Store-Independent Orai1/3 Channels Activated by Intracrine LeukotrieneC ₄

Abstract

Rationale Through largely unknown mechanisms, Ca2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca2+ entry (SOCE) has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca2+ entry pathways, its upregulation during VSMC remodeling and its contribution to neointima formation remain unknown. Objective The goal of this study was to determine the agonist-evoked Ca2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon-injury of rat carotid arteries. Methods and Results Ca2+ imaging and patch clamp recordings showed that while the platelet-derived growth factor (PDGF) activates the canonical Ca2+ release-activated Ca2+ (CRAC) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca2+-selective channel contributed by Orai1, Orai3 and STIM1 in the same cells. Unexpectedly, Ca2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling and in vivo Orai3 knockdown inhibits neointima formation. Conclusions These results demonstrate that distinct native Ca2+-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Emerging roles of Orai3 in pathophysiology

“…82,84 We recently reported that Orai3 proteins are also upregulated when VSMC switch to a synthetic phenotype. 21 Interestingly, Orai3 did not contribute to Ca 2+ entry activated by either passive store depletion or physiological stimulation with PDGF; instead, Orai3 was shown to play a crucial role in mediating Ca 2+ entry upon stimulation with another VSMC agonist, thrombin. 21 In synthetic VSMCs, thrombin activated a store-independent pathway contributed by both Orai3 and Orai1 and requiring STIM1.…”

“…21 Interestingly, Orai3 did not contribute to Ca 2+ entry activated by either passive store depletion or physiological stimulation with PDGF; instead, Orai3 was shown to play a crucial role in mediating Ca 2+ entry upon stimulation with another VSMC agonist, thrombin. 21 In synthetic VSMCs, thrombin activated a store-independent pathway contributed by both Orai3 and Orai1 and requiring STIM1. This ARC-like store-independent pathway was highly Ca 2+ selective and required intracellular leukotrieneC 4 (LTC 4 ) produced by LTC 4 synthase downstream of thrombin receptor ligation.…”

“…This ARC-like store-independent pathway was highly Ca 2+ selective and required intracellular leukotrieneC 4 (LTC 4 ) produced by LTC 4 synthase downstream of thrombin receptor ligation. 21 Furthermore, using an in vivo model of vascular injury we showed that Orai3-mediated Ca 2+ selective currents were required for neointimal hyperplasia, suggesting the potential use of Orai3 or LTC 4 -regulated Ca 2+ (LRC) channels as potential targets for therapy of vascular occlusive diseases. The exact stoichiometry of LRC channels, the mechanisms of actions of LTC 4 , how STIM1 regulates LRC channel function and the downstream pathways and transcriptions factors activated by LRC-mediated Ca 2+ signals are important questions that remain unanswered.…”

“…[15][16][17][18][19] Although mammals possess three Orai proteins (Orai1/2/3), 6 Orai1 was uniformly shown to mediate native SOCE and CRAC currents in various cell types. Orai3 is an exclusively mammalian protein 20 that contributes, along with Orai1, to store-independent conductances discussed below 21,22 ( Fig. 1).…”

“…50 We have recently reported a store-independent ARC-like Ca 2+ selective entry channel contributed by Orai1 and Orai3 and activated by the agonist thrombin in primary vascular smooth muscle through generation of the second messenger leukotrieneC 4 (LTC 4 ) downstream receptor stimulation. 21 This LTC 4 -regulated Ca 2+ (LRC) channel requires STIM1 downstream of LTC 4 . LTC 4 acts on LRC channels from the cytosolic side in a manner analogous to a second messenger; the pool of STIM1 involved, the exact role of STIM1 in the activation of this channel and whether LTC 4 acts directly or through intermediary factors remain unknown.…”