Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

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Article

Reference

Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent,

TRPC3/PLC/PKC-mediated pathways in human endothelial cells

ANTIGNY, Fabrice, et al .

Abstract

The ER Ca²+ sensor STIM1 and the Ca²+ channel Orai1 are key players in store-operated Ca²+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca²+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca²+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca²+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca²+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca²+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca²+ entry by more [...]

ANTIGNY, Fabrice, et al . Thapsigargin activates Ca²+ entry both by store-dependent,

STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells. Cell Calcium , 2011, vol. 49, no. 2, p. 115-27

DOI : 10.1016/j.ceca.2010.12.001 PMID : 21193229

Available at:

http://archive-ouverte.unige.ch/unige:40242

Disclaimer: layout of this document may differ from the published version.

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Cell Calcium49 (2011) 115–127

Contents lists available atScienceDirect

Cell Calcium

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / c e c a

Thapsigargin activates Ca ²⁺ entry both by store-dependent,

STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

Fabrice Antigny

^a

, Hélène Jousset

^a

, Stéphane König

^b

, Maud Frieden

^a,∗

aDepartment of Cell Physiology and Metabolism, University of Geneva Medical School, 1 rue Michel Servet, 1211 Geneva 4, Switzerland

bBasic Neurosciences, University of Geneva Medical School, 1 rue Michel Servet, 1211 Geneva 4, Switzerland

a r t i c l e i n f o

Article history:

Received 17 September 2010 Received in revised form 11 November 2010 Accepted 2 December 2010 Available online 28 December 2010

Keywords:

Store-operated Ca²⁺entry STIM

Orai TRPC PLC PKC

Endothelial cells

a b s t r a c t

The ER Ca²⁺sensor STIM1 and the Ca²⁺channel Orai1 are key players in store-operated Ca²⁺entry (SOCE).

In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926.

siRNA directed against STIM1 or Orai1 reduced Ca²⁺entry by about 50–60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca²⁺entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca²⁺entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCetais required for Ca²⁺

entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca²⁺entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca²⁺entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca²⁺entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.

1. Introduction

Store-operated Ca²⁺entry (SOCE) is a ubiquitous mechanism of regulated Ca²⁺inﬂux, which is getting activated upon Ca²⁺store depletion[1]. By contributing to cytosolic Ca²⁺elevation and to the replenishment of the ER Ca²⁺ compartment[2,3], SOCE is a key regulator of many Ca²⁺dependent physiological processes[4].

Under physiological conditions, SOCE is induced by the activation of cell surface receptors coupled to e.g. phospholipase C (PLC) that leads to the generation of inositol 1,4,5-trisphophate (IP3), and the release of Ca²⁺from the endoplasmic reticulum (ER)[5].

The extent to which SOCE is engaged during agonist stimulation might greatly vary depending on the level of ER Ca²⁺ depletion [6,7]. Experimentally, SOCE is frequently stimulated by blocking the sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCA), with drugs

Abbreviations: SOCE, store-operated Ca²⁺ entry; IP3, inositol 1,4,5- trisphosphate; ER, endoplasmatic reticulum; SERCA, sarco-endoplasmic reticulum Ca²⁺ATPase; TG, thapsigargin; PKC, protein kinase C; DAG, diacylglycerol; PLC, phospholipase C; TRPC, canonical transient receptor potential.

∗Corresponding author. Tel.: +41 22 379 5198; fax: +41 22 379 5338.

E-mail address:maud.frieden@unige.ch(M. Frieden).

like thapsigargin (TG) that passively deplete the ER. During the last 20 years, the family of TRPC (canonical transient receptor potential) channels has been proposed as candidates for SOCE. Number of reports has shown a reduced SOCE activity when TRPC expression was knocked down or knocked out[8–11]. When exogenously expressed some TRPC channels displayed SOCE activity[12–14].

However, numerous studies also demonstrated that TRPC channels did not operate as SOCE channels, but rather are gated by sec- ond messengers like Ca²⁺or diacylglycerol (DAG;[15–17]). Hence, the involvement of TRPC channels in the SOCE mechanism is a controversial issue, and appears to dependent on the cell types investigated, and importantly on the expression level of the TRPC [14,18,19].

More recently, two major SOCE components have been iden- tiﬁed: the Stromal Interacting Molecule 1 (STIM1) [20–22]

predominantly located in the ER membrane and that senses the luminal Ca²⁺concentration, and the Ca²⁺channel Orai1 (also known as CRACM;[23–25]). Upon Ca²⁺store depletion, STIM1 oligomer- izes, translocates close to the plasma membrane and aggregates into punctate structures[2,21,26]. The interaction between STIM1 and Orai1 leads to channel opening and Ca²⁺entry[23,25,27]. An additional level of complexity emerged as several recent reports showed that STIM1 binds to TRPC1, TRPC4 and TRPC5 [28]and indirectly regulates TRPC3 and TRPC6 [29]. Furthermore, it was 0143-4160/$ – see front matter© 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ceca.2010.12.001

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Table 1

Sequences of siRNA and primers used for the real time RT-PCR (all sequences are oriented 5–3).

Sense Antisense From

siRNA

STIM1 GGCUCUGGAUACAGUGCUCtt GAGCACUGUAUCCAGAGCCtt Ambion

Orai1 CGUGCACAAUCUCAACUCGtt CGAGUUGAGAUUGUGCACGtt Ambion

TRPC1 CGGACUUCUAAAUAUGCUAtt UAGCAUAUUUAGAAGUCCGAAtt Oiagen

TRPC3 GCAGCAGCUCUUGACGAUCUGGUAU AUACCAGAUCGUCAAGAGCUGCUGC Invitrogen

TRPC4 UGUCUAUGUUGGAGAUGCUCUAUUA UAAUAGAGCAUCUCCAACAUAGACA Invitrogen

TRPC5 CCCAAGUCAUUUCUAUACCUUGGUA UACCAAGGUAUAGAAAUGACUUGGG Invitrogen

TRPC6 CCCAAGGAAUAUUUGUUUGAGUUGU ACAACUCAAACAAAUAUUCCUUGGG Invitrogen

PKC␩ CCAGAUUUGUGGCUUAUAAtt UUAUAAGCCACAAAUCUGGtt Qiagen

PKC␦ GCCGGGACACUAUAUUCCAtt UGGAAUAUAGUGUCCCGGCUtt Ambion

PCR primers

STIM1 TGACAGGGACTGTGCTGAAG AAGAGAGGAGGCCCAAAGAG Invitrogen

Orai1 GCTCATGATCAGCACCTGCAC GGGACTCCTTGACCGAGTTG Invitrogen

TRPC1 GAGGTGATGGCGCTGAAGG GCACGCCAGCAAGAAAAGC Invitrogen

TRPC3 CTGGCTCTGCTTGTGTTCAATG AGTCAGTAACTGTGATATTGGATATTG Invitrogen

TRPC4 TCAGCACATCGACAGGTCAGAC CCACGGTAATATCATCCACTCGAC Invitrogen

TRPC5 CTCTGATTGCGGAAGCCTCT GCAACGAACTTAAAATGTTGGATATTG Invitrogen

TRPC6 AAGTATAAGGAGCTCAGAAATTTCCAT TCTGATATTGTCTTGGAGGATTATTGA Invitrogen

GusB CCACCAGGGACCATCCAAT AGTCAAAATATGTGTTCTGGACAAAGTAA Invitrogen

TBP GCCCGAAACGCCGAATATA CGTGGCTCTCTTATCCTCATGA Invitrogen

GAPDH GCACAAGAGGAAGAGAGAGACC AGGGGAGATTCAGTGTGGTG Invitrogen

EE-EF1 AGCAAAAATGACCCACCAATG GGCCTGGATGGTTCAGGATA Invitrogen

reported that Orai1 and TRPC proteins form complexes together with STIM1 that participate to Ca²⁺entry[30–35].

In endothelial cell lines, several studies have demonstrated that TRPC1[36]and TRPC4[37,38]participate to SOCE. On the contrary, a recent study showed in primary human umbilical vein endothelial cells (HUVECs) that SOCE is mediated almost exclusively by the STIM1/Orai1 machinery[39].

In the current study, we investigated the molecular candidate of SOCE in a human umbilical vein endothelial cell line, the EA.hy926.

To this aim, we evaluated the involvement of STIM1, Orai1 and TRPCs proteins in TG-induced Ca²⁺entry. We showed that STIM1, Orai1 or TRPC3 invalidation reduced Ca²⁺entry by about 50%, while the combination of STIM1 and TRPC3 knockdown reduced the entry by 85%. Moreover, indirect evidence is provided that TRPC3 is getting activated following a PLC/PKC signaling pathway initiated by the addition of TG.

2. Material and methods 2.1. Materials

DMEM, penicillin and streptomycin were obtained from Invit- rogen. Fetal calf serum (FCS) was from PPA Laboratories (Linz, Austria). Thapsigargin, U73122, U73343, PP1, Src inhibitor-1, staurosporine, chelerythrine, rottlerin, PMA and OAG were obtained from Sigma. Acetoxylmethyl ester form of Fura-2 (Fura-2/AM) was from Molecular probes Europe (Leiden, the Netherlamds).

The myristoylated peptide PKCeta was from Calbiochem and GF103209X from Tocris.

2.2. Cell culture and transfection

Experiments were performed on the human umbilical vein endothelial cells derived cell line EA.hy926 at passages > 45. Cells were grown in DMEM containing 15% FCS, 0.5% fungizone, 1%

HAT (5 mM hypoxanthin, 20␮M aminopterin, 0.8 mM thimidine), 50 units/ml penicillin, 50␮g/ml streptomycin, and were main- tained at 37^◦C in 5% CO₂atmosphere.

2.3. siRNA knockdown

EA.hy926 cells were transfected in suspension by incubating 3×10⁵cells in a solution containing 6␮l of lipofectamine RNAiMax

(Invitrogen) and 100 nM of a speciﬁc siRNA (Ambion, Invitro- gen or Qiagen) according to manufacturer protocols (Invitrogen).

Experiments using one gene invalidation (Ca²⁺imaging, QRT-PCR, Western blot analysis) were performed 48 h post-transfection, while for the double STIM1/TRPC3 knockdown experiments, the siSTIM1 was transfected 24 h before siTRPC3 (72 h and 48 h post- transfection for STIM1 and TRPC3, respectively). Sequences of the different siRNA are summarized inTable 1. The siRNA scramble from Ambion was used as negative control. The siRNA transfection efﬁciency was approximately 90%, estimated by Block-iT Alexa Fluor Red Fluorescent Oligo (Invitrogen).

2.4. Quantitative real-time PCR

Real-time experiments were performed at the genomics plat- form of the NCCR Frontiers in Genetics (Geneva). For each PCR reaction, 1/20th of the cDNA template was PCR ampliﬁed in a 7900HT SDS System using Power SYBR Green PCR master mix (both from Applied Biosystems, Foster City, CA, USA). We obtained raw threshold-cycle (Ct) values using SDS 2.0 software (Applied Biosytems). A mean quantity was calculated from triplicate PCR reactions for each sample, and this quantity was normalized to the average of four endogenous control genes (glucuronidase B, GAPDH, TBP and EE-EF1) as described by[40]. Primers used for the real-time PCR are described inTable 1.

2.5. Cytosolic calcium measurements

For Ca²⁺imaging, cells were plated on 30 mm glass cover slips.

The changes in cytosolic Ca²⁺concentration were measured with Fura-2. Cells were loaded with 2␮M Fura-2/AM plus 1␮M pluronic acid for 30 min in the dark at room temperature in a medium containing (in mM): 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 Hepes acid, 2.6 NaHCO3, 0.44 KH2PO4, 10 glucose with 0.1% vitamins and 0.2%

amino acids, pH adjusted to 7.45 with NaOH. Cells were washed twice and equilibrated for 10–15 min in the same buffer to allow de-esteriﬁcation. Ratiometric images of Ca²⁺signals were obtained using a microscope (Axio Observer, Zeiss) equipped with a Lambda GD4 illumination system (Sutter Instrument Company, Novato, CA, USA), which rapidly changed the excitation wavelengths between 340 nm (340AF15; Omega Optical) and 380 nm (380AF15; Omega Optical). Emission was collected through a 415DCLP dichroic mir-

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F. Antigny et al. / Cell Calcium49 (2011) 115–127 117

Fig. 1.Effect of STIM1 and Orai1 knockdown on TG-induced Ca²⁺entry. (A) Cytoplasmic Ca²⁺was assessed on EA.hy926 cells loaded with 2␮M Fura-2. SOCE was triggered by depletion of the stores with 1␮M thapsigargin (TG) in the absence of external Ca²⁺(1 mM EGTA). 2 mM Ca²⁺was then re-added to the extracellular medium as indicated.

The control experiment without stimulation, showed a minimal Ca²⁺entry. (B) Cells were transiently transfected with control siRNA (scramble, siControl), or siRNA against STIM1 or Orai1. Each trace represents the Ca²⁺entry phase following store depletion, and is the mean of a representative coverslip. (C) Quantiﬁcation of the effects of siSTIM1, siOrai1 and siSTIM1/Orai1 on the initial slope of ratio increases, after Ca²⁺re-addition. Bars are mean±SEM. The number of cells is written on the bar graph.

ror, and a 510WB40 filter (Omega Optical), by a cooled, 12-bit CCD camera (CoolSnap HQ, Ropper Scientific, Trenton, NJ, USA). Image acquisition and analysis were performed with the Metafluor 6.3 software (Universal Imaging, West Chester, PA, USA). Experiments

were performed at room temperature in Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. The Ca²⁺-free solution contained 1 mM EGTA instead of 2 mM CaCl2. Due to the high trans-

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Fig. 2.Effect of STIM1 and Orai1 knockdown on mRNA and protein levels. (A) C mRNA levels were assessed by quantitative RT-PCR 48 h after transfection with siSTIM1 (A) or siOrai1 (C) (mean±SEM,n= 3 or 4 independent experiments). (B and D, left panels) Western blots showing the decrease of the STIM1 or Orai1 bands 48 h after the transfection with siSTIM1 (B) or siOrai1 (D).␣-Tubulin was used as a loading control. Right panels: quantiﬁcation of Western blots from 4 to 5 different experiments.

fection efﬁciency, cytosolic Ca²⁺measurements were performed on the whole cell population.

2.6. Western blots

Western blots were performed as previously described[41].

Brieﬂy, the endothelial cells were lysed using modiﬁed NP40 cell lysis buffer (Invitrogen), 50 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4and 1% Nonidet P40.

Total proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated in T-TBS (0.1% Tween 20, 20 mM Tris–HCl, pH 7.5, 137 mM NaCl) and 5% nonfat milk. Blots were incubated with the primary antibodies diluted in T-TBS and nonfat milk as follows: rabbit polyclonal anti-STIM1 antibody (1:1000; Sigma), rabbit polyclonal anti-Orai1 antibody (1:1000; Sigma), rabbit polyclonal anti-PKCeta antibody (1:700;

Santa Cruz Biotechnology), rabbit polyclonal anti-TRPC3 antibody (1:1000; Sigma) and mouse monoclonal antibody against ␣- tubulin (clone DM1A, Sigma) 1:10,000. Blots were incubated with horseradish peroxydase (HRP)-conjugated goat anti-mouse diluted 1:10,000 (BioRad) or with HRP-conjugated goat anti-rabbit diluted 1:10,000 (BioRad), respectively. Antibodies were revealed using ECl reagents and hyperﬁlm ECL (Amersham Biosciences). Image-J Software was used to quantify the level of protein expression.

2.7. Statistics

Results are expressed as mean±SEM of individual cells. Sets of data were compared with a Student’sttest. Differences were considered statistically signiﬁcant whenP< 0.05. ns: non signiﬁ- cant difference, *P< 0.05, **P< 0.01, ***P< 0.001. All statistical tests were performed using GraphPad Prism version 5.0 for Windows (Graphpad Software).

3. Results

3.1. STIM1 and Orai1 are involved in store-operated Ca²⁺inﬂux

To measure the store-operated Ca²⁺entry (SOCE), the cells were stimulated with 1␮M thapsigargin (TG) in the absence of external Ca²⁺, and 2 mM Ca²⁺was re-added to the extracellular medium to elicit Ca²⁺entry (Fig. 1A). Only a very small response was detected in the absence of TG stimulation (Fig. 1A). To compare Ca²⁺inﬂux between different conditions, we measured the initial slope of ﬂu- orescence increase (Fig. 1C).

First, we investigated the role of STIM1 and Orai1 proteins on SOCE, by transiently transfecting the EA.hy926 cells with the respective siRNA designed against STIM1 and Orai1. The basal Ca²⁺

entry, in the absence of TG stimulation, was not different between

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Fig. 3.Role of phospholipase C in TG-induced Ca²⁺entry. (A) EA.hy926 cells were loaded with Fura-2 to monitor cytosolic Ca²⁺changes. The Ca²⁺entry phase is depicted and each trace is the mean of a representative coverslip. Cells were transiently transfected either with control siRNA or with siSTIM1. The PLC blocker U73122 (2␮M) or the inactive analog U73343 (5␮M) was added 5 min before TG stimulation. (B) The bar graphs show aggregate data of the effect of U73122 and U73343 on the initial slope of ratio increase on STIM1, Orai1 and STIM1/Orai1 silenced cells (nranges from 41 to 335 cells). The bars representing the control experiments (for control, siSTIM1, siOrai1 and siSTIM1/Orai1) are the same as presented inFig. 1.

control cells, and cells invalidated for STIM1 or Orai1 (data not shown). The addition of TG in Ca²⁺-free medium elicited similar responses in control and STIM1 or Orai1 knockdown cells (data not shown), whereas the response elicited by Ca²⁺re-addition was inhibited by 60% in STIM1 knockdown cells (Fig. 1B and C). In Orai1 silenced cells, the SOCE was reduced by 43% (Fig. 1B and C). These results conﬁrmed that STIM1 and Orai1 proteins contribute to SOCE in this endothelial cell line.

3.2. Effect of STIM1 and Orai1 knockdown on mRNA level and protein expression

We performed a quantitative RT-PCR to evaluate the extend of gene silencing upon siRNAs transfection. As shown inFig. 2A, STIM1 mRNA level was decreased by 86% in cells transfected with siRNA against STIM1 (n= 4) when compared to cells exposed to scramble siRNA (noted siControl). The amount of STIM1 protein measured by Western blot 48 h after transfection was reduced by 67% in cells treated with siSTIM1 (n= 4; Fig. 2B) conﬁrming the efﬁciency of STIM1 silencing. Orai1 mRNA levels were decreased by 99% in Orai1 silenced cells (n= 3) (Fig. 2C) when compared

with control cells. The amount of Orai1 protein 48 h after transfection was reduced by 81% in cells treated with siOrai1 (n= 5;

Fig. 2D) confirming that Orai1 silencing was highly efficient. In double (STIM1/Orai1) knockdown cells, the level of STIM1 mRNA was decreased by about 67% and 99% for Orai1 mRNA (n= 2;Fig. S1). The double knockdown (Orai1/STIM1) did not reduce the SOCE more than siRNA against STIM1 alone (Fig. 1C), suggesting that STIM1 is the limiting player in SOCE mechanism. Altogether, these data confirmed the role played by STIM1/Orai1 machinery in TG-induced Ca²⁺entry, but suggest also the involvement of other proteins in this process.

3.3. The phospholipase C inhibitor (U73122) blocks the residual Ca²⁺inﬂux in STIM1 or Orai1 knockdown cells

Several studies have shown that PLC is involved in TG-induced Ca²⁺entry[42–44]. We thus used the membrane-permeable phospholipase C (PLC) inhibitor U73122 to evaluate the implication of PLC in TG-induced Ca²⁺entry. In control cells, a 5 min preincu- bation (before TG stimulation) with 2␮M U73122 resulted in a reduction of the Ca²⁺inﬂux by 20% (Fig. 3B). In STIM1 silenced

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Fig. 4.Role of protein kinase C (PKC) in TG-induced Ca²⁺entry. (A) Cells were transiently transfected with siSTIM1 or control siRNA. The PKC inhibitor staurosporine was added 2 min before TG stimulation. The Ca²⁺entry phase is depicted and each trace is the mean of a representative coverslip. (B) Statistic evaluation of the effect of staurosporine and chelerythrine on TG-induced Ca²⁺inﬂux in control and STIM1 silenced cells (nranges from 36 to 335 cells). (C) The addition of 1␮M PMA or 100␮M OAG, 5 min before TG, restored the Ca²⁺entry in cells treated with the PLC inhibitor U73122. Bar graphs are the quantiﬁcation of the effect of PMA and OAG on the initial slope of ratio increase, after Ca²⁺re-addition (nranges from 41 to 335 cells). The bars of panel B and C, representing the control experiments (for control, siSTIM1, siOrai1 and siSTIM1/Orai1) are the same as presented inFig. 1.

cells, the U73122 pretreatment completely prevented the residual Ca²⁺influx (Fig. 3A and B). The remaining Ca²⁺influx in Orai1 and Orai1/STIM1 knockdown cells was also completely abolished after PLC inhibition (Fig. 3B). On the contrary, the inactive analog U73343 (5␮M) did not affect the Ca²⁺influx activated by TG (Fig. 3). Neither

U73122 nor U73343 had an effect on the ability of TG to release calcium from the stores (data not shown). Interestingly, the addition of 2␮M U73122 just prior the Ca²⁺ re-addition phase or during the Ca²⁺re-addition phase did not prevent Ca²⁺inﬂux (Fig. S2).

This suggests that this compound does not act as a direct channel

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Fig. 5.Inhibition or invalidation of PKC␩reduced TG-induced Ca²⁺entry. (A) The myristoylated peptide MPI-PKC␩was preincubated 30 min before TG stimulation. The bar graphs represent the slope of ratio increase following Ca²⁺re-addition (nranges from 65 to 130 cells). (B) Statistical evaluation of the effect of siRNA against PKC␩on TG-induced Ca²⁺entry. The bars represent the slope of the ratio increase after Ca²⁺re-addition (nranges from 72 to 130 cells). (C, left panel) Western blot showing the decrease expression level of STIM1 and PKC␩, 48 h after transfection, either alone or in combination.␣-Tubulin was used as a loading control. Right panel: quantiﬁcation of the Western blot, showing also the combination of siOrai1 with siPKC␩from 2 to 4 different experiments.

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blocker, and that PLC is essential for the activation but not for the maintenance of SOCE activity.

3.4. The protein kinase C (PKC) pathway is involved in TG-induced Ca²⁺

The activation of PLC induces the cleavage of phosphatidyli- nositol 4,5-bisphosphate into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)[5]. In turn, DAG is an activator of the

“classical” and “novel” PKC isoforms[45]. To investigate the potential involvement of PKC in endothelial SOCE, we ﬁrst used two types of PKC inhibitors: chelerythrine (a widely generic inhibitor of PKC)[46,47]and staurosporine[48,49]. Each inhibitor was added 2 min before TG stimulation, in control and STIM1 knockdown cells.

In control cells chelerythrine and staurosporine reduced the Ca²⁺

influx by about 45% (Fig. 4B), while in STIM1 knockdown cells, the Ca²⁺influx was inhibited by 70% for staurosporine (Fig. 4A and B) and by 90% for chelerythrine (Fig. 4B). The same inhibitory effect of PKC blockers was observed in Orai1 and Orai1/STIM1 silenced cells (data not shown). Like for the PLC inhibitor U73122, the PKC blockers had no effect on Ca²⁺influx when they were added before or during the Ca²⁺re-addition phase (Fig. S3), suggesting that these compounds did not act as direct channel blockers. In order to further support our hypothesis that the PLC/PKC pathways is involved in TG-induced Ca²⁺entry, we performed rescue experiments on

cells treated with the PLC inhibitor U73122. In this condition, the addition of either the PKC activator PMA (phorbol myristate acetate 1␮M), or the membrane-permeable DAG analog OAG (1-oleoyl-2- acetyl-sn-glycerol 100␮M) restored the Ca²⁺entry in control cells and STIM1 or Orai1 knockdown cells (Fig. 4C). These data suggest that the PLC metabolite DAG activates the PKC, limiting the possible PKC isoforms engaged in the Ca²⁺entry as those that belong to the “classical” (activated by Ca²⁺and DAG) or “novel” family (activated by DAG but nor by Ca²⁺). On EA.hy926 cells, the PKC␣and PKC␤, from the “classical” family, as well as the PKC␦, PKC␧and PKC␩from the “novel” family are present[50]. Thus we applied inhibitors directed against different isoforms of PKC. GF103209X that preferentially blocks the PKC␣/␤did not reduced Ca²⁺entry (Fig. S4A). On the contrary, rottlerin (Fig. S4B) and the myristoylated peptide MPI-PKC␩(Fig. 5A), which inhibit PKC␦and PKC␩, respectively, reduced by more than 50% TG-induced Ca²⁺entry, both in control cells, and in cells invalidated for STIM1 or Orai1. To conﬁrm these data, we transiently transfected the cells with siRNA directed against these two PKCs isoforms. The invalidation of the PKC␦reduced by about 15% the Ca²⁺entry in control cells, but did not affect Ca²⁺entry in STIM1 or Orai1 invalidated cells (Fig. S5A).

On the contrary, the knockdown of PKC␩reduced TG-induced Ca²⁺

entry by 20% in control cells, and by about 40% in STIM1 and Orai1 invalidated cells (Fig. 5B). To measure the efﬁciency of gene silencing, we performed Western blots 48 h after transfection. The siRNA

Fig. 6.Effect of TRPCs knockdown on TG-induced Ca²⁺entry. (A) EA.hy926 cells were loaded with 2␮M Fura-2 to monitor cytosolic Ca²⁺changes. Traces are the mean of a representative coverslip showing the Ca²⁺entry phase. Cells were transiently transfected with control siRNA or siRNA against TRPC3. (B) Quantiﬁcation of the effects of siTRPC1, siTRPC3, siTRPC4, siTRPC5 and siTRPC6 expressed as percent of the initial slope of ratio increase compared to control cells. Bars are mean±SEM (nranges from 50 to 335 cells). (C) mRNA levels were assessed by quantitative RT-PCR 48 h after transfection (mean±SEM, 2–4 independent experiments). For each TRPC invalidation, the control was set to 1, and the mRNA level of each TRPC was expressed as compared to its own control. (D, left panel) Western blot showing the disappearance of the TRPC3 bands 48 h after the transfection with siTRPC3.␣-Tubulin was used as a loading control. Right panel: quantiﬁcation of Western blots from 4 different experiments.

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Fig. 7.Effect of STIM1 and TRPC3 knockdown on TG-induced Ca²⁺entry. (A) EA.hy926 cells were loaded with 2␮M Fura-2 to monitor cytosolic Ca²⁺changes. Traces are the mean of a representative coverslip showing the Ca²⁺entry phase. Cells were transiently transfected with control siRNA, siRNA against STIM1, or siRNA against STIM1 together with siRNA against TRPC3; siSTIM1 was transfected 24 h before siTRPC3 (72 h and 48 h after transfection for STIM1 and TRPC3, respectively). (B) The bars represent the slope of the ratio increase after Ca²⁺re-addition in siControl, siSTIM1 and siSTIM1/siTRPC3 silenced cells. Bars are mean±SEM (nranges from 46 to 190 cells). (C) Western blots showing the disappearance of the STIM1 protein and the TRPC3 protein expression after the transfection with siSTIM1/siTRPC3.␣-Tubulin was used as a loading control. (D) Quantiﬁcation of Western blots from 2 different experiments.

against PKC␩was efﬁcient in reducing protein levels, both on single transfection, and in combination with STIM1 and Orai1 silencing.

The same efﬁciency on protein levels was obtained with the siRNA against PKC␦(Fig. S5B), suggesting that PKC␦is not essential for TG-induced Ca²⁺entry.

3.5. Involvement of canonical transient receptor potential in TG-induced Ca²⁺entry

We next tested the involvement of TRPC in TG-induced Ca²⁺

entry. To this aim, we designed siRNA against each of the five isoforms expressed in this endothelial cells line (TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6). Interestingly, only TRPC3 appear to be involved in TG-induced Ca²⁺entry, as shown by the reduction of the initial slope of Ca²⁺influx by 45% (Fig. 6A and B). The efficiency of each siRNA in silencing its messenger RNA was verified by quantitative RT-PCR, and the mRNA levels were reduced between 50% and 90%

(Fig. 6C). At the protein level, TRPC3 was decreased by around 70%

(Fig. 6D). We conﬁrmed this result by using two other siRNA against TRPC3, which gave similar reduction of the Ca²⁺entry (Fig. S6A).

The combination of siRNA against STIM1 and TRPC3 reduced Ca²⁺

entry by 85% (Fig. 7A and B). In these conditions, the efficiency of TRPC3 and STIM1 invalidation was between 70 and 85% at the protein level (Fig. 7C and D). These data confirmed the involvement of STIM1 and TRPC3 protein in TG-induced Ca²⁺influx in endothelial cells.

3.6. Effect of PLC, PKC or Src inhibitors on TRPC3 knockdown cells

To explore the putative target of the PKC pathway, we tested the PLC and PKC inhibitors on TRPC3 knockdown cells. U73122 reduced by about 12%, and chelerytrine by 25% TG-induced entry (Fig. S6B). Staurosporine on the other hand did not inhibit Ca²⁺

entry in TRPC3 knockdown cells (Fig. S6B), and neither OAG nor PMA rescued the Ca²⁺ entry after U73122 treatment in siTRPC3 treated cells (Fig. S6C). The MPI-PKC␩reduced by about 25% the slope of Ca²⁺inﬂux, while the siRNA against PKC␩did not reduce, and even paradoxically increased the entry (Fig. 8A). The absence (staurosporine, siPKC␩) or small inhibitory effect (U73122, chelerythrine, MPI-PKC␩) of the PLC/PKC pathway inhibitors on TRPC3 knockdown cells, suggested that TRPC3 is likely the target of the pathway engaged upon TG stimulation. The non-receptor tyrosine

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Fig. 8.Effect of PKC␩and Src inhibition on TG-induced Ca²⁺entry. (A) Statistical evaluation of the effect on TG-induced Ca²⁺entry, of MPI-PKC␩and siPKC␩. The bar graphs represent the initial slope of the ratio increase following Ca²⁺re-addition (nranges from 37 to 130 cells). (B) Effect of two Src inhibitors, PP1 and Src inhibitor-1 (Src1) on TG-induced Ca²⁺entry. The Src inhibitors were incubated 30 min before TG stimulation. Experiments were performed in STIM1, Orai1 or TRPC3 invalidated cells. Bars are mean±SEM of the initial slope following Ca²⁺re-addition (nranges from 24 to 141 cells). The bars representing the control experiments (for control, siSTIM1, siOrai1) are the same as presented inFig. 5.

kinase Src was shown to be essential for TRPC3 activation[51]. We thus tried two different Src inhibitors, PP1 and Src inhibitor-1 on TG-induced Ca²⁺entry. As shown inFig. 8B, Src inhibition reduced Ca²⁺entry in STIM1 and Orai1 silenced cells, but not in TRPC3 invalidated cells, conﬁrming again TRPC3 as a probable target of the PLC/PKC signaling pathway.

4. Discussion

In the present work, we studied the molecular identity and the mechanism of TG-induced Ca²⁺ entry in human umbilical vein endothelial cell line, the EA.hy926. By siRNA approach, we assessed the involvement of STIM1, Orai1 and TRPCs proteins in this process. We showed that TG-induced Ca²⁺entry is partially mediated by STIM1 and Orai1, and partially by TRPC3, and the double STIM1/TRPC3 invalidation reduced the entry by 85%. Interestingly, the residual STIM1/Orai1-independent Ca²⁺entry is fully abolished following inhibition of PLC and/or PKC, while the residual TRPC3- independent entry is almost not affected by PLC or PKC inhibition.

These data strongly suggested that PLC is getting activated upon TG stimulation, and that the downstream signaling pathway, involving

PKC and TRPC3, account for a large part in TG-induced Ca²⁺entry in endothelial cells.

Since 2005 and the discovery of the major role played by STIM1 and Orai1 proteins in SOCE, the function of TRPCs isoforms in this mechanism remained to be firmly established. In this study, we confirm that STIM1 and Orai1 are important players of SOCE, as invalidation of these proteins reduced the Ca²⁺entry by 45% (Orai1) to 65% (STIM1). Interestingly, even though the silencing efficiency was of between 65 and 80% at the protein level, an important residual Ca²⁺entry persisted upon store depletion. Such a “partial” SOCE reduction was already observed, for instance in smooth muscle cells [52–54]or HEK cells[20], but in most cell types, the inhibition of SOCE upon STIM1 invalidation is very pronounced[3,39,55,56]. This finding prompted us to investigate the mechanism of the residual TG-induced Ca²⁺entry.

Several studies have reported a reduction of SOCE following inhibition of the PLC by U73122[42], or after PLC␥knockdown [43,44]. In our hands, U73122 reduced by only 20% the SOCE in control cells, while in STIM1 and/or Orai1 knockdown cells, the Ca²⁺

entry was completely abolished. To investigate whether the downstream products of PLC activation might affect Ca²⁺entry after TG

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stimulation, we used two PKC inhibitors, staurosporine and chelerythrine. Both compounds reduced by about 45% TG-induced Ca²⁺

entry in control cells, and completely inhibited Ca²⁺entry in STIM1 and/or Orai1 knockdown cells. The involvement of PKC was further conﬁrmed by using PMA to directly activate PKC on cells treated with the PLC blocker. This treatment restored the Ca²⁺inﬂux to levels achieved without blocking the PLC, both in control cells, and in cells invalidated for STIM1 or Orai1. Additionally, the DAG analog OAG also restored Ca²⁺entry upon PLC inhibition. It thus appears that the lipase activity of PLC is required for TG-induced Ca²⁺entry, in particular through the production of DAG that activates the PKC.

In previous studies reporting the importance of PLC for SOCE, it was shown that either the lipase activity of the enzyme was not required [44], or the production of DAG was not important[42,43]. On the contrary, in our cellular system, the generation of DAG seems to be essential for TG-induced Ca²⁺entry. Next, we wanted to determine which isoform of the PKC was involved in the Ca²⁺entry. The PKC family consists of ten isozymes[57]. Based on their mechanism of activation, the PKC are divided into three subfamilies, the

“conventional” (or “classical”), the “novel”, and the “atypical” PKC.

“Conventional” PKCs contain the isoforms␣,␤I,␤II, and␥. They require Ca²⁺, DAG, and phospholipids such as phosphatidylserine for their activation. “Novel” PKCs include the␦,␧,␩, and␪isoforms require DAG, but not Ca²⁺to be activated. Thus, “conventional” and

“novel” PKCs are activated downstream of the PLC signal trans- duction pathway. The “atypical” PKCs (including protein kinase M␨ and␫/␭isoforms) neither require Ca²⁺nor diacylglycerol for their activation.

GF103209X, an inhibitor of the classical PKC had no inhibitory effect on Ca²⁺ entry. On the contrary, rottlerin and MPI-PKC␩ that inhibit PKC␦and PKC␩, respectively, markedly prevented TG- induced Ca²⁺entry. Caution had to be taken by using PKC inhibitors, in particular rottlerin that was shown to have serious side effects [58]. We thus used siRNA against PKC␦and PKC␩to conﬁrm our data. While the knockdown of PKC␦did not inhibit Ca²⁺ entry, the invalidation of PKC␩negatively impacted on TG-induced Ca²⁺

entry, both in control and STIM1 and Orai1 silenced cells. Alto- gether, these results strongly point to a role played by the “novel”

PKC family (likely PKC␩), in TG-induced Ca²⁺entry.

The inhibitory effect of PLC and PKC blockers was observed in control cells, but also in cells invalidated for STIM1 or Orai1.

Hence, it is unlikely that STIM1/Orai1 proteins are the target(s) of PKC, even though we could not completely rule this out, as the silencing of these two proteins was not complete. We thus investigated what other proteins could be implicated in TG-induced Ca²⁺ entry, focusing on the TRPC channel family and found out that only TRPC3 invalidation reduced the Ca²⁺entry. The double STIM1/TRPC3 invalidation almost completely prevented Ca²⁺entry, strongly suggesting that TG-induced Ca²⁺entry in endothelial cells was constituted by the STIM1/Orai1 machinery for one part, and for the other part by TRPC3. Interestingly however, when TRPC3 was knocked-down, the efﬁciency of PLC of PKC inhibition was much less pronounced, with an absence of signiﬁcant inhibition using staurosporine or siPKC␩, and a modest reduction of the Ca²⁺

entry of about 15–20%, upon U73122, chelerythrine and MPI-PKC␩ application. In addition, when TRPC3 was down-regulated, neither OAG nor PMA potentiate the Ca²⁺ entry. We thus propose that TRPC3 channel is likely a target of the PLC/PKC pathway activation. PKC was shown to phosphorylate TRPC3 on Ser 712, resulting in channel inhibition[59,60], whereas in our study PKC inhibition decreased Ca²⁺entry. Hence, in our cellular system, it is unlikely that PKC directly phosphorylates TRPC3. PKC might phosphorylate an intermediate protein that in turn positively regulates the activity of TRPC3. It was shown that the non-receptor tyrosine kinase Src is essential for TRPC3 activation[51], by phosphorylating the channel on a tyrosine at position 444[61]. Using two different Src

inhibitors, we showed that both reduced Ca²⁺entry in STIM1 and Orai1 knockdown cells, while not in TRPC3 invalidated cells. This result suggests that TRPC3 is the target of phosphorylation by Src.

We can reasonably postulate that Src is getting activated indirectly by PKC, as it was reported in smooth muscle cells[62]. The precise mechanism between PKC activation and TRPC3 opening remains however to be ﬁrmly established, but our data suggested that Src is, at least partially, involved in this process. Considering that the inhibition of the Ca²⁺entry varied between about 40 and 60% upon src inhibition or siRNA treatment against PKC␩, it is possible that for a part, TRPC3 is also getting directly activated by DAG, like it was already reported[15,63,64]. This point, however, remains to be further investigated.

Moreover, recent papers showed that STIM1 directly or indirectly binds to all TRPC (except TRPC7) and activated them[28,29].

Orai1 was also shown to be associated with the complex formed by STIM1 and TRPC. Hence, the idea emerged that the function of TRPC channels might well depend on their association or not with the STIM/Orai molecules[30–35]. Our experimental evidence suggested that TRPC3 function independently from STIM/Orai and that its activity is not linked to the Ca²⁺content of the store.

In summary, we showed that in EA.hy926 cells, the inﬂux elicited upon TG application has two components, one being the

“classical” STIM1 and Orai1 machinery, and the other one that com- prises TRPC3 being activated by a rather unusual mechanism: TG stimulation leads to PLC activation and the production of DAG that in turns activates a “novel” PKC (likely PKC␩). This presumably results in an activation of Src that phosphorylates TRPC3 leading to Ca²⁺entry. This chain of event is unexpected following TG stimulation, even though PLC was already reported to be involved in SOCE.

While we can certainly call the STIM1/Orai1-dependent Ca²⁺entry SOCE, the machinery involving PLC/PKC/Src/TRPC3 is not necessar- ily store-operated. Indeed, we do not know whether the depletion of the ER per se leads to PLC activation, or whether the conse- quence of the depletion, for instance the cytosolic Ca²⁺elevation, is responsible for this activation. In any instance, in EA.hy926 cells, TG stimulation engaged more molecules than STIM1 and Orai1, and surprisingly activated a Ca²⁺signaling pathway usually triggered by agonist stimulation.

Conﬂict of interest

There is no conﬂict of interest

Acknowledgements

We thank Cyril Castelbou for his excellent technical assis- tance, and Dr. C.J.S. Edgell for the EA.hy926 cells. We thank Dr.

Michelangelo Foti for the PKC antibodies, and Drs. R. Malli, W.

F. Graier, C. Vandebrouck and N. Demaurex for critically reading the manuscript. This work was supported by the Swiss National Science Foundation, Grant 310000-120186/1, and the foundation Carlos and Elsie de Reuter.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.ceca.2010.12.001.

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