Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways

Abstract

Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications.Methods and Results: Myocardial transcriptomes of Cops8flox/flox, Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways.Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.

Role of the COP9 Signalosome (CSN) in Cardiovascular Diseases

“…Furthermore, the autophagosomal phenotype of Csn8 -deficient hearts of adult mice was linked with increased levels of oxidized proteins, necrotic cardiomyocytes, and morphological and functional changes characteristic of dilated cardiomyopathy [206]. To further understand how Csn8 -deficiency leads to cardiomyocyte necrosis, a perinatal myocardial transcriptome analysis was performed in another study with a focus on differentially expressed genes and different pathways including the CRL substrate receptors (SRs), chromatin remodeling genes, autophagy, vesicle trafficking, endocytosis, and cell death, which compare homozygous myocardial-restricted Csn8 -deficient mice with corresponding heterozygous mice and a control group [207]. That study and another one indicated that myocardial CSN8 not only acts through post-translational pathways but also on a transcriptional level [207,208].…”

The COP9 Signalosome Suppresses Cardiomyocyte Necroptosis

“…Indeed, our prior transcriptome analysis has revealed that Nrf2 target genes are markedly upregulated in Cops8 CKO hearts. 43 Here our further work detected that myocardial protein levels of total Nrf2 and Ser40-phosphorylated Nrf2 (pS40-Nrf2) were significantly increased in Cops8 CKO mice at 2 and 3 weeks of age, compared with littermate controls ( Figure 8A~8C ). Phosphorylation of Nrf2 by protein kinase C (PKC) at Ser40 is known to promote Nrf2 nuclear translocation and increase its target gene expression; 44 hence, the increases in pS40-Nrf2 are consistent with increased Nrf2 transactivation in Cops8 deficient hearts as we previously detected via transcriptome profiling.…”

“…A surprising discovery of this study is that the sustained activation of Nrf2 in CMs promotes CM necroptosis and mouse premature death in the Cops8 CKO mice. Our prior transcriptome analysis has revealed a marked upregulation of Nrf2 target genes in Cops8 CKO hearts at both 2 and 3 weeks of age, 43 indicative of Nrf2 activation by Cops8 deficiency. The sustained activation of Nrf2 is reflected further by increases in both pS40-Nrf2 (an active form of Nrf2) and total Nrf2 protein levels in Cops8 CKO mouse hearts at both 2 and 3 weeks of age ( Figure 8A ~ 8C ) and by increased proteins and mRNA expression of BCL2 ( Figure 7D, 7E ), a known Nrf2 target gene.…”

“…Phosphorylation of Nrf2 by protein kinase C (PKC) at Ser40 is known to promote Nrf2 nuclear translocation and increase its target gene expression; 44 hence, the increases in pS40-Nrf2 are consistent with increased Nrf2 transactivation in Cops8 deficient hearts as we previously detected via transcriptome profiling. 43…”

Screening and Authentication of Key Genes and Prognostic Value Analysis in Oligodendroglial Tumors

“…The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases [32]. At the same time, some ndings reported obviously challenge this theory.…”