Effects Of CPNE5 On The Apoptosis And Its Mechanism

The copines are a novel family of ubiquitous Ca2+-dependent, phospholipid-binding proteins. Copines are comprised of two N-terminal C2 domains and a C-terminal A domain (AD). The C2 domains of copines are responsible for calcium and phospholipid-binding. C2 domains also present in proteins such as protein kinase C, phospholipase C and synaptotagmin. Because of A domain, copines may also be involved in protein-protein interactions since they contain a domain similar to the protein-binding'A domain'of integrins. CPNE5 is mapped on chromosome 6p21.1. CPNE5 contains a complete open reading frame of 1782bp which encoding a 593 amino acid protein. Here, we show the studies on CPNE5 gene function, including its regulation in TNF-a induced NF-kB transcriptional activation and the possible mechanisms, and its role in apoptosis.In HEK293 cells, TNF-a treatment significantly increased CPNE5 mRNA expression. Dual-Luciferase Reporter Assay showed that exogenous expressed CPNE5 inhibited the transcriptional activity of NF-κB induced by TNF-a. RT-PCR results showed that overexpression of CPNE5 inhibited mRNA expression of NF-κB downstream genes such as Bcl-2. To investigate the role of C2 domains in the inhibition of NF-κB transcriptional activity, we constructed an AD-GFP expression vector with two C2 domain deletion. We found high expression of AD can significantly inhibit the transcriptional activity of NF-κB induced by TNF-a. The inhibition is dose-dependent with the amount of AD transfection. This indicates that AD domain plays an important role during the inhibition of NF-κB transcriptional activity.To further investigated the inhibitory mechanism, we established the tetracycline induced expression system (Tet-on) of CPNE5 in HEK293 cells. After Zeocin selection, we picked seven pcDNATM 4/TO-CPNE5 T-Rex-293 cell clone. CPNE5 was differentially expressed after 12h induction by tetracycline in these cells. We used the cell clones with the highest CPNE5 expression level to investigate the mechanism of CPNE5 inhibiting NF-κB activity. The translocation of NF-κB subunit P65 from cytoplasm to nucleus is the typical activation of NF-κB. We first detected whether CPNE5 overexpression inhibit the translocation of P65. Both western blot and imunofluorescence results showed that CPNE5 overexpression had no effect on translocation of P65 induced by TNF-a. These results indicate that CPNE5 did not suppress NF-κB transcriptional activity through the classical NF-κB regulatory pathway. However, electrophoretic mobility shift assay (EMSA) result showed that CPEN5 overexpression repressed NF-κB DNA-binding activity. Immunoprecipitation experiments showed that exogenous expressed CPNE5 interact with NF-κB (P65) directly.TNF-a induced activation of NF-κB plays important role in cell survival. To study whether exogenous expression of CPNE5 induces apoptosis through inhibiting the transcriptional activity of NF-κB, we explored the effect of exogenous CPNE5 on apoptosis induced by TNF-a. HEK293 cells were transfected with pcDNA3.1 and pcDNA3.1-CPNE5 respectively and treated with TNF-a, the apoptosis rate were detected by FACS analysis. Compared to the cells transfected with pcDNA3.1, cells transfected with pcDNA3.1-CPNE5 showed higher apoptsis rate. The cells expressing exogenous CPNE5 showed characteristic morphological changes of apoptosis, such as cell shape fuzzy, cell shrinkage, reduced cell volume and so on. Further investigation into the apoptosis signaling showed that PARP, a cutting substrate of caspase, is involved in the above process. Western blot result showed that exogenous expression of CPNE5 promoted PARP cleavage. These results showed that CPNE5 could promote TNF-a induced apoptosis to some degree. Exogenous expression of CPNE5 had no significant effect on CHX (TNF-a–/+) induced apoptosis. CHX is a most common inhibitor of eukaryotic protein translation, which can inhibit the 60S ribosomal subunit peptide transferase activity to prevent protein synthesis. These results showed that exogenous expression of CPNE5 regulated TNF-a induced apoptosis process involved with synthesis of protective protein.Further investigation of the expression of NF-κB downstream genes, we found an interesting result. Without TNF-a induced activation of NF-κB, overexpressed CPNE5 upregulate Bcl-xL expression significantly. Bcl-xL, a member of the Bcl-2 family, is also known to inhibit TNF-a-induced apoptosis. We cloned the cDNA and constructed the eukaryotic expression vector of Bcl-xL. Overexpression of Bcl-xL inhibited the transcriptional activity of NF-κB. Downregulate the expression of Bcl-xL with SiRNA-Bcl-xL weaken the inhibition of NF-κB activity by CPNE5. These results indicate that Bcl-xL may play an important role in inhibition of NF-κB transcriptional activation by CPNE5. This research summarized as: 1. exogenous expression of CPNE5 inhibited the TNF-a induced NF-κB transcriptional activity; A domain of CPNE5 also had this inhibitory effect in dose-dependent manner; 2. CPNE5 had no effect on translocation of P65; CPNE5 did not suppress NF-κB transcriptional activity through the classical NF-κB regulatory pathway, but CPEN5 inhibited DNA binding activity of NF-κB and interacted with P65; 3. CPNE5 promoted TNF-a induced apoptosis but had no effect on apoptosis induced byCHX or TNF-a/CHX; 4. overexpressed CPNE5 upregulate Bcl-xL expression; overexpression of Bcl-xL inhibited NF-κB activity; SiRNA-BclxL weaken the inhibition of NF-κB activity by CPNE5, these indicated that Bcl-xL may be involved in inhibition of NF-κB activity by CPNE5.Our results for the first time revealed that CPNE5 inhibit NF-κB transcriptional activity. CPNE5 might block the DNA binding activity of NF-κB through interacting with p65. In addition, we also found that overexpression of CPNE5 enhance cell apoptosis induced by TNF-a. The upregulation of Bcl-xL expression by CPNE5 might play a certain role in this process.