Store-independent Orai1/3 channels activated by intracrine leukotriene C4: role in neointimal hyperplasia

Abstract

Rationale: Through largely unknown mechanisms, Ca(2+) signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca(2+) entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca(2+) 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 Ca(2+) entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries.

Methods and results: Ca(2+) imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca(2+) release-activated Ca(2+) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca(2+)-selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca(2+) 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 Ca(2+)-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 Ca(2+) signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Figure 1. Thrombin-activated Ca²⁺ entry and currents are additive to SOCE and CRAC, and are store-independent

a; Ca²⁺ imaging experiments showing addivity between thrombin- (100nM) and PDGF-(100ng/mL) activated Ca²⁺ entry pathways. b; Whole- cell patch clamp electrophysiology shows additivity of CRAC currents (activated by dialysis of 20mM BAPTA through the patch pipette for 6min) and thrombin-activated currents (Na⁺ I/V depicted in c and statistics in d). e; ER-Ca²⁺ levels were measured using the ER-targeted FRET sensor Cameleon-D1ER, before and after stimulation with maximal concentrations of thapsigargin (TG 4μM; n=12) and Thrombin (Th 500nM; n=9); only thapsigargin caused a significant decrease in ER Ca²⁺ levels. Whole-cell patch clamp electrophysiology showing the development of PDGF-activated CRAC currents with typical depotentiation in DVF solutions (PDGF 100ng/mL; f). Heparin (3mg/mL) dialysis through the patch pipette for 6 minutes completely abrogated PDGF-activated CRAC (g, h). Heparin dialysis failed to inhibit the development of thrombin-activated currents (i, k). Na⁺ I/V relationships taken from traces (f, g, i) where indicated by the color-coded asterisks are depicted in j.

Figure 2. Thrombin-activated Ca²⁺ entry is mediated by STIM1, Orai1 and Orai3

Efficiency of STIM1, Orai1 and Orai3 protein knockdown after shRNA infection was documented by western blot (a). Representative Ca²⁺ imaging traces in response to 100nM thrombin in VSMCs infected for 7days with lentivirus-encoding a non-targeting shRNA (shNT, control for shOrai3) or an shRNA targeting fly luciferase (shLuc, control for shSTIM1 and shOrai1; see online methods for details), or shRNA targeting either Orai1, Orai3 or STIM1 (b); VSMCs were also transfected (and assayed after 36 hours) with either pore mutants of Orai1 (O1-E106Q), Orai3 (O3-E81Q) or GFP vectors (control) (b). All control traces (shNT, shLuc, GFP) show comparable Ca²⁺ entry to wild type cells and only one trace, shLuc is shown. A representative image showing membrane expression of Orai1-E106Q construct is shown in inset d1. Representative Ca²⁺ imaging traces in response to 100nM thrombin in VSMCs transfected with siRNA sequences targeting Orai2, Orai3 and STIM1 and non-targeting control siRNA (c). d; Statistical summary on several independent Ca²⁺ imaging experiments, including experiments described in b and c. Statistics on Ca²⁺ imaging experiments evaluating the contributions of TRPC1/4/6 channels (the only isoforms expressed in rat VSMCs) to thrombin-activated Ca²⁺ entry are also shown (d). Whole-cell patch clamp electrophysiological recordings showing no additivity between arachidonic acid (AA; 8μM) and thrombin (100nM; e–g). For experiments depicted in e, I/V relationships are also shown (f). Origins of I/V curves on the current traces are indicated by the color-coded asterisks. The statistical summary is also included in g.

Figure 3. Thrombin-activated Ca²⁺ entry and currents are mediated through LTC₄ production

Whole-cell patch clamp electrophysiological recordings testing for additivity between LTC₄ and thrombin (a–c). I/V relationships and statistical summary are shown in b and c respectively. d; Competitive ELISA measurements of LTC₄ concentrations from VSMC cultures of either control cells (t=0) or cells stimulated with 500nM thrombin for a duration of 5 or 15 minutes. e; Cells transfected with siRNA against LTC₄S show significant knockdown of LTC₄S protein and abrogation of thrombin-activated Ca²⁺ entry (f, g). f; Representative Ca²⁺ imaging traces and statistical analysis (g) from cells transfected with either control siRNA or LTC₄S siRNA and stimulated with thrombin. Whole-cell patch clamp electrophysiology in VSMCs infected with lentivirus carrying either non targeting control (shNT) or LTC₄S shRNA (shLTC₄S) showed that dialysis of LTC₄ (100nM) through the pipette was able to activate currents indistinguishable from thrombin-activated currents in control cells and LTC₄S-depleted cells (h, i). Na⁺ I/V relationships (j) and statistical analysis (k) are shown for LTC₄-activated currents. Origins of I/V curves on the current traces are indicated by the color-coded asterisks.

Figure 4. STIM1 is required for LTC₄-activated currents

Whole-cell patch clamp recordings of LTC₄- activated currents (a–d) in VSMCs infected with lentiviral vectors encoding either control shRNA against luciferase (shLuc) or STIM1 shRNA (shSTIM1). Depletion of STIM1 completely abrogated currents activated by inclusion of LTC₄ in the patch pipette (b). Na⁺ I/V relationships are shown for LTC₄- activated currents (c). Statistics on current data are shown in d. Na⁺ I/V relationships are taken from current traces where indicated by the color-coded asterisks.

Figure 5. Orai1 and Orai3 are required for LTC₄-activated currents

Whole-cell patch clamp electrophysiology in VSMCs infected with lentivirus carrying either shRNA against luciferase (shLuc), or shRNA targeting Orai1 (shOrai1). Orai1 knockdown completely abrogated LTC₄-activated Na⁺ currents (b) as compared to control (a). Na⁺ I/V relationships (c) confirm the requirement of Orai1 for LTC₄-activated currents in VSMCs. Statistical analysis is shown in d. Representative western blots showing that shRNA targeting Orai3 does not affect Orai1 protein levels (e, f) while significantly abrogating Orai3 protein expression (g, h). Whole-cell patch clamp electrophysiology in VSMCs infected with lentivirus-encoding either non-targeting control shRNA (shNT) or shRNA targeting Orai3 (shOrai3). Orai3 knockdown completely abrogated LTC₄-activated currents (j) as compared to control (i). The I/V relationships are shown in k. Statistical analysis of patch clamp data is shown in l. Values for current densities represented as mean± range and number of independent recordings for shRNA Control, shRNA Orai1 and shRNA Orai3 are reported in Table 1.

Figure 6. Orai3 and LRC currents are upregulated in VSMC after vascular injury

Whole-cell patch clamp electrophysiological recordings on VSMCs freshly isolated from media of non-injured carotids (a, n=4) or from either media or neointima of injured carotid arteries. Dialysis of LTC₄ through the patch pipette activated Ca²⁺ selective LRC currents only in VSMC isolated from either media (b) or neontima (c) of injured vessels 14 days post-injury. Na⁺ I/V relationships are taken from data points where indicated by asterisks and shown in d. Statistical summary for this experiment is also shown in (e). Lentiviral infection with shRNA targeting Orai3 (shOrai3) after balloon injury prevented up-regulation of Orai3 in injured carotid artery with no significant effect on Orai1 (f–h; n=5). Control non-targeting shRNA (shNT) and Orai3 shRNA (shOrai3) lentiviruses efficiently infected carotid arteries as evidenced by GFP expression in the protein lysate of media and neointima from left (injured) carotid arteries 14 days after injury and infection (f); no GFP signal was detected in the protein lysate from the right (non-injured and non-infected) carotid artery.

Figure 7. In vivo knockdown of Orai3 inhibits LRC currents and neointima formation

Whole-cell patch clamp electrophysiological recordings of VSMCs freshly isolated from the media (a, b) or neointima (c, d) of injured carotid arteries two weeks post injury and transduction treatment with viral particles carrying either a control shRNA sequence (shNT) or a sequence targeting Orai3 (shOrai3). As shown in figure 6 for injured vessels, carotid vessels injured and treated with shNT are characterized by the emergence of a Ca²⁺-selective LRC current activated by intracytoplasmic LTC₄ in both medial (a; n=4) and neontimal VSMCs (c; n=4). This Ca²⁺ selective LRC current was reduced upon Orai3 knockdown with shRNA by ~55% in medial cells (b; n=5) and ~48% in neointimal cells (d; n=5). Na⁺ I/V relationships are taken from traces where indicated by the color-coded signs and are shown in (e, f). Statistical summary is shown in (g). h; H&E staining on vessel cross-sections from control left carotid isolated from a sham-operated rat, and from injured left carotid arteries infected with either control shNT or shOrai3 (scale bar = 200μm). Fourteen days after injury, neointimal growth was evident in left carotids injured and infected with shNT compared to left control non-injured vessels from sham-operated animals. This neointima (N) was visibly inhibited by shOrai3 as compared to shNT. i; The neointimal (N), intimal (I) and medial (M) areas of the carotid cross sections were measured from left injured and virus-treated carotids and from right non-injured and non-infected carotids from the same animals using Image J software and statistical analyses on areas (mm²) are shown. The media/neointima (N/M) ratios for left-injured and virus-treated carotids or intima/media (I/M) ratios of right non-injured and non-infected right carotids from the same animals (j) from 5 independent rats per condition are shown. Statistics on Western blots of medial and neointimal VSMCs and quantification of neointima were performed on 5 rats per condition.