Vascular smooth muscle RbFox2 regulates the cytoskeleton and arterial stiffness by a RhoBTB1/Cullin-3 mechanism

The RhoBTB1/Cullin-3 (CUL3) pathway in smooth muscle cells (SMCs) controls the ubiquitination and proteasomal degradation of target proteins that regulate vasodilation, vasoconstriction, and the actin cytoskeleton and, through this, blood pressure (BP) and arterial stiffness. Using proximity labeling coupled with mass spectrometry in A7R5 SMCs, we identified proteins that bound to the C-terminal half of RhoBTB1, which functions as an adaptor to deliver substrates to CUL3. We examined the physiological relevance of one of these substrates, RbFox2. Coimmunoprecipitation validated the interaction of RbFox2 with RhoBTB1. RbFox2 expression was elevated in response to inhibition of the ubiquitination-proteasomal pathway, CUL3 deficiency, and RhoBTB1 inhibition by either siRNA or angiotensin II (ANG). RbFox2 was ubiquitinated in a RhoBTB1- and CUL3-dependent manner, suggesting its regulation through the RhoBTB1/CUL3-dependent ubiquitin-proteasome pathway. Inhibition of RbFox2 impaired the actin cytoskeleton in A7R5 cells and in primary SMCs from RbFox2fl/fl mice and decreased the levels of globular and filamentous actin. ANG increased BP and arterial stiffness of RbFox2fl/fl mice, but the progression of arterial stiffness was halted after SMC-specific RbFox2 deletion despite a continued rise in BP. We conclude that RhoBTB1 and RbFox2 are important regulators of arterial stiffness through a mechanism that influences cytoskeletal integrity.