Study On The Anti-apoptotic Activity Of PAI-2 Against TNF-α

Plasminogen activator inhibitor-2 (PAI-2), a member of the serine protease inhibitor (serpin) super family, is a multifunctional protein that has been involved in the regulation of fibrinolysis, bacterial infection, development of immunity system, invasion and metastasis of cancer cells, and in regulation of apoptosis induced by TNF-α. But the exact molecular mechanism by which PAI-2 repress apoptosis triggered by TNF-α still remains unclear. Emerging evidences revealed that urokinase -type plasminogen activator (uPA), receptor of TNF-α (TNFR) and bcl-2 have no relationship with anti-apoptotic activity of PAI-2. And some recent studies have also indicated that PAI-2 is not the substrate of caspase 1, 3, 8, but the activity of serine proteinase inhibition is necessary for PAI-2 to inhibit apoptosis. Additionally, the interhelical region of C and D of PAI-2 and the regulation of tissue transglutaminase (tTG) also seem to play most important roles in mediating the anti-apoptotic activity of PAI-2.Since apoptosis protection by PAI-2 is dependent on a 33 amino acids fragment between helix C and D of PAI-2, through which PAI-2 could interact with some intracellular proteins, we used the yeast two-hybrid system to screen the proteins that could bind PAI-2 during apoptosis. Firstly, a Hela cells cDNA library was constructed during apoptosis induced by TNF-α and amplified according to the manufacture's instructions before use. Secondly, we used the fragment between helix C and D of PAI-2 as the bait to screen this cDNA library. 40 positive clones survived in the Trp/Leu/His/Ade deficient selective mudium and 36 clones showed their β-galactosidase activities verified by the colony-lift filter assay. After the speciality test, 24 positive clones were considered to be the candidates of PAI-2 partners. The results of sequence analysis were submitted to Genbank and the results of homology comparison indicated that 5 proteins could bind PAI-2, including IRF-3, COX7C, PRPF8, PSMB1, PPPSV3. After obtained the full-length cDNA of IRF-3 through RT-PCR, we used co-immunoprecipitation experiments to confirm the interaction between PAI-2 and IRF-3 in HeLa cells. In addition, we have constructed the deletion mutant of PAI-2 (PAI-2-ΔCD), which encodes PAI-2 protein without the interhelicalregion of C and D. The data suggested that this mutant can't interact with IRF-3. Thus we proved that PAI-2 could interact with IRF-3 via its interhelical region of C and D in mammalian cells as well as in yeast.IRF-3 is a transcription factor that can be phosphorylated rapidly and be driven from cytoplasm to nuclear after stimulated, where it associates with the transcriptional coactivator CBP/p300, and stimulates the DNA binding and transcriptional activates virus-inducible genes. But we found IRF-3 still remained in the nucleus during apoptosis induced by TNF-α, and that indicated IRF-3 could not be phosphorylated after treatment with TNF-α. Then, whether the interaction between PAI-2 and IRF-3 will affect the phosphorylation of IRF-3, whether the interaction between them will be related with the antiviral function of PAI-2, and whether this relationship is associated with the anti-apoptotic activity of PAI-2, all these interesting questions warrant further study.Under nomal environments, intracellular PAI-2 spreads evenly throughout the cytoplasm and nuclei in HeLa cells, and there exists no conservative nuclear localization signal (NLS) in its sequence. Thus it is of importance to study how it enters the nucleus and whether the antiapoptotic activity of PAI-2 could be affected by its cellular distribution. In this paper, a PAI-2 mutant, C-terminal of which was inserted with a classical NLS comprising 5 amina acids named Lys-Lys-Lys-Arg-Lys(KKKRK), was generated and cloned in-frame into eukaryotic expression vector, pEGFPC3, producing pEGFPC3-PAI2-NLS.After transfected into Hela cells, different distributions of PAI-2 and PAI2-NLS mutant in Hela cells were observed through fluorescence microscopy. Guided by the nuclear localization...