CYLD Deubiquitinates Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Contributing to Adventitial Remodeling

Transdifferentiation of adventitial fibroblasts (AFs) into myofibroblasts plays a critical role during the vascular remodeling that occurs during atherosclerosis, restenosis, and aortic aneurysm. The ubiquitination/deubiquitination regulatory system is essential for the quality control of proteins. The involvement of ubiquitination/deubiquitination during AF transdifferentiation remains largely unknown. In this study, we determined the role of cylindromatosis (CYLD), a deubiquitinase, in the process of AF differentiation and activation in vitro and in vivo.

Transforming growth factor-β1 and homocysteine, 2 known inducers of AF transdifferentiation, greatly upregulated CYLD expression in a time- and dose-dependent manner. The silencing of CYLD significantly inhibited AF transdifferentiation and activation as evidenced by the expression of contractile proteins, the production of the proinflammatory cytokines MCP-1 (monocyte chemotactic protein 1) and IL-6 (interleukin-6), the deposition of extracellular matrix, and cell migration. We further asked whether CYLD mediates AF activation via the regulation of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) as it is an essential factor during AF transdifferentiation. Indeed, the silencing of CYLD repressed transforming growth factor-β1-induced and homocysteine-induced Nox4 upregulation and reactive oxygen species production, whereas Nox4 overexpression greatly rescued the inhibitory effect on AF activation by CYLD silencing. Most interestingly, transforming growth factor-β1 and homocysteine repressed Nox4 ubiquitination and prolonged the half-life of Nox4. Moreover, Nox4 was deubiquitinated via a direct interaction with the ubiquitin-specific protease domain of CYLD. In accordance, hyperhomocysteinemia significantly increased adventitial CYLD and Nox4 expression, promoted AF transdifferentiation, and aggravated CaPO₄-induced abdominal aortic aneurysm in mice. These effects were abolished in CYLD^-/- mice.

CYLD contributes to the transdifferentiation of AFs via deubiquitinating Nox4 and may play a role in vascular remodeling.