα, (E) Raptor/Raptor, (F) p-TSC2/TSC2, (G) p-p70S6K/p70S6K, (H) p-S6/S6, and (I) p-eIF4B/eIF4B. Signals were normalized to actin. Data are the mean values from 3 separate experi- ments. *P<0.05; **P<0.01 (t-test).
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haematologica | 2018; 103(3)