Abstract 191: Cops5-mediated Nuclear Exclusion Of P27 Is Responsible For The Promotion Of Vascular Smooth Muscle Cells Proliferation By Cops8 Hypomorphism

Abstract

Background: The COP9 signalosome (CSN) holocomplex is formed by 8 stoichiometric protein subunits: COPS1 through COPS8. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. Our prior data show that vascular injury significantly increases Cops8 proteins in wild-type mice and induces more severe neointimal thickening and increased VSMC proliferation in Cops8 hypomorphic (Cops8-hypo) mice compared to non-hypomorphic littermates (CTL), suggesting an important role of the CSN in vascular pathology. The present study was carried out to further elucidate the mechanism underlying this phenomenon. We hypothesize that increased COPS5-mediated nuclear exclusion of p27 contributes to the promotion of VSMC proliferation by COPS8 hypomorphism. Methods and Results: We isolated and cultured VSMCs from the COPS8-hypo and CTL mice and used the treatment with platelet-derived growth factor (PDGF-BB) to mimic vascular injury. In response to PDGF-BB, COPS8-hypo VSMCs displayed increased hyperproliferation compared to CTL VSMCs. We detected markedly smaller nuclear to cytoplasmic ratio for p27 and COPS5; and native gel electrophoresis revealed increases in the cytoplasmic COPS5 mini-complex in hypomorphic VSMCs. Nuclear export inhibition with leptomycin attenuated the increases in PCNA and cytoplasmic p27 by COPS8 hypomorphism. Interestingly, CSN deneddylase inhibitor (COPS5i-3) did not have an effect on the proliferation of COPS8-hypo VSMCs but suppressed the PDGF-BB induced PCNA increases in CTL VSMCs. We then, constructed plasmids expressing full-length COPS5 harboring a mutation either in its nuclear export sequence (NES) or in its MPN domain. We found that genetically disabling the nuclear-export but not the deneddylase activity of COPS5 suppressed the hyperproliferation and restored p27 nuclear localization of hypomorphic VSMCs. These data suggest that promoting the nuclear export of p27 by COPS5 rather than the deneddylation activity of COPS5 is responsible for the greater PDGF-BB induced proliferation of COPS8-hypo VSMCs. Conclusion: COPS8 hypomorphism promotes the proliferation of VSMCs in injured vessels through the increased nuclear exclusion of p27 mediated by COPS5.