Calpain 6 Expression And Biological Functions Regulated By PI3K-Akt Pathway And Its Molecular Mechanism

The calpains are a family of cysteine proteases implicated in various biological processes whose activities are highly dependent on Ca2+. The family includes classical calpains and non-classcial calpains. The genes and the encoded calpain proteins by the classical calpains include a penta-EF-hand type of calcium-binding domain with sequence relatedness to domain IV of calpain 1 or 2 and domain VI. This group includes calpainl,2,3,8,9, and 11. These "classical" calpains are ubiquitously expressed and are implicated in various cellular functions, such as migration, apoptosis, cell growth, and cell cycle progression. Non-classical calpains, lacking a penta-EF-hand domain, includes calpain 5,6,7,10,12 and 14. Calpain 6 (CAPN6), one member of non-classical calpains, is located on the X chromosome, expressed during embryogenesis and its activity is rapidly down-regulated after birth. Recent reports show that CAPN6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization and supports carcinogenesis by inhibiting apoptosis and facilitating angiogenesis. Additionally, emerging studies show that CAPN6 expresses in tumor tissues. In addition, it lacks the active-site cysteine residues requisite for protease activity, which determines that its function and regulation may be different from those of the classical calpains. Our previous study showed that CAPN6 was up-regulated in the Akt transformed MEF cells by cDNA chip and suggests that CAPN6 may be related to Akt. So we investigated the mechanism underlying elevated CAPN6 expression by PI3K-Akt signaling pathway and its biological functions.In the first section, to determine the contribution of PI3K-Akt in CAPN6 protein expression, HeLa,7404 and 293T cells were treated with the PI3K inhibitor LY294002, and we found a time-dependent decrease in CAPN6 protein levels concomitant with an inhibition of PI3K activity as revealed by the loss of phosphorelated Akt (pS473). We further verified that the suppression of PI3K by LY294002 in many cancer cells led to the down-regulation of CAPN6 expression. Next, we used insulin-like growth factor-1 (IGF-1) potently activating Akt in cancer cells, and observed increased CAPN6 protein levels concomitantly with enhanced Akt phosphorylation. Conversely, inactivation of PTEN by RNAi, which resulted in elevated Akt activity, led to up-regulation of CAPN6 in HeLa cells. We found that CAPN6 protein decreased in 7404 cells of Akt 1/2 deletion using lentivirus mediated RNAi stably or an Akt inhibitor and the MEF cells over-expressed Akt led to CAPN6 up-regulation. To verify whether PI3K-Akt influences CAPN6 expression on mRNA level, we observed that CAPN6 mRNA increased strongly in MEFs stably expressing Akt using real time RT-PCR and decreased in cells treated with LY294002. The results were verified in many human cancer cell lines. Next, we determinated how PI3K-Akt regulated CAPN6 expression mechanistically. We found that inhibition of PI3K by LY294002 decreased CAPN6 mRNA. We also observed that CAPN6 mRNA decreased after activation of either Aktl or Akt2 by RNAi in 7404 cells. We also found that inhibition of GSK-3?induced down-regulation of CAPN6 protein.To address whether the PI3K-Akt pathway affects the stability of endogenous CAPN6 protein, in the second section, we treated the cells with CHX (a protein synthesis inhibitor) in various time, respectively. We found that inactivation of PI3K with LY294002 shortened the half-life of endogenous CAPN6. To further investigate whether Akt plays a role in CAPN6 protein stability, cells were infected with lentivirus of Akt 1/2 shRNA, and the result showed that Akt did not influence the process. CAPN6 protein increased in 7404 cells treated with MG132 (an inhibitor of both proteasome and calpain), and LY294002 or LiCl promoted the degradation of CAPN6, but rapamycin did not. The results suggest that PI3K-Akt inhibites the degradation of CAPN6 protein through proteasome by GSK-3?.To define the CAPN6 promoter region, in the third section, we constructed a series of CAPN6 promoter plasmids, induced them into the 293T and 7404 cells and found that the -93/+200 construct had the highest reporter activity measured by dual-luciferase reporter system, and the others showed less reporter activity. These supported that the -93/+200 fragment represented the core promoter of CAPN6, and potentially implied the presence of additional upstream silencing elements. We found that LY294002 could inhibit the promoter activity, so did Akt down-regulation by RNAi. The data further verified the PI3K-Akt pathway could regulate CAPN6 promoter activity on the transcriptional level. And then we searched the transcriptional factors by TFSEARCH and Match softwares that may regulate the CAPN6 gene promoter. According to the predicted transcriptional factors, we mutated the potential binding sites including API, VBP, FOXD3, OCT-1, HNF-3?, and ZEB1. The mutated promoters of API, FoxD3, OCT-1, and HNF-3?showed greatly decreased activity compared with the-93/+200 construct, the others showed less decrease than the above mutated ones. To determine the functional significance of transcription factors including API, FoxD3 and Oct-1 for the CAPN6 core promoter (-93bp/+200bp) activity, we employed RNAi to knockdown API, Oct-1 or FoxD3 in HeLa and 7404 cells respectively, and performed luciferase reporter assays. Transfection of API, Oct-1, or FoxD3 siRNAs together with the-93/+200 construct led to an approximately different decrease in reporter activity as compared with the control. We also could observe a stronger activity decrease when any of FoxD3, Oct-1, and API knocking down combined in decreasing the promoter than any of them alone to decrease the promoter activity. The results suggest that not only one transcription factor played roles in CAPN6 gene transcription. And then we found that CAPN6 mRNA and protein were down-regulated by knocking down API, Oct-1, and FoxD3 respectively and the three transcription factors were binded with CAPN6 gene promoter by Chip. These results clearly indicate that the three transcription factors are the activators of transcription of the CAPN6 gene.To clarify the role of CAPN6 regulated by PI3K-Akt in carcinogenesis, in the fourth section, cancer cellular biological functions were performed. The results showed that 7404 and HeLa cells with CAPN6 down-regulation were less proliferative than their parent cells by the method of colony formation. The cell cycle analysis showed S phase decreased in the cells with CAPN6 down-regulation compared with their parent ones and the cell cycle regulatory protein cyclinDl decreased, which suggested that CAPN6 could promote cellular proliferation. Cell apoptosis was performed using AnnexinV by flow cytometry, and the results showed that CAPN6 increased apoptosis resistance, and the apoptosis regulatory proteins Bcl-2 was up-regulated, caspase 3 decreased. The results of cell migration showed that there was no significant change in cells with CAPN6 over-expression or down-regulation comparing with their parent ones. The EMT regulatory proteins such as E-cadherin or N-cadherin did not change. These indicate that CAPN6 may not involve in migration and EMT.