The Functional Analysis Of CXXC5 In Poptosis

Apoptosis is a widespread basic biological phenomena in biological world which plays a role in individual shape-formation, morphological changes, the constancy of adult maintenance and other organisms in defense. The human CXXC5 gene, cloned by the previous work in our lab was found to be involved in inducing apoptosis. This article is to further study the function of the CXXC5 gene in apoptosis.The CXXC5 polyclonal antibody was first prepared. The research results using HEK293T cell line as a model-showed that:1) Hochest33258 anlysis detected that overexpression of the gene can induces apoptosis.2) Western blot analysis showed that overexpression of the gene can activate the expression of TNF-a, indicating the CXXC5 protein induced apoptosis through TNF-a membrane receptor pathway.3) As the marker gene in mitochondrial pathway-P53 protein’s expression didn’t change when overexpressing CXXC5 and analyzed by Western blot analysis which indicated that the apoptosis induced by CXXC5 was not through the mitochondrial pathway.4)With a death domain, FADD is a regulatory molecule that brigdes the connection receptors and Caspase-8 in the apoptosis pathway, Western blot analysis showed that CXXC5 didn’t bind with FADD, nor activate the expression of FADD, but the RNA interference to expression of endogenous CXXC5 would reduce the expression of FADD.5) When "knocking out" CXXC5 expression, Western blot analysis showed that inactivation of CXXC5 protein resulted in down-expression of Caspase3. Together with the preliminary work in our laboratory that overexpression of CXXC5 would up-regulate the expression of Caspase8 and Caspase3, we confirm that CXXC5 caused a cascade reaction of Caspases by regulating protein expression Caspase3 and Caspase8.6) Western blot analysis of the expression of BID, cytochrome C and Caspase9, which are the marker genes in mitochondrial pathway, indicated that overexpression of CXXC5 wouldn’t affected their expression, indicating that after the activation of Caspase8 and then triggering of cascade reaction of Caspase, CXXC5 didn’t induce apoptosis through mitochondrial pathway.7) By luciferase reporter analysis of subcellular co-localization experiments, separation of nucleus and cytoplasm detection, Western blot analysis and COIP experiments, we confirmed that CXXC5 interacted with SMAD3 and SMAD4. Western blot analysis showed that CXXC5 activated the expression of the phosphorylated SMAD3 protein, but didn’t activate the non-phosphorylated SMAD3 and SMAD4 expression. Further analysis indicated that co-transfection SMAD3, SAMD4 and CXXC5 together would activated TNF-a transcriptional activity.These results above suggest that CXXC5 interacted with SMAD3 and SMAD4, and reduced apoptosis through the TNF-a membrane receptors pathway.