For confocal analyses, images were acquired using a C1Si confocal laser-scanning microscope (Nikon) and analyzed using EZ-C1 software (v3.20) (Nikon) and MetaMorph software (v6.0) (MetaMorph software, GE Healthcare). of this pathway is not sufficient to suppress detachment-induced autophagy in MECs. Instead, inhibition of IKK, as well as its upstream regulator, MAP3K7/TAK1, significantly attenuates detachment-induced autophagy in MECs. Furthermore, function-blocking experiments corroborate that both IKK activation and autophagy induction result from decreased ITGA3-ITGB1 (31 integrin) function. Finally, we demonstrate that pharmacological IKK inhibition enhances anoikis and accelerates luminal apoptosis during acinar morphogenesis in three-dimensional culture. Based on these results, we propose that the IKK complex functions as a key mediator of detachment-induced autophagy and anoikis resistance in epithelial cells. and in MEFs to test whether activation of MTORC1 suppresses autophagy induction during ECM detachment. AMG-47a or MEFs were cultured attached or in suspension for 24 h to assay autophagic flux. Although increased LC3-II conversion and turnover was observed in suspended MEFs, LC3-II conversion and turnover were potently inhibited in cells (Fig.?1A). To more rigorously validate these findings, we performed a rescue experiment and stably reintroduced either wild-type human TSC2 or a mutant version of TSC2N1643I into MEFs. TSC2N1643I contains a point mutation in its GTPase Sirt6 activating protein (Space) domain name that abolishes the Space activity toward RHEB, thereby rendering it unable to modulate MTORC1 activity. As shown in Physique?1A, wild-type TSC2 but not TSC2N1643I rescued autophagy induction during ECM detachment in MEFs. Importantly, the rescued autophagy induction also correlates with the ability of TSC2 to downregulate MTORC1 activity as monitored by RPS6 phosphorylation (Fig.?1B). These results support the idea that loss of MTORC1 activity functionally contributes to ECM detachment-induced autophagy in fibroblasts. Open in a separate window Physique?1. Activation of PI3K-AKT-MTORC1 pathway suppresses ECM detachment-induced autophagy in mouse embryonic fibroblasts (MEFs). (A) Top: Lysates from or MEFs produced attached (A) or suspended (S) for 24 h were immunoblotted with anti-LC3 and anti-tubulin (TUBA) antibodies. Where indicated, E64d and pepstain A (E/P) were added 6 h prior to harvesting. Bottom: MEFs stably expressing TSC2N1643I or wild-type TSC2 (TSC2-WT) were produced attached or suspended for 24 h were immunoblotted with anti-LC3 and anti-TUBA AMG-47a antibodies. Where indicated, E64d and pepstain A (E/P) were added 6 h prior to harvesting. (B) Lysates from MEFs stably expressing vacant vector (Emp), TSC2-WT or TSC2N1643I were produced attached (A) or in suspended (S) for 24 h were subject to immunoblotting with antibodies against TSC2, phospho-RPS6 ribosomal protein (Ser240,244) [pRPS6(Ser240,244)] and RPS6 ribosomal protein (RPS6). (C) Wild-type MEFs stably expressing vacant vector (Emp), activated PIK3CA (PIK3CA*), or myristoylated AKT (myrAKT) were produced attached (A) or suspended (S) for 24 h. Cell lysates were subject to immunoblotting with antibodies against phospho-AKT(Ser473) [pAKT(Ser473)], AKT, phospho-p70 RPS6 Kinase(Thr389) [pRPS6KB1(Thr389)] or RPS6KB1. (D) Wild-type MEFs stably expressing vacant vector (Emp) and PIK3CA* (top) or myrAKT (bottom) were produced attached (A) or suspended (S) for 24 h were immunoblotted with anti-LC3 and anti-TUBA antibodies. Where indicated, E64d and pepstain A (E/P) were added 6 h prior to harvesting. In response to growth factors, AKT directly phosphorylates multiple sites on TSC2 that suppress the inhibitory effect of TSC2 toward RHEB and MTORC1.16,17 During ECM detachment, we also observed decreased AKT activity in suspended cells (Fig.?1C). To investigate whether AKT and its upstream regulator PI3K contribute to autophagy regulation during ECM detachment, we stably expressed activated forms of PIK3CA* (PIK3CAE545K) and AKT (myrAKT) in wild-type MEFs. Cells expressing PIK3CA* and myrAKT exhibited higher levels of AKT and RPS6KB1 phosphorylation during both attachment and suspension, indicative of AMG-47a potently sustained activation of the AKT-MTORC1 pathway. At the same time, upon matrix detachment, LC3 conversion and LC3-II turnover were significantly reduced in PIK3CA* and myrAKT cells compared with empty vector controls (Fig.?1D). Together, these data corroborate that in fibroblasts, reduced activation of the PI3K-AKT-MTORC1 pathway plays a key role in autophagy induction during ECM detachment. PI3K-AKT-MTORC1 activation does not inhibit autophagy in detached mammary epithelial cells Next, we examined whether autophagy was regulated similarly in mammary epithelial cells. MCF10A cells were transfected with siRNA oligonucleotide pools targeting endogenous TSC2 and.
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