Afadin Promotes Vascular Smooth Muscle Cell Contraction by Interacting With Phospholipase C to Enhance Ca2+ Signaling for Blood Pressure Regulation.

Abstract

Background: Vascular smooth muscle cells (VSMCs) regulate vascular tone and blood pressure. Stimulation of VSMCs with vasoconstrictors, such as AngII (angiotensin II) or norepinephrine, activates the G-protein-coupled receptor-mediated cascade, leading to a hypercontractile state and vascular remodeling. Afadin, an intracellular adaptor protein that mainly localizes at cell-cell junctions, regulates various biological phenomena. However, its role in VSMCs remains unclear.

Methods: VSMCs were isolated from newly generated VSMC-specific afadin conditional knockout mice. A small peptide (7 amino acids) designed in silico to inhibit the afadin-PLC (phospholipase C) β association was administered to the mouse VSMCs and aortic media using adeno-associated virus.

Results: Unlike control mice, afadin conditional knockout mice did not exhibit AngII- or norepinephrine-induced elevation in blood pressure. VSMCs isolated from afadin conditional knockout mice were less responsive to AngII- or norepinephrine-induced cell contractility compared with control VSMCs, as evidenced by reduced release of intracellular Ca²⁺ resulting from lowered production of AngII- or norepinephrine-induced inositol 1,4,5-trisphosphate. Mechanistically, the PDZ (disk large tumor suppressor, zonula occludens-1) domain of afadin was shown to associate with the C terminus of PLCβ, providing support for the localization of PLCβ on the plasma membrane, where it generates inositol 1,4,5-trisphosphate. Furthermore, the newly designed small peptide, which inhibited the afadin-PLCβ association, attenuated AngII-induced cell contractility and intracellular Ca²⁺ release in vitro and blocked AngII-stimulated blood pressure elevation in vivo.

Conclusions: Afadin expression in VSMCs promotes cell contraction by interacting with PLCβ to enhance Ca²⁺ signaling and has potential as a novel molecular target for blood pressure regulation.