CSN5‐mediated nuclear exclusion of p27 in vascular smooth muscle cells contributes to the exacerbation of neointimal hyperplasia by CSN8 hypomorphism
Authors: Giri Samiksha , Suo Chao , Martin Douglas , Wang Xuejun 
Published: May 2022

Background

Neointimal hyperplasia is a common pathological response to vascular injury and is caused by the proliferation and migration of vascular smooth muscle cells (VSMCs). The COP9 signalosome (CSN) is a highly conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8) that functions as the deneddylase for Cullin and thereby regulates ubiquitination. However, CSN subunits may have deneddylation‐independent function. Downregulation of CSN8 was shown to accelerate the growth rate of cultured mouse embryonic fibroblasts but impaired cardiac protein quality control in mice. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. To fill this critical gap, we performed the present study to test the hypothesis that increased CSN5‐mediated nuclear exclusion of p27 contributes to the promotion of VSMC proliferation in injured vessels by CSN8 hypomorphism (Figure 1).

Methods and Results

To produce neointimal hyperplasia in vivo, we subjected adult CSN8 hypomorphic (CSN8‐hypo) and the littermate non‐hypomorphic control mice (CTL) to left common carotid artery (LCCA) ligation and collected the LCCA segment proximal to the ligation for analyses. LCCA ligation induced significant increases in CSN8 proteins in wild type mice (p=0.0109), suggesting an important role of the CSN in vascular pathology. Compared to CTL, the CSN8‐hypo mice displayed more severe neointimal thickening at both 1‐ and 4‐week after ligation, a markedly greater increase in PCNA (proliferating cell nuclear antigen) as revealed by western blot analyses (2.05±0.46 vs. 1.37±0.098, p=0.0214), and a significantly greater prevalence of Ki67‐positive VSMCs as detected by double immunostaining for Ki67 and Sm‐22 at 1 week after LCCA ligation (p=0.005). In cell cultures, in response to platelet derived growth factor (PDGF‐BB), CSN8‐hypo VSMCs displayed an accelerated G0/G1 progression (p=0.04) and greater proliferation as assessed by measuring PCNA (p=0.015) and MTT assays (p=0.009), compared to CTL VSMCs. Furthermore, nuclear fractionation revealed that the nuclear to cytoplasmic ratio for p27 and CSN5 proteins were markedly smaller in hypomorphic mice (p=0.0038; p= 0.0246) and hypomorphic VSMCs (p=0.0045; p=0.027); and native gel electrophoresis revealed a significant increase in the cytoplasmic CSN5 mini complex or free CSN5 by CSN8 hypomorphism (in vivo, in vitro; p<0.05). Interestingly, significant increases in PCNA by CSN8 hypomorphism was attenuated by nuclear export inhibitor leptomycin B (LMB) (p=0.04) but not by CSN deneddylase inhibitor (CSN5i‐3). CSN5i‐3, however, suppressed the PDGF‐BB induced PCNA increases in control VSMCs. This suggests that nuclear export (of p27) rather than changes in CSN deneddylation activity is responsible for greater PDGF‐BB induced PCNA increases in CSN8‐hypo VSMCs.

Conclusion

CSN8 hypomorphism increases VSMC proliferation and promotes neointimal hyperplasia in injured arteries likely through the nuclear exclusion of p27 protein that is mediated by CSN5.