The Pro-apoptotic Role Of Porcine BCL-G And Its Interaction With JAB1or MELK

Apoptosis is an ordered and orchestrated cellular process that occurs normally during development and aging and acts as a homeostatic mechanism to maintain cell populations in adult tissues. The process is also a defense mechanism in immune reactions or when cells are damaged by disease or noxious agents. Many pathways by which induction of apoptosis occurs are known but only two, the intrinsic and extrinsic pathways, have been demonstrated in detail. Bcl-2family genes, which operate immediately upstream of mitochondria, regulate the intrinsic pathway. BCL-G, an important member of BCL-2family, is involved in systemic lupus erythematosus, mammary cancer and prostate cancer. In recent years, BCL-G gene has been studied in human and mouse. However, porcine BCL-G (pBCL-G) has little been studied. Pigs are more optimal animals used as medical models than mice. Therefore, the study on pBCL-G gene will be helpful to study human BCL-G gene and its related diseases. In this study, we studied the function of pBCL-G and mainly focused on its spatial expression pattern, subcellular localization, interactions with porcine JAB1(pJAB1) or MELK (pMELK), and its enhancement to apoptosis. The results were shown as follows.1. The spatial expression of the pBCL-G mRNA was examined by quantitative real-time PCR (qRT-PCR) in RNA samples isolated from different porcine tissues. The obtained results revealed differential expression of pBCL-G mRNA in different porcine tissues, with the highest level in heart, and the lowest level in kidney. The difference in the level of pBCL-G transcripts between these two tissues exhibited about6-fold. A medium expression level was observed in abdominal lymph node, hilar lymph node, mandibular lymph node, mesenteric lymph node, and spleen, whereas most other porcine tissues exhibited a low pBCL-G transcript level.2. Two mutant genes expressing pBCL-G proteins lacking BH2or BH3domain respectively were created and subcloned into the pEGFP-C1vector to generate pEGFP-pBCL-G-BH2-deficient or pEGFP-pBCL-G-BH3-deficient vector. Our results by confocal microscopy analysis showed that the mutant pBCL-G protein lacking the BH2domain was distributed in a punctate cytosolic pattern whereas the mutant pBCL-G protein lacking the BH3domain and the complete pBCL-G protein were both distributed throughout PK-15cells. 3. By transient transfection and selection with G418, two clones of SUVEC cells stably expressing GFP-pBCL-G or GFP protein were obtained respectively. By fluorescence analysis, GFP signal was detected in more than90%cells of the selected clones. By western blot analysis, the expression of GFP-pBCL-G or GFP protein was detected in two clones of cells respectively.4. By light microscopy analysis, compared with control group, a series of typical apoptotic features were observed in cells transiently expressing GFP-pBCL-G protein induced by poly I:C or staurosporine (STS), such as shrinkage of cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells. By staining with Hoechst33342and fluorescence analysis, compared with control group, chromatin condensation and nuclear fragmentation were observed in cells transiently expressing GFP-pBCL-G protein induced by poly I:C or STS. Annexin V-PE/7-AAD staining and flow cytometry analysis showed that pBCL-G significantly enhanced apoptosis induced by poly I:C or STS.5. Porcine JAB1gene with8exons encoding334amino acids was cloned by reverse transcription PCR. Bioinformatics analysis showed that JAB1was highly conserved among different species. By qRT-PCR analysis, pJAB1transcripts were detected in all examined organs and tissues, with the highest levels in heart, which was consistent with pBCL-G gene. Co-immunoprecipitation assay showed that pBCL-G protein could interact with pJAB1or pMELK protein and pMELK protein was degraded based on the interaction. In addition, the subcellular localization of pBCL-G protein could be affected by pJAB1or pMELK protein.6. Annexin V-PE/7-AAD staining and flow cytometry analysis showed that stably expressed pBCL-G protein significantly enhanced apoptosis induced by STS and transiently expressed pJAB1protein also significantly enhanced apoptosis induced by STS. pBCL-G and pJABl protein synergistically enhanced apoptosis induced by STS. By caspase activity assay and western blot analysis, caspase-8, caspase-9and caspase-3were activated in the SUVEC cells over-expressing pBCL-G protein induced by STS and the activation of these three proteins was significantly increased compared with control cells; Only caspase-9and caspase-3were activated in the cells over-expressing pJABl protein induced by STS and the activation of these two proteins was also significantly increased compared with control cells.In conclusion, we firstly determined the spatial expression of the pBCL-G mRNA, analyzed the key domains related with its subcellular localization, identified its enhancement to apoptosis induced by poly I:C or STS, confirmed the interaction of pBCL-G protein with pJAB1or pMELK protein, characterized the synergistical enhancement of pBCL-G and pJAB1to apoptosis induced by STS, and explored the mechanism of apoptosis enhanced by pBCL-GorpJAB1.