DRAM1Regulate Apoptosis Through BAX

Aim：To investigated if DRAM1can promote apoptosis via upregulation of BAX.Methods:(1)To evaluate whether the elevation of BAX was regulated by DRAM1,cells were treated with Doxorubicin with or with not DRAM1siRNA. Cells were alsotransfected with DRAM1-pCDNA4.(2)RT-PCR amplification was used to test if BAXupregulation was dependent of transcription.(3)To examine the fate of BAX, cells weretreated with Ubiquitin-proteasome pathway inhibitor or autophagy-lysosome pathwayinhibitor. The effects of ATG5siRNA on BAX protein level was also tested.(4) To furtherconfirm the degradation of BAX, A549cells and A549-DRAM1RNAi cells were treatedwith rapamycin to activate autophagy.(5)To examine whether DRAM1can prevent BAXdegradation, cells transfected with DRAM1-pCDNA4was treated with or with notautophagy inhibitor. The half-life of BAX in A549cells and those stably expressingDRAM1were tested.(6)To examine if DRAM1can interact with BAX and prevent it fromdegradation, co-IP and GST-pull down experiment were taken.(7)The lysosomallocalization of BAX was detected with immunohistochemical analysis. And the lysosomeswere fractionated to test whether the lysosomal localization of BAX will induce the releaseof lysosomal enzyme.(8) To evaluate whether BAX was involved in lysosome-mediatedapoptosis, cells were treated with3-NP with or with not BAX siRNA, and the cleavage ofBID, the release of cyto-C, the activation of caspase-3were tested.Results: We have successfully established a DRAM1stably overexpression cell line.Data showed overexpressing of DRAM1can robust increase the protein level of BAX,while DRAM1siRNA can blocked Doxorubicin-induced BAX upregulation. RT-PCRamplification of DRAM1RNA and BAX RNA revealed that overexpressing of DRAM1did not increase BAX mRNA level, indicating that BAX upregulation was independent oftranscription. Western blot analysis showed that BAX protein levels did not increase whenUPS was inhibited. Western blot analysis also showed a progressively increase in BAXprotein levels when autophagy was inhibited and a progressively decrease in BAX proteinlevels when autophagy was activated. Data showed both transfection and autophagyinhibitor can induce BAX protein levels, however, autophagy inhibitor couldn’t induce a higher level of BAX protein when DRAM1was overexpression, and results showed theoverexpression of DRAM1lead to a longer half-life of BAX. Co-IP and GST-pull downdemonstrated DRAM1interacts with BAX, and immunohistochemical analysis showedpartially co-localization of BAX with LysoTracker after treatment of3-NP. Western blotanalysis also showed that BAX protein levels increased in the lysosome enriched fractionsafter treatment of3-NP, and cathepsin B was released from lysosome after treatment of3-NP or incubated with BAX protein. Further investigation demonstrated that BAX wasinvolved in lysosome-mediated apoptosis, as data showed BAX RNAi can inhibit thecleavage of BID, the release of cyto-C, the activation of caspase-3induced by3-NP.Conclusions: DRAM1can increase the protein level of BAX, but this BAXupregulation was independent of transcription. BAX can be degraded by autophagy,DRAM1can prevent BAX degradation and prolong the half-life of BAX. DRAM1caninteract with BAX and recruit BAX to lysosomes. The lysosomal localization of BAX willinduce the release of cathepsin B and further induce the lysosome-mediated apoptosis.