Knockdown of CSN3 inhibits skeletal muscle differentiation and alters cardiomyocyte hypertrophy
AuthorBa, Mariam A.
AdvisorValencik, Maria L.
Biochemistry and Molecular Biology
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Cardiac hypertrophy is an early marker of heart failure, and understanding the signaling pathways involved in stimulating the pathological hypertrophic process may lead to the discovery of novel therapeutic strategies to prevent or attenuate the development/prevalence of heart disease. The COP9 signalosome (CSN) is a highly conserved multifunctional complex consisting of 8 subunits. It has been hypothesized that CSN is involved in the ubiquitin-dependent degradation of regulators of cardiac/striated muscle gene expression/protein stability. Our lab has shown that CSN3, the third subunit of CSN, binds β1D-integrin in adult cardiac myocytes and is up regulated during muscle differentiation. CSN3 also binds to kinases that phosphorylate c-Jun and nuclear factor kappa of activated B cells (NF-κB). Additionally, CSN3 has been shown to be phosphorylated on serine residues 410, 421 and 423 and although, the physiological significance of these phosphorylations is unclear, they may play a role in the regulation of CSN function. The main goal of this study is to investigate the role of CSN3 in skeletal muscle differentiation and cardiac hypertrophy.To determine the role of CSN3 in skeletal muscle differentiation, lentiviral RNA interference was used to generate two CSN3 stable knockdowns expressing low (7%) and intermediate (43%) levels of CSN3 as well as a stable non-target control C2C12 cell line. Cells expressing low and medium levels of CSN3 are referred to as shCSN3-Low and shCSN3-Med respectively. The effects of CSN3 knockdown on myoblast fusion, the expression of molecular markers (myogenin and myosin heavy chain) of differentiation and skeletal muscle maturation were evaluated. Non-target control cells formed thick elongated multinucleated myotubes, accompanied by up regulation of MHC protein levels indicative of skeletal muscle maturation. Whereas, shCSN3-Med cells showed thinner shorter myotubes with fewer nuclei and shCSN3-Low cells showed high levels of myogenin but did not fuse. Interestingly, both CSN3 knockdowns failed to express MHC throughout the differentiation period indicating impaired myotube maturation. Knockdown of CSN3 resulted in the destabilization of other CSN subunits including CSN1, CSN2, CSN5 and CSN8. These results demonstrate that knockdown of CSN3 destabilized the CSN holo-complex and impaired skeletal muscle differentiation. We also investigated whether CSN3 was involved in C2C12 proliferation and cell cycle progression. Indeed, knockdown of CSN3 decreased C2C12 proliferation rate and led to cell cycle arrest in S phase. We further examined if CSN3 signals via integrin pathways that activates the AP-1 and/or NF-κB. Preliminary phosphorylation data indicated that knockdown of CSN3 reduces the phosphorylation levels of focal adhesion kinase (FAK) and NF-κB, but favors c-Jun phosphorylation.To investigate the involvement of CSN3 in integrin-mediated cardiac hypertrophy, adenoviruses encoding CSN3 shRNA and a negative control shRNA were generated and used to infect neonatal rat ventricular myocytes (NRVMs). Cardiac hypertrophy was induced with phenyephrine (PE), an α1 adrenergic agonist, and hypertrophy was evaluated based on cell size, atrial natriuretic peptide (ANF) expression and cytoskeletal reorganization. Prior to PE treatment, CSN3 knockdown cells showed increased ANP expression and increased cytoskeletal organization compared to the non-target viral controls. PE stimulation caused an increase in ANP expression and increased cytoskeletal reorganization in NT viral control cells. However ANP expression, cytoskeletal reorganization and cell size remained unchanged in CSN3 knockdown cells in response to PE treatment. Surprisingly, infection of NRVMs with non-target adenovirus alone was sufficient to increase cell size and ANP production, making it difficult to determine the effects of CSN3 on cell size. These findings suggest that knockdown of CSN3 in the absence of PE caused some of the changes associated with cardiomyocyte hypertrophy.To investigate if CSN3 phosphorylation regulated the β1D-CSN3 interaction described by Hunter et al., we introduced mutations into all potential CSN3 phosphoserine sites. These mutations involved the substitution of serine residues at position 410, 421 or 423 to alanine or aspartic acid to create CSN3 phosphoresistent and phosphomimetic constructs respectively. In the future we hope to use these constructs to evaluate the effects of CSN3 phosphorylation on the CSN3-β1D integrin interaction. Our results show for the first time that CSN3 participates in muscle differentiation. These studies are crucial for a better understanding of integrin-mediated pathways leading to striated muscle differentiation.