Homer binds to Orai1 and TRPC channels in the neointima and regulates vascular smooth muscle cell migration and proliferation

Abstract

The molecular components of store-operated Ca2+ influx channels (SOCs) in proliferative and migratory vascular smooth muscle cells (VSMCs) are quite intricate with many channels contributing to SOCs. They include the Ca2+-selective Orai1 and members of the transient receptor potential canonical (TRPC) channels, which are activated by the endoplasmic reticulum Ca2+ sensor STIM1. The scaffolding protein Homer assembles SOC complexes, but its role in VSMCs is not well understood. Here, we asked whether these SOC components and Homer1 are present in the same complex in VSMCs and how Homer1 contributes to VSMC SOCs, proliferation, and migration leading to neointima formation. Homer1 expression levels are upregulated in balloon-injured vs. uninjured VSMCs. Coimmunoprecipitation assays revealed the presence and interaction of all SOC components in the injured VSMCs, where Homer1 interacts with Orai1 and various TRPC channels. Accordingly, knockdown of Homer1 in cultured VSMCs partially inhibited SOCs, VSMC migration, and VSMC proliferation. Neointimal area was reduced after treatment with an adeno-associated viral vector expressing a short hairpin RNA against Homer1 mRNA (AAV-shHomer1). These findings stress the role of multiple Ca2+ influx channels in VSMCs and are the first to show the role of Homer proteins in VSMCs and its importance in neointima formation.

TRPC Channels in the SOCE Scenario

“…The activation of TRPC1 requires electrostatic interaction between highly positively charged lysines ( 684 KK 685 ) located in polybasic lysine-rich domain (K-domain) of the STIM1 C-terminus with the conserved, negatively charged, aspartate residues in TRPC1 ( 639 DD 640 ) and equivalent residues in other TRPC channels [25]. However, there is no evidence about the domains of Orai1 and TRPC1 involved in their interaction, suggesting that TRPC1-Orai1 binding could be indirectly mediated by STIM1 or still unidentified adaptor proteins [68,69].…”

Distinct and Overlapping Expression Patterns of the Homer Family of Scaffolding Proteins and Their Encoding Genes in Developing Murine Cephalic Tissues

“…Ca 2+ signaling is a fundamental mechanism involved in a multitude of cellular processes, including proliferation, differentiation, apoptosis, gene transcription and specialized cell functions such as neuronal activity, secretion, contraction [62] and taste perception [63]. Homer proteins are known to play key roles in various types of Ca 2+ signaling by directly or indirectly modulating the activities of Ca 2+ -handling proteins in a wide variety of cell types, including neurons [7,8,13,14,37,38,64], acinar cells of the pancreas [32] and parotid glands [33], platelets [39], striated [34,58] and smooth [40] muscle, T cells [65], osteoblasts [66] and osteoclasts [67].…”

“…Furthermore, it has been shown that the stratum intermedium and papillary layer express PMCA1 and that ameloblasts express both PMCA1 and PMCA4 [42]. (3) Previous studies have shown that Homer proteins bind to IP3Rs [7,12], that Homer1 interacts directly with Stim1 and indirectly with Orai1 in a Ca 2+ -dependent way during agonist-induced SOCE activation in human platelets [39] and that in rat balloon-injured arteries Homer1 binds Orai1 channels and is required for SOCE in vascular smooth muscle cells [40]. (4) Finally, Homer proteins have been shown to bind PMCAs [33,37,80] and to modulate PMCA-mediated Ca 2+ -clearance in rat hippocampal neurons [38] and in mouse salivary gland acinar cells [33].…”

“…It is noteworthy that endothelial cells respond to pro-angiogenic signals by increasing intracellular Ca 2+ concentration as a result of activation of the SOCE mechanism and other Ca 2+ channels, including TRPC channels [100]. Besides directly interacting with key proteins of SOCE [39,40,95], Homer proteins also bind and modulate the activity of TRPC channels [11,101,102]. Other binding targets of Homer proteins include Drebrin [75,76], an F-actin-binding protein with a key role in maintenance of endothelial integrity [103].…”

“…In addition to binding and modulating the activities of IP3Rs, RyRs and TRPC channels [7,8,14,36], Homer proteins also interact physically and/or functionally with other Ca 2+ -handling proteins, such as the plasma-membrane Ca 2+ -ATPase (PMCA) pumps [33,37,38] as well as with Orai1 and Stromal interaction molecule 1 (Stim1), key proteins of the store-operated Ca 2+ entry (SOCE) system [39,40], a ubiquitous physiological process with a crucial role in the regulation of extracellular Ca 2+ influx into cells and a major mechanism of Ca 2+ influx in non-electrically excitable cells [8,41].…”