The Anti-apoptotic Biological Function And Mechanism Of TRAF1 Induced Aggregation Of Mitochondria

TARF1 is a member of the tumor necrosis factor receptor-associated factor (TRAF) family. The TRAF family is a group of adapter proteins that link a wide variety of cell surface receptors, including the TNF and IL-1 receptor superfamily and mediate diverse signaling cascades, which ultimately lead to the activation of NF-kB(nuclear factor kB) and JNK(Jun N-terminal kinase) and so on. Unlike other members of TRAF family, TRAF1 does not have a RING finger and the zinc finger domain near the RING finger. TRAF1 can recruit a number of distinct members of the TNFR superfamily, including TNFR2, CD30, CD27, and TRANCE-R. The overexpression of TRAF1 in transgenic animals demonstrates the inhibitory role of TRAF1 on the antigen-induced apoptosis of CD8+ cells. TRAF1 is also reported to achieve its anti-apoptotic effect via cIAP recruitment.Our previous work discovered the phenomenon of the aggregation and colocation of mitochondria and TRAF1 when recombinants of pDsRed-TRAF1 was transfected into COS-7 cells. This phenomenon has a certain universality. And TRAF1 also has the effect of anti-apoptosis and NF-κB activation in HepG2 cell. Then, it was found that the overexpression of TRAF1 can increase mRNA level of mitochondria fusion protein Mfn2, suggesting that the mitochondria aggregation induced by TRAF1 may be associated with the fusion mechanism of mitochondria.In order to further study the mechanism and function of TRAF1 induced mitochondria aggregation, we use Realtime-PCR method to detect mRNA level changes of another mitochondria fusion protein Mfn1 after Hela cells being transfected with TRAF1. We construct a model of apoptosis of Hela cell induced by Adriamycin. Then a cytotocity , mitochondrial membrane potential, caspase-9 and IκB activation assay have been studied based on the model. 1. The overexpression of TRAF1 can increase mRNA level of Mfn1Transfect respectively Hela cell with pDsRed-TRAF1 and pDsRed, non-transfected cells for the negative control, the RT-PCR results show that Mfn1 expression have a certain degree of increase in the transfected pDsRed-TRAF1 group, compared with the transfected pDsRed group and control group respectively, which increased 2.48 times, 2.18 times (P<0.01). It is indicated that TRAF1 induce the mitochondria aggregation by increasing the expression of Mfn1.2. The overexpression of TRAF1 can resist the injury of AdriamycinTreating the TRAF1 transfected cell and non-transfected cell by Adriamycin 1μM for 48 hour, using the MTT assay to detect the different cytotocity to the cells, we find that TRAF1 transfected cell can resist the killing of Adriamycin.3. The overexpression of TRAF1 (before or after Adriamycin stimulation) induce mitochondria aggregationTreating the TRAF1 transfected cell and non-transfected cell by Adriamycin 1μM for 24 hour, using the Fluorescence microscope to observe the morphology of mitochondria, we find that the mitochondria of TRAF1 transfected cell is still aggregated before or after Adriamycin stimulation.4. The overexpression of TRAF1 (before or after Adriamycin stimulation) can maitain the stability of mitochondrial membrane.Treating the TRAF1 transfected cell and non-transfected cell by Adriamycin 1μM for 24 hour, using the mitochondrial membrane potential detection kit, we find that TRAF1 can increase the mitochondrial membrane potential before or after Adriamycin stimulation.5. The overexpression of TRAF1 (before or after Adriamycin stimulation) can decrease the activation of caspase-9Treating the TRAF1 transfected cell and non-transfected cell by Adriamycin 1μM for 24 hour, using western blot to detect the activation of caspase-9, we find that the activation of caspase-9 was decreased in TRAF1 transfected cell before or after Adriamycin stimulation. 6. The overexpression of TRAF1 (before or after Adriamycin stimulation) can decrease the degradation of IκBTreating the TRAF1 transfected cell and non-transfected cell by Adriamycin 1μM for 24 hour, using western blot to detect the activation of caspase-9, we find that the degradation of IκB was decreased in TRAF1 transfected cell, suggesting that the activation of NF-kB was increased before or after Adriamycin stimulation.Conclusion: TRAF1 can induce the mitochondria aggregation by increasing the expression of mitochondria fusion protein Mfn1. And, TRAF1 may resist apoptosis by maitaining the stability of the mitochondrial membrane, decreasing the activation of caspase-9 and increasing the activation of NF-kB. All these results make a basis for a thoroughly study about the function of TRAF1 and the relationships of TRAF1 and mitochondria.