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(Circulation Research. 2008;103:1289.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

Iskandar F. Abdullaev^*, Jonathan M. Bisaillon^*, Marie Potier, Jose C. Gonzalez, Rajender K. Motiani, Mohamed Trebak

From Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA

Correspondence to Mohamed Trebak, PhD, Cardiovascular Sciences, MC8, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, NY 12208. E-mail trebakm{at}mail.amc.edu

	Abstract

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Recent breakthroughs in the store-operated calcium (Ca²⁺) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore forming subunit of the highly Ca²⁺-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca²⁺ release-activated Ca²⁺ current (I_CRAC), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na⁺ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I_CRAC in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I_CRAC. Ectopic expression of Stim1 in ECs increased their I_CRAC to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

Key Words: CRAC currents • endothelial cell • Orai1 • SOC channels • Stim1 proliferation

	Introduction

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Store-operated calcium (Ca²⁺) entry (SOCE) is a common and ubiquitous mechanism of regulating Ca²⁺ influx into cells. In virtually all cell types, depletion of endoplasmic reticulum (ER) Ca²⁺ content using sarcoplasmic/ER Ca²⁺ ATPase inhibitors such as thapsigargin activates SOCE.^1,2 Under physiological conditions SOCE is initiated by inositol^1,4,5 triphosphate (IP₃)-mediated depletion of ER Ca²⁺ in response to a plethora of stimuli acting through phospholipase C-coupled receptors. The best characterized SOC current is the Ca²⁺ release-activated Ca²⁺ current (I_CRAC), first recorded in rat basophilic leukemia (RBL) mast cells,³ and later described in other cell types.⁴ SOCE is necessary for the replenishment of ER Ca²⁺ content and is a key regulator of many Ca²⁺-dependent physiological processes.⁴ Recently, high-throughput RNA silencing (siRNA) screens by several laboratories have identified 2 molecules, Stim1 and Orai1, as key components of the I_CRAC pathway in mast cells, lymphocytes, and HEK293 cells.^5–8 On ER Ca²⁺ depletion, Ca²⁺-sensing Stim1 proteins translocate to close proximity of the plasma membrane, where they aggregate into multiple puncta. Strikingly, Orai1 molecules also translocate to the same Stim1-containing structures on store depletion, where they open to mediate Ca²⁺ influx.^9–12

In endothelial cells (ECs), SOCE in response to passive store depletion was reported for several EC types, including human umbilical vein endothelial cells (HUVECs),¹³ bovine/rabbit aorta,^14,15 and bovine pulmonary artery.¹⁶ The electrophysiological profile of the SOC conductance in ECs is unclear with an early study reporting small (<0.5 pA/pF at –80 mV) Ca²⁺-selective I_CRAC-like currents,¹⁷ and others describing larger currents (>5pA/pF at –80 mV).^18,19 Studies reporting endothelial I_CRAC are very scarce, likely because of extremely low current densities (6- to 10-times lower than those reported in Jurkat or RBL cells¹⁷). The molecular composition of the SOC channels in many cell types, and in ECs in particular, remains a highly controversial topic. Several studies have proposed members of the transient receptor potential canonical (TRPC) family, either TRPC1^18–22 or TRPC4,^23–25 to mediate SOCE in ECs. However, it is not clear how nonselective TRPC channels can encode the highly Ca²⁺-selective I_CRAC. In the light of the recent discovery of Stim1 and Orai1 as key players in I_CRAC in mast cells and lymphocytes, we evaluated their involvement in endothelial SOCE and their contribution to EC proliferation. We show that: (1) store depletion in ECs activates the highly Ca²⁺-selective SOC current, I_CRAC, that displays similar electrophysiological characteristics to that recorded from RBL cells; (2) SOCE and I_CRAC in ECs are inhibited by low concentrations of lanthanides and by 2-aminoethoxydiphenyl borate (2-APB); (3) SOCE and I_CRAC are mediated by Stim1 and Orai1, whereas TRPC1 and TRPC4 are not involved; (4) the small I_CRAC in ECs is attributable to low levels of Stim1 in these cells, and Stim1 overexpression generated I_CRAC of similar amplitude to that recorded in RBL cells, demonstrating that Stim1 is limiting in ECs and explaining the sporadic success in reliably recording these currents in the past; and (5) knockdown of Stim1 and Orai1 markedly reduced EC proliferation by inducing cell cycle arrest at S and G2/M phase.

	Materials and Methods

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Cell culture and transfections, Western blots, Ca²⁺ imaging, and patch clamp were performed as previously described,^26–28 and the protocols and compositions of various solutions and buffers are listed in the online supplementary material (http://circres.ahajournals.org). All experiments were performed using standard or slightly modified protocols as described. Details of these protocols along with concentrations of reagents, drugs, compositions of media, and solutions are provided in the online supplement. The list of primers, siRNA and shRNA sequences, and antibodies is also provided.

	Results

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HUVECs and Human Pulmonary Artery ECs Exhibit Classical SOCE
To characterize pharmacological properties of SOCE in ECs, we used Fura-2 Ca²⁺ imaging and thapsigargin (2 µmol/L) to activate SOCE. In the absence of extracellular Ca²⁺, thapsigargin induces a passive Ca²⁺ leak from the ER (Figure 1). When Ca²⁺ was restored to the bath, Ca²⁺ entry through SOC channels occurred. Thapsigargin-induced SOCE was completely inhibited by low concentrations of lanthanides (10 µmol/L Gd³⁺) or by 30 µmol/L 2-APB, reminiscent of SOCE in HEK293²⁹ (Figure 1A and 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1. Thapsigargin activates SOCE in HUVECs. A and B, Store depletion by 2 µmol/L thapsigargin (TG) induces SOCE that is inhibited by 30 µmol/L 2-APB (A) and 10 µmol/L Gd3+ (B). C and D, Thrombin (100 nmol/L) induces SOCE and is inhibited by similar concentrations of 2-APB (C) and Gd3+ (D). E and F, Similar results were obtained with vascular endothelial growth factor (100 ng/mL). Data in each panel are an average of 4 to 12 cells and representative of at least 3 independent experiments.

Physiological stimuli acting through phospholipase C-coupled receptors also activate SOCE in ECs. Thrombin, stimulating a G-protein-coupled receptor, and vascular endothelial growth factor, operating through a receptor tyrosine kinase, activate isoforms of phospholipase C and cause IP₃-mediated Ca²⁺ store depletion. Application of 100 nmol/L thrombin elicited fast and transient cytosolic Ca²⁺ release from the ER (Figure 1C and 1D). Reintroduction of extracellular Ca²⁺ induced typical SOCE that was blocked by Gd³⁺ and 2-APB. Preincubation with the same concentrations of Gd³⁺ and 2-APB induced a complete block of SOCE (Figure II; see supplementary figures at http://circres.ahajournals.org). Similar results were obtained when HUVECs were stimulated by 100 ng/mL of vascular endothelial growth factor (Figure 1E and 1F). Similar results were obtained with another primary EC type; SOCE in human pulmonary artery ECs induced by either thrombin or thapsigargin had the same pharmacological profile (Figure III). We conclude that thapsigargin and phospholipase C-coupled agonists activate SOCE with similar characteristics.

I_CRAC in HUVECs
I_CRAC have a unique set of electrophysiological features that are easily distinguishable from other Ca²⁺ currents.⁴ These currents are very inwardly rectifying, are inhibited by low concentrations of lanthanides (1 to 10 µmol/L Gd³⁺), potentiated by low concentrations of 2-APB (5 µmol/L), and inhibited by higher concentrations (30 to 50 µmol/L 2-APB). I_CRAC is highly Ca²⁺-selective and is negatively regulated by cytosolic Ca²⁺. A standard method for I_CRAC activation in whole-cell mode is intracellular dialysis by high concentrations of the pH-independent, fast Ca²⁺ chelator BAPTA.³⁰ As previously shown,³ passive store depletion by BAPTA led to the activation of typical I_CRAC in RBL cells with a magnitude of 1.25±0.25 pA/pF at –100 mV (n=5). This current was inhibited by low concentrations of Gd³⁺ (10 µmol/L; Figure 2A and 2B). Similar inward currents, although of a much smaller magnitude, developed on intracellular dialysis of HUVECs by BAPTA (0.26±0.04 pA/pF at –100 mV; n=5; Figure 2C and 2D), or extracellular application of thapsigargin (0.36±0.1 pA/pF at –100 mV; n=4; Figure 2E and 2F). These currents were also inhibited by Gd³⁺ (Figure 2C and 2E). Figure 2G shows a statistical comparison of the amplitudes of I_CRAC in RBL and those in HUVECs.

View larger version (18K): [in this window] [in a new window]   Figure 2. ICRAC in HUVEC and RBL cells. A and C, ICRAC activation on intracellular dialysis with BAPTA in RBL and HUVEC, respectively. E, ICRAC activation on extracellular application of thapsigargin (TG; 2 µmol/L). Ramps from –100 mV to +60 mV were taken every 3 seconds, and data points from each ramp were taken at –100 mV and plotted. B, D, and F, Representative ramps taken at time points marked by asterisks in A, C, and E, respectively. *Before application of Gd3+; *after application of 10 µmol/L Gd3+. G, Statistical analysis of maximal current values at –100 mV.

Given the small size of I_CRAC in HUVECs, we sought to amplify its magnitude by performing whole-cell patch clamp in divalent-free (DVF) bath solutions. In DVF conditions, I_CRAC readily conducts Na⁺, mediating a significantly larger conductance.^31–33 These large Na⁺ currents exhibit the unique property of being fast-inactivating over the course of tens of seconds, a process called depotentiation.³⁴ Switching to DVF solution in RBL cells induced large (9.5±1.3 pA/pF at –100 mV; n=6), Gd³⁺-sensitive, 2-APB-sensitive, and rapidly inactivating inward Na⁺ currents (Figure 3A, 3B, and 3G). Using this protocol in HUVECs, we observed a relatively large (1.2±0.3 pA/pF at –100 mV; n=5) Na⁺ inward-current with current voltage relationship typical of I_CRAC (Figure 3D and 3E). As expected, these Na⁺ currents were blocked by Gd³⁺ and 2-APB (Figure 3D, 3E, and 3H). In HUVECs, however, we observed a small remaining linear current (0.50 pA/pF ±0.09 at –100 mV; n=9) that was insensitive to Gd³⁺, possibly representing a leak current (see I/V relationship before and after subtraction in supplementary Figure IV). In addition, Na⁺ currents in both RBL and HUVECs showed the typical depotentiation characteristic of I_CRAC³³ (Figure 3A and 3D; for close-ups see Figure 3C and 3F). We conclude that HUVECs display the archetypical I_CRAC identical to that found in RBL but of a much smaller density (6-fold smaller than RBL).

View larger version (23K): [in this window] [in a new window]   Figure 3. ICRAC in DVF conditions. A and D, Na+ currents recorded in response to repetitive pulses (approximately every min) of DVF bath solutions in RBL and HUVEC, respectively. After whole-cell mode, ramps from –100 mV to +60 mV were applied, and data points taken at –100 mV and plotted. 10 µmol/L Gd3+ was added where indicated. B and E, Representative ramps of DVF-induced Na+ currents in RBL (B) and HUVEC (E). Ramps shown were taken at time points indicated by * in A and D, respectively. C and F, Close-up of DVF-induced Na+ currents (*A and *D) in RBL and HUVECs. G and H, Mean values of maximal Na+ currents at –100 mV before, after application of 10 µmol/L Gd3+, and 30 µmol/L 2-APB in RBL (G) and HUVECs (H).

Molecular Players of SOCE in HUVECs
Recent studies have identified 2 conserved genes that are required for SOCE in lymphocytes, mast cells, and HEK293 cells, Stim1 and Orai1.^5–8,35,36 Stim1 is the Ca²⁺ sensor in the ER that somehow signals the activation of Orai1, a pore-forming subunit of the SOC channel. However, most studies on ECs have suggested either TRPC1 or TRPC4 as SOC channel components. We used siRNA to assess the involvement of Stim1, Orai1, TRPC1, and TRPC4 in endothelial SOC. Knockdown of either Stim1 or Orai1 in HUVECs was achieved using 2 different shRNA and 2 different siRNA sequences (Table I) used individually. SiRNA sequences induced a marked decrease in their target mRNA levels (76.6%±2.09 for Stim1 #1 and 78.6%±3.63 for Orai1 #1; n=3; Figure 4A). SiRNA against either Stim1 or Orai1 lead to 74.2%±6.6 and 58.0%±5.7 decreases in Stim1 and Orai1 proteins levels, respectively, as assessed by Western blotting (Figure 4B). This is likely an underestimation of the knockdown at the single cell level because transfection efficiency of siRNA in HUVECs is unlikely 100%. We assessed the off-targets effect of Stim1 and Orai1 siRNA sequences on the mRNA of TRPC1, TRPC4, Stim2, Orai2, and Orai3. As expected, siRNA targeting either Stim1 or Orai1 induced a decrease in their respective mRNA with no statistically significant effect on other genes (Figure IVB and IVC).

View larger version (26K): [in this window] [in a new window]   Figure 4. SOCE in HUVECs is mediated by Stim1 and Orai1. A, Quantitative polymerase chain reaction data showing decreased expression of Orai1 and Stim1 mRNA in silenced cells compared to control cells (scrambled siRNA), 72 hours after transfection (n=3; 1-way ANOVA, *P<0.05). B, Western blots and densitometry showing significant downregulation of Stim1 (left) and Orai1 (right) proteins 72 hours after siRNA transfection. C, Stim1 and Orai1 knockdown inhibits SOCE in response to thapsigargin. D, SOCE in Stim1-silenced and Orai1-silenced cells can be rescued by overexpression of eYFP-Stim1 and CFP-Orai1, respectively. All traces shown represent average data from 7 to 25 cells from at least 3 independent experiments.

Interestingly, downregulation of either Stim1 or Orai1 using siRNA significantly suppressed both thapsigargin-induced (Figure 4C) and thrombin-induced (not shown, but see Figure V, panel B in online supplement at http://circres.ahajournals.org) SOCE in HUVECs. SOCE was greatly reduced when transfecting cells with siRNA against either Stim1 or Orai1 (data shown for siStim1#1 and siOrai1#1; representative of 2 independent siRNA sequences) by comparison to cells transfected with a scrambled control siRNA (Figure 4C). Similar results were obtained using 2 shRNA sequences used independently (supplementary Figure VA and VB). Similar results were obtained when knockdown of either Stim1 or Orai1 was achieved in human pulmonary artery ECs (supplementary Figure VC). Furthermore, the effect of Stim1 or Orai1 downregulation on SOCE was successfully rescued by ectopic expression of either eYFP-Stim1 or CFP-Orai1, respectively (Figure 4D). Surprisingly, during this rescue experiment, expression of eYFP-Stim1 yielded a much greater SOCE compared to CFP-Orai1. This result implies that in HUVECs the level of Stim1 proteins is limiting. This in fact turned out to be the case as described later.

We determined the effect of Stim1 and Orai1 siRNA on membrane currents activated by store depletion using whole-cell patch clamp. As expected in scrambled control siRNA-transfected cells, store depletion activated I_CRAC in DVF conditions that were sensitive to 10 µmol/L Gd³⁺ (Figure 5A). Either Stim1 or Orai1 silencing by siRNA led to a dramatic reduction of I_CRAC densities, although Orai1 was somewhat more efficient (scrambled siRNA, 1.57±0.34 pA/pF; siStim1, 0.26±0.03 pA/pF; siOrai1, 0.11±0.1 pA/pF at –100 mV; n=3; Figure 5A and 5C). Figure 5B shows typical I/V relationship of I_CRAC recorded in DVF bath solutions from control cells and from cells transfected with either siStim1 or siOrai1.

View larger version (23K): [in this window] [in a new window]   Figure 5. Stim1 and Orai1 mediate ICRAC in HUVECs. A, siRNA against either Stim1 or Orai1 in HUVECs greatly reduced ICRAC recorded under DVF conditions compared to control scrambled siRNA-transfected cells. B, Representative ramps taken at time points indicated by * in A. C, Summary of the whole-cell data recorded from control-siRNA, Stim-1#1-siRNA and Orai1#1-siRNA transfected HUVECs. D, mRNA expression of Stim and Orai isoforms in HUVECs. E, Ca2+ imaging showing thapsigargin-activated SOCE, its potentiation with 5 µmol/L 2-APB, and subsequent inhibition by 50 µmol/L 2-APB; trace is average of 22 cells and representative of 4 independent experiments (1-way ANOVA, *P<0.05).

HUVECs also express mRNA encoding Orai2 and Orai3 (Figure 5D), and it is therefore possible that these 2 proteins contribute subunits to a heteromultimeric SOC channel in HUVECs. We found that thapsigargin-activated SOCE in HUVECs resembles that in HEK293 and RBL cells: it is potentiated by low concentrations of 2-APB (5 µmol/L) and inhibited by high concentrations (50 µmol/L). Only Orai1 possess this peculiar characteristic,³⁷ arguing against an involvement of Orai2 and Orai3.

Small SOCE and I_CRAC in HUVECs Attributable to Limiting Stim1 Levels
Figure 4D suggested that the very small densities of I_CRAC in HUVECs could be attributable to limiting levels of Stim1. Western blots analysis showed that Stim1 protein levels in HUVECs are 8-fold less than those of RBL cells (Figure 6A), providing a possible explanation for the smaller I_CRAC in HUVECs. Indeed, eYFP-Stim1 overexpression in HUVECs was verified by fluorescence microscopy showing typical fibrillar staining (inset in Figure 6B) and by Western blotting (supplementary Figure VI) and shown to induce a large increase in SOCE and 5.7-fold increase in I_CRAC densities at –100 mV (6.89±0.5 pA/pF, n=3 vs 1.2±0.3 pA/pF for control, n=5; Figure 6C and 6D). These data strongly suggest that Stim1 is the limiting factor for SOCE and I_CRAC in ECs.

View larger version (27K): [in this window] [in a new window]   Figure 6. Small ICRAC in HUVECs are attributed to low expression levels of Stim1. A, Western blots showing low Stim1 protein levels in HUVECs compared with RBL cells. Ectopic expression of 1 µg of eYFP-Stim1 cDNA plasmid (B; see inset) in HUVECs dramatically increased SOCE (B) and ICRAC (C). D, Representative ramps of ICRAC in wild-type (WT) and eYFP-Stim1–expressing HUVECs were taken from C, when indicated by *.

TRPC1 and TRPC4 Are not Involved in SOCE and I_CRAC in ECs
Previous data suggested that SOC channels in ECs are encoded by either TRPC1 or TRPC4.^21,25 Two siRNA sequences against either TRPC1 or TRPC4 used separately induced substantial decrease in their respective mRNA levels (56%±3.9 for siTRPC1 #1 and 83%±1.8 for siTRPC4#1; n=3; Figure 7A) and a drastic knockdown of protein levels (88%±1.7 for siTRPC1 and 91±3.2 for siTRPC4; n=3; Figure 7B and 7C). The integral version of the Western blots membranes is shown in supplementary Figure VII, showing antibody recognition of specific bands at the expected molecular weights. Given the previously published data regarding TRPC4 as SOC,²³ and the controversy surrounding the antibodies from Alomone Labs, anti-TRPC4 antibody was further validated by overexpression of human TRPC4 in HUVECs (Figure VIII). However, knockdown of either TRPC1 or TRPC4 failed to affect SOCE (Figure 7D) and I_CRAC (Figure 7E). Figure 7F summarizes data of the amplitude of Ca²⁺ entry using Fura2 imaging (control, 0.57±0.03 ratio units; siTRPC1, 0.61±0.05; siTRPC4, 0.63±0.04; based on 91, 62, and 77 total cells from control, siTRPC1, and siTRPC4, respectively; 12 independent recordings each) and I_CRAC at –100 mV (control, 1.2±0.3 pA/pF; siTRPC1, 1.5±0.5 pA/pF; siTRPC4, 1.4±0.4 pA/pF; n=5) showing no statistical difference between control, siTRPC1, and siTRPC4.

View larger version (32K): [in this window] [in a new window]   Figure 7. TRPC1 and TRPC4 knockdown has no effect on SOCE and ICRAC in HUVECs. A, Quantitative polymerase chain reaction showing decreased expression of TRPC1 and TRPC4 mRNA in silenced cells compared to control cells (scrambled siRNA), 72 hours after transfection. B and C, Western blots and statistical analysis of densitometry on TRPC1 and TRPC4 protein knockdown. D, TRPC1 and TRPC4 silencing has no effect on SOCE in response to thapsigargin in HUVECs. Data are representative of 12 independent experiments. E, ICRAC recorded in DVF conditions (from top to bottom) control-siRNA, TRPC1#1-siRNA, and TRPC4#1 siRNA transfected HUVECs. F, Statistical analysis of Ca2+ entry measured by Fura2 (top) and ICRAC (bottom) from control-siRNA, TRPC1-siRNA and TRPC4-siRNA transfected HUVECs (1-way ANOVA, *P<0.05).

Stim1 and Orai1 Are Involved in EC Proliferation
In human lymphocytes, SOCE is believed to be the sole Ca²⁺ entry involved in response to antigen receptor stimulation and is vital for lymphocyte proliferation.⁵ Therefore, we evaluated the involvement of Stim1 and Orai1 in EC proliferation. Protein knockdown of Stim1, Orai1, or both was achieved using siRNA and EC proliferation in complete media was evaluated by counting cells different days after transfection after trypan blue exclusion. Figure 8A and 8B show that at 96 hours after knockdown, Stim1 inhibited cell proliferation by 23.3%±5.39, whereas Orai1 had a much greater effect (68.8%±3.8); knockdown of both proteins caused a comparable inhibitory effect to that of Orai1 knockdown alone (75.5%±1.7). Propidium iodide staining on day 3 after silencing revealed that Orai1 knockdown increased the proportion of cells at the S and G2/M of the cell cycle (15.25% compared to 7.95% for control; Figure 8C and 8E). Stim1 knockdown had a much smaller effect than Orai1 knockdown (10.53%; Figure 8D). Knockdown of both Stim1 and Orai1 produced an effect similar to that seen with Orai1 knockdown alone (15.67%; Figure 8F).

View larger version (28K): [in this window] [in a new window]   Figure 8. Stim1, Stim2, and Orai1 knockdown inhibit proliferation in HUVECs. A, Four groups of cells were transfected with the following siRNA: control, Stim1, Orai1, and Stim1 plus Orai1. At time 0, 10 000 cells were seeded per well (9.6 cm2) in triplicate. At times indicated, cells were detached and total number of cells per well counted after trypan blue exclusion. B, Data in (A) are represented as fold-increase of cell number compared to time 0. Cell cycle analysis was performed 3 days after transfection with siRNA using propidium iodide staining with flow cytometry in control-siRNA (C), Stim1#1-siRNA (D), Orai1#1-siRNA (E), and Stim1#1 + Orai1#1-siRNA (F) transfected HUVECs. Control not stained with propidium iodide is shown in (C). G, Quantitative polymerase chain reaction showing Stim2 mRNA knockdown, 72 hours after transfection with specific siRNA. H, EC proliferation at 72 hours after transfection with indicated siRNA using the same protocol described in (A). Data are representative of 4 independent experiments (1-way ANOVA, *P<0.05).

Given the relatively smaller effect of Stim1 knockdown on EC proliferation compared to Orai1, we tested whether Stim2 might mediate some of Orai1 actions on EC proliferation. We used 2 siRNA sequences independently against Stim2 (Table I) that substantially decreased Stim2 mRNA levels as measured by quantitative polymerase chain reaction (74.3%±6.0 inhibition for Stim2 siRNA #1; Figure 8G). Figure 8H shows that Stim2 knockdown induced a significant inhibition of EC proliferation 72 hours after transfection (28.8%±1.7 for siStim2 compared to 19.4%±2.4 for siStim1). However, knockdown of both Stim proteins produced a smaller inhibition compared to that of Orai1 knockdown (34.1%±2.3 for siStim1 plus siStim2 compared to 47.7%±1.02 for siOrai1), suggesting that part of the role of Orai1 on EC proliferation is Stim-independent. Similar experiments were performed to assess the role of TRPC channels in EC proliferation. As depicted in Figure IX, Specific siRNA against TRPC1, TRPC4, or TRPC6 substantially inhibited EC proliferation (60.7%±1.2 for siTRPC1; 73.1%±1.21 for siTRPC4; 51.1%±3.18 for siTRPC6), suggesting an important role for these channels in EC proliferation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although SOCE using Ca²⁺ dyes was reported for several EC types, SOC currents, however, are not extensively characterized because of technical difficulties in detecting extremely low current densities in these cells.¹⁷ Here we report that I_CRAC is functionally present in ECs and has similar kinetics, reminiscent of I_CRAC in RBL cells. Although I_CRAC in ECs has a very small density (6-fold smaller than RBL cells), it could be amplified in DVF solutions, as previously shown in other cell types.^4,30,31 Rapid time-dependent inactivation of inward Na⁺ currents (termed depotentiation) on removal of extracellular divalents, strong inward rectification, and inhibition by low concentrations of lanthanides and 2-APB are typical properties of I_CRAC⁴. We propose that I_CRAC is mediating SOCE in HUVECs.

We showed that Stim1 and Orai1 are required for I_CRAC and SOCE in ECs. Endothelial I_CRAC and SOCE were drastically inhibited by silencing of Orai1 and Stim1. SOCE was rescued by exogenous expression of Stim1 and Orai1. Stim1 rescue led to the development of an unusually bigger SOCE compared to Orai1 rescue. Similarly, overexpression of eYFP-Stim1 in HUVECs generated a bigger SOCE and markedly increased I_CRAC. Furthermore, Stim1 protein levels were found much lower in HUVECs compared to RBL cells, strongly suggesting that Stim1 is limiting in the activation of I_CRAC and SOCE in HUVECs.

In this study, we did not observe an involvement of TRPC1 or TRPC4 in SOCE despite complete knockdown of their protein expression. Previous studies on endothelial SOC suggested that TRPC channels can participate in endothelial SOCE.^18–25 Nonselective TRPC1 and TRPC4 were reported to play some role in an endothelial conductance that displayed unusually large currents (>5 pA/pF at –80mV).^18,19,25 In these and other studies, currents were activated by inclusion of either IP₃,^{20, 21, 38} thapsigargin,^19,25,39 EGTA,^19,25,39 low concentrations of the chelator BAPTA (1 mmol/L)²² in the patch pipette, or a combination of these. Whereas I_CRAC is strongly inhibited by intracellular Ca²⁺, TRPC channels are activated downstream of phospholipase C and are positively regulated by IP₃ and IP₃ receptor.⁴⁰ Although our data suggest that endothelial SOC currents are I_CRAC-like and are not mediated by TRPC, we can speculate that under certain patch clamp recording conditions, TRPC1, TRPC4, or both might mediate currents that are activated secondarily as a result of phospholipase C activation in response to cytoplasmic Ca²⁺ increase or by IP₃ included in the patch pipette in the absence of a strong buffer, as suggested by Zarayskiy et al⁴¹ for IP₃-mediated activation of TRPC1. Most of the evidence suggesting a role of TRPC in SOCE is either correlative or based on experiments with blocking peptides or anti-TRPC antibodies.^{19,21,22,25,38,39} Two recent studies on TRPC1 knockout mice have questioned the specificity of anti-TRPC1 antibodies and the role of TRPC1 as a component of SOC channels in smooth muscle⁴² and platelets.⁴³ One study, however, showed that ECs from mice display a store depletion-activated current similar to I_CRAC, and that TRPC4 knockout mice lack this CRAC current in ECs.²³ The reason for the discrepancy between these data and ours is unknown. It is worthwhile to draw an analogy between the results on TRPC4^–/– mice and the data by the Mori group⁴⁴ obtained with DT40 B lymphocytes, where the TRPC1 gene was genetically disrupted. In these cells, SOCE and I_CRAC were lost in the majority of cells (80%). This result suggests that perhaps in the long-term TRPC channels might play an important role in maintaining the components of I_CRAC. Alternatively, the discrepancy could be explained by differences in the protocols or the type of cells used. The study on TRPC4^–/– mice was performed in ECs from a different vascular bed in a different species in which primary cultures of mouse aortic ECs were established using an explant method, with ECs growing out from small pieces of mouse aorta placed on growth factor-enriched Matrigel.⁴⁵

Our results do not conflict with the conclusions of previous studies^{18,19,22,25,46} reporting a role of TRPC1 or TRPC4 in endothelial permeability. Instead, we show that the Stim1/Orai1 pathway is important for cell proliferation. Orai1 knockdown inhibits cell proliferation, reflecting growth arrest at S and G2/M phases. Stim1 and Stim2 knockdown yielded a smaller effect as compared to Orai1 knockdown. This is likely a reflection of a Stim-independent role of Orai1 in controlling EC proliferation. Clearly, further studies are needed to understand the role of Stim, Orai, and TRPC proteins in EC function.

	Acknowledgments

 
The authors gratefully acknowledge Meryem Zannagui for performing the calcium imaging experiments depicted in Figure II.

Sources of Funding

Research in the authors’ laboratory is supported by an NIH early career grant (K22ES014729) to Mohamed Trebak.

Disclosure

None.

	Footnotes

 
*Both authors contributed equally to this work.

I.F.A. and J.M.B. contributed equally to this work.

Original received June 16, 2008; revision received September 27, 2008; accepted September 30, 2008.

	References

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