Articles in PresS. Am J Physiol Cell Physiol (January 27, 2010). doi:10.1152/ajpcell.00325.2009
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Essential Role for STIM1/Orai1-Mediated Calcium Influx in PDGF-
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Induced Smooth Muscle Migration
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By
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Jonathan M. Bisaillon1, Rajender K. Motiani1, José C. Gonzalez-Cobos1, Marie Potier2,
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Katharine E. Halligan, Wael F. Alzawahra, Margarida Barroso, Harold A. Singer, David
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Jourd’heuil, and Mohamed Trebak3
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From
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The Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave, Albany
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NY 12208 USA
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Equal contribution
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Current address: INSERM U921 Nutrition Croissance Cancer; Faculté de Médecine , Bat
Dutrochet, 10 Bd Tonnellé, 37032 Tours; Email: [email protected]
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47 New Scotland Ave, Albany NY 12208. Phone (518)262-4682; Fax (518)262-8101; Email:
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[email protected]
Address correspondence to: Mohamed Trebak, PhD. Center for cardiovascular Sciences, MC8,
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Running Title: STIM1/Orai1 and PDGF-mediated smooth muscle migration
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Copyright © 2010 by the American Physiological Society.
Bisaillon et al.
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ABSTRACT
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We recently demonstrated that thapsigargin-induced passive store depletion activates calcium
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entry in vascular smooth muscle cells (VSMC) through STIM1/Orai1, independently of TRPC
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channels. However, under physiological stimulations, despite the ubiquitous depletion of IP3-
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sensitive stores, many VSMC PLC-coupled agonists (e.g. vasopressin, Endothelin etc) activate
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various store-independent Ca2+ entry channels. The platelet-derived growth factor (PDGF) is an
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important vascular smooth muscle cell (VSMC) pro-migratory agonist with an established role in
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vascular disease. Nevertheless, the molecular identity of the calcium channels activated by
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PDGF in VSMC remains unknown. Here we show that inhibitors of store-operated Ca2+ entry
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(Gd3+ and 2-APB at concentrations as low as 5 μM) inhibit PDGF-mediated Ca2+ entry in
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cultured rat aortic VSMC. Protein knockdown of STIM1, Orai1 and PDGF receptor β (PDGFRβ)
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impaired PDGF-mediated Ca2+ influx while Orai2, Orai3, TRPC1, TRPC4 and TRPC6
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knockdown had no effect. Knockdown of STIM1, Orai1 or PDGFRβ inhibited PDGF-mediated
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VSMC migration assayed using scratch wound assay while STIM2, Orai2 and Orai3 knockdown
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were without effect. The levels of STIM1, Orai1 and PDGFRβ mRNA were upregulated in vivo in
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VSMC from balloon-injured rat carotid vessels compared to non-injured controls. The protein
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levels of STIM1 and Orai1 were also upregulated in medial and neointimal VSMC from injured
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carotids compared to non-injured vessels as assessed by immunofluorescence microscopy.
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These results establish STIM1/Orai1 as important components for PDGF-mediated Ca2+ entry
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and migration in VSMC that are upregulated in vivo during vascular injury and provide insights
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linking PDGF to STIM1/Orai1 during neointima formation.
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Key Words: SOC channels, TRPC channels, PDGF, Smooth muscle dedifferentiation,
neointima formation
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Abbreviations: PDGF, platelet-derived growth factor; STIM1, stromal interaction molecule 1;
TRPC, transient receptor potential canonical; VSMC, vascular smooth muscle cells; PLC,
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Phospholipase C; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; SR, sarcoplasmic
reticulum; siRNA, silencing RNA
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INTRODUCTION
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Vascular smooth muscle cells (VSMC) are one of the major cell types in blood vessels that play
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a central role in controlling the vascular tone and maintaining the integrity of the vessel wall(19,
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49). Stimulation of VSMC membrane receptors to vasoactive compounds (e.g. vasopressin,
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angiotensin II) and growth factors (e.g. PDGF, FGF) that typically couple to isoforms of
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Phospholipase C (PLC) induces phosphatidylinositol-4,5-bisphosphate (PIP2) breakdown into
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two second messengers: inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 causes
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Ca2+ release through IP3 receptors from the sarcoplasmic reticulum (SR)(9). Since the amount
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of Ca2+ in the internal stores is finite, Ca2+ entry across plasma membrane channels is typically
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activated to replenish the empty stores and maintain a sustained increase in cytoplasmic Ca2+
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necessary for signaling downstream to the nucleus. One of the major means for Ca2+ entry into
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cells is through store-operated Ca2+ entry (SOC) channels that are activated as a result of
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internal Ca2+ store depletion(36). Depletion of internal Ca2+ stores induces the ER/SR-resident
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Ca2+ store sensor STIM1 to translocate to areas near the plasma membrane to signal the
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activation of SOC channels encoded by Orai1 proteins(13-14, 17, 35). However, in addition to
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Ca2+ selective Orai1 (33-34), diverse SOC conductances encoded by TRPC proteins have been
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described in many VSMC types(2-3, 42). We and others have recently demonstrated the
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upregulation of STIM1/Orai1 proteins in vitro in synthetic cultured VSMC compared with
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quiescent contractile freshly isolated VSMC(8, 34). We have also showed that passive store
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depletion by thapsigargin activates calcium entry in VSMC through STIM1/Orai1, independently
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of Orai2, Orai3 and TRPC channels(34) and showed the presence of store depletion-activated
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ICRAC-like currents in cultured synthetic migratory VSMC encoded by STIM1/Orai1(34). Golovina
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and coworkers subsequently confirmed that store-operated Ca2+ entry is mediated by Orai1,
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independently of Orai2 and Orai3 and provided evidence for a functional association between
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Orai1, the Na+/Ca2+ exchanger (NCX) and the plasma membrane associated Ca2+ ATPase
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(PMCA) pump in proliferating smooth muscle cells(6). However, under physiological agonist
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stimulation such as the platelet-derived growth factor (PDGF), the exact Ca2+ entry route
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activated remains uncertain. In fact, despite concomitant depletion of IP3-sensitive stores,
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ligation of many PLC-coupled receptors activate instead distinct Ca2+ entry channels that do not
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depend on the state of filling of the internal stores(11, 39). For instance, the store-independent
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arachidonic acid-activated Ca2+ entry pathway mediated by arachidonic-acid activated channels
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(ARC) is activated in response to PLC-coupled receptor stimulation under certain agonist
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conditions (39-40). Recently, ARC channels were shown to depend on plasma membrane
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STIM1 and to be contributed by Orai1 and Orai3 proteins(27-28). Furthermore, stimulation of α1
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adrenergic receptors activates store-independent Ca2+ entry through DAG-activated transient
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receptor potential canonical 6 (TRPC6) channel (22). Heteromultimeric TRPC6-TRPC7
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channels were shown to contribute to vasopressin-activated Ca2+ entry in A7r5 smooth muscle
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cells(26) and increased Ca2+ entry induced by endothelin-1, another PLC-coupled receptor
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vasoactive agonist, was attenuated by TRPC3 knockdown(50).
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Expression of PDGF can occur in all resident cells of the artery and in inflammatory cells that
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infiltrate the artery during disease(37). PDGF and its receptors are expressed at low or
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undetectable levels in normal vessels but see their expression increased during vascular injury
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and during atherosclerosis(37). It is now clearly established that PGDF plays a prominent role in
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migration of vascular smooth muscle cells (VSMC) into neointima following acute vessel injury
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or during atherosclerotic lesions(37). PDGF mediates its effects through binding to its receptor
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with subsequent Ca2+ mobilization. However, the molecular identity of the Ca2+ influx channels
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activated by PDGF in general and in VSMC in particular remains unknown.
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In this study, we investigated the contribution of several Ca2+ channel candidates, known to be
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activated downstream of PLC isoforms, to the molecular composition of PDGF-activated Ca2+
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entry channels in VSMC. We used protein knockdown using silencing RNA against PDGFRβ,
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the Ca2+ sensor STIM1, all three Orai isoforms and the three TRPC isoforms expressed in
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primary VSMC (TRPC1/4/6). We show that PDGF activates Ca2+ entry across the plasma
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membrane through the STIM1/Orai1 pathway, independently of TRPC1/4/6 and Orai2/3
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proteins. We also show that STIM1 and Orai1 knockdown inhibits VSMC migration in response
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to PDGF in vitro and that STIM1 and Orai1 are upregulated in vivo in neointimal VSMC during
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vascular injury.
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METHODS
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Reagents
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Gadolinium (Gd3+) was purchased from Acros Oragnic; Recombinant rat PDGF-BB was from
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RD Biosystems. 2-aminoethoxydiphenyl borate (2-APB) was purchased from Calbiochem. All
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siRNAs sequences were from Dharmacon. All siRNA sequences and specific primers for rat
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STIM, Orai and TRPC are described elsewhere(34). For rat PDGFRβ, the primers used are:
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Forward:
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siRNA
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ACAUCAAAUACGCGGACAU. Anti-STIM1 was from BD Biosciences, anti- β-actin-NT was from
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Sigma, and Anti-Orai1 (extracellular; cat#ACC-060) from Alomone. Western blot and
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immunofluorescence data shown were generated using the Anti-Orai1 against the extracellular
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domain (Alomone) and similar results were obtained using Anti-Orai1 antibodies from Sigma
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(cat#O8264) and Prosci Inc (cat#4041). All other chemical products were from Fisher Scientific
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unless specified otherwise.
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VSMC dispersion and culture
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Male rats were euthanized by suffocation in a CO2 chamber. Aortas were dissected out into ice
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cold PSS. The fat tissues and endothelium were removed. The artery was cut into small pieces
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and digested with a papain solution for 20 min at 37° followed by a second digestion with a
CGCATGGCCCAACCTGCTCA;
against
rat
PDGFRβ
are:
reverse:
siRNA#1:
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TCGGAGGCTGTGGAGACCCG.
GUAGAAAUCCUUGGGUAUA;
The
siRNA#2:
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mixture of Collagenase II and Collagenase for 15 min at 37°. After removal of digestion solution
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and washes, cells were gently liberated with fire-polished glass pipette and transferred into
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culture plates. Isolated VSMC undergo phenotypic modulation in culture that is complete within
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30 hours(18). Isolated VSMC were maintained in culture (45% DMEM, 45% HAM F12 with 10%
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FBS supplemented with L-glutamine) at 37°C , 5% CO2 and 100% humidity, passaged
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(synthetic) and used within 1-6 passages in all experiments.
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RT-PCR and real time PCR.
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Detailed protocol and sequences of specific primers used were described previously(34).
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Expressions of TRPC, STIM or Orai were compared to the housekeeping gene GAPDH and
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were measured using comparative threshold cycle values as previously described(1, 34).
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Cell transfections
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Transfections in VSMC were done using Nucleofector device II (Amaxa Inc. MD) according to
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the manufacturer’s instructions. As a marker of cell transfection, 0.5 µg GFP was co-transfected
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with siRNA for identification of successfully transfected cells during experiments. As a control,
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we used a scrambled non-targeting sequence as reported previously(34). For calcium imaging
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experiments, cells were transfected with 10µg of siRNA of choice per 1x106 cells, seeded to
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either round glass coverslips (for imaging) or plates (for various assays) and used 72 hours post
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transfection.
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Ca2+ measurements
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Ca2+ measurements were performed as described previously(1, 45, 47). Briefly, coverslips with
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attached cells were mounted in a Teflon chamber and incubated at 37°C for 45 min in culture
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media (DMEM with 10% fetal bovine serum) containing 4 µM Fura-2 AM (Molecular Probes,
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Eugene, OR). Cells were then washed and bathed in HEPES-buffered saline solution (in mM:
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NaCl 140, MgCl2 1.13, KCl 4.7, CaCl2 2, D-glucose 10, and HEPES 10, adjusted to pH 7.4 with
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NaOH) for at least 10 min before Ca2+ measurements were made. For Ca2+ measurements,
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fluorescence images of several cells were recorded and analyzed with a digital fluorescence
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imaging system (InCyt Im2; Intracellular Imaging Inc., Cincinnati, OH). Fura-2 fluorescence at an
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emission wavelength of 510 nm was induced by exciting Fura-2 alternately at 340 nm and
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380 nm. The 340/380 ratio images were obtained on a pixel by pixel basis. All experiments were
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conducted at room temperature.
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Western blots
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Cells were lysed using RIPA lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100,
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0.5% sodium deoxycholate, 0.1% SDS, and 0.2 mM EDTA) and 20-50µg of proteins in
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denaturing conditions were subjected to SDS-PAGE (7.5%) and then electrotransferred onto
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polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk (NFDM) dissolved in
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Tris-buffered saline containing 0.1% Tween 20 (TTBS) for 2 h at room temperature, Blots were
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given 3 washes with TTBS for 5 min. each and probed overnight at 4°C, with specific primary
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antibodies in TTBS containing 2% NFDM. The primary antibodies used were anti-STIM1 (1:250),
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anti-Orai1 (Alomone; 1:1000) and β-actin-NT domain (1:2000). Next day membranes were
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washed with TTBS (3 washes of 5 min. each) and were incubated for 45 min at room
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temperature with a horseradish peroxidase-conjugated anti-mouse (1:10,000; Jackson) for
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STIM1 and Actin or anti-rabbit IgG for Orai1 (1:20,000; Jackson) in TTBS containing 2% NFDM.
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Detection was performed using the enhanced chemiluminescence reagent (ECL Western
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blotting detection reagents; Amersham).
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Migration Assays
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VSMC were transfected with scrambled control or target siRNA, seeded and allowed to form a
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monolayer for 48 hours. A 100µL pipette tip was used to scrape across the dish and the
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resulting wound was washed with PBS. Culture medium containing 0.4% FBS with or without
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100ng/ml PDGF was added to the cells followed by incubation in a 37°C incubator with 5% CO2.
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VSMC showed very little or no migratory response in the absence of PDGF. At different time
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intervals bright field images were captured using a Leica DM IRB at 10X microscope.
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images were analyzed using Adobe Photoshop CS3 and the total number of pixels in empty
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The
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spaces inside the wound were counted and normalized to the control (scrambled siRNA) to
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gauge changes associated with knockdown of specific proteins. Data is represented as
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percentage of migration relative to the scrambled siRNA control condition.
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Balloon injury of rat carotid arteries
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The use of rats for these experiments has been reviewed and approved by the Institutional
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Animal Care and Use Committee at the Albany Medical College Animal Resource Facility which
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is licensed by the USDA and N.Y.S. Department of Public Health, Division of Laboratories and
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Research, and is accredited by the American Association for the Accreditation of Laboratory
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Animal Care. Male Sprage-Dawley rats (350-400 g; Taconic Farms, Germantown, NY) were
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anesthetized with Xylazine 5 mg/kg and Ketamine 70 mg/kg via intraperitoneal injection and
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balloon angioplasty carried out essentially as previously described(20). Briefly, a 2F Fogarty
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balloon embolectomy was inserted through a small arteriotomy in the external carotid artery and
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passed into the common carotid artery. After balloon inflation at 2 atm of pressure (6X), the
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catheter was partially withdrawn and reinserted 3 times. After recovery from operation and
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anesthesia, animals received a postoperative dose of the analgesic Buprenex (0.02 mg/kg SC).
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Sham animals were operated using a similar procedure but without balloon insertion.
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Immunofluorescence
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Rats were euthanized by asphyxiation with CO2 and injured or non-injured carotids were
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harvested and embedded in OCT and frozen by submersion in liquid nitrogen. Tissue sections
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(7 μm) were cut using a Leica CM3050 cryostat and the sections mounted onto glass slides and
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stored at -80 °C. The carotid sections were fixed with pre-cooled acetone for 10 min at 4 ºC and
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rinsed with 1 x PBS. The sections were then incubated in a PBS washing buffer containing
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0.1% Triton X-100 for 10 min. The sections were incubated for 30 min in blocking buffer (1 x
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PBS/5 % goat serum/0.5 % fish gelatin/0.1 % Triton X-100). The sections were then treated
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overnight with primary antibody at 4 °C.
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antibody (Dilution 1:25; BD biosciences) or three rabbit polyclonal anti-Orai1 antibodies used
Either a mouse monoclonal anti-STIM1 primary
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independently (Anti-Orai1 extracellular, cat# ACC-060(6) from Alomone; Orai1-NT from Prosci-
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inc, cat# 4041(34) and Anti-Orai1 from Sigma, Cat # O8264) were diluted 1:25, 1:10 and 1:25
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respectively in blocking buffer. All three Orai1 antibodies showed similar results with robust
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upregulation of Orai1 in injured vessel sections. The sections were rinsed with washing buffer
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and then incubated with either anti-mouse or anti-rabbit secondary antibodies for 1 h at room
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temperature. For STIM1 staining, an FITC-coupled anti-mouse secondary antibody (Molecular
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Probes) was diluted 1:300 in blocking buffer. For Orai1 staining, an FITC-coupled anti-rabbit
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secondary antibody (Molecular Probes) was diluted 1:300 in blocking buffer. Staining conditions
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with secondary antibodies alone were conducted for control purposes. Finally, the sections were
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mounted with a mounting media containing DAPI.
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LSM510 META confocal microscope. Images were collected using a 63x oil immersion lens and
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0.7x zoom (low magnification or 2x (high magnification). LSM software was used to drive the
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hardware and image acquisition. Images collected at low magnification show intensity levels
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without modification, whereas images collected at high magnification were processed using
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Image J and Photoshop to a higher level of contrast for better visualization of protein subcellular
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distribution. Vertical sections were collected at high magnification every 0.5µm with pinhole
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settings at airy units =1. Image J volume viewer plugin was used to generate a vertical cross-
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section image using trilinear interpolation.
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For immunofluorescence in cultured synthetic VSMC, cells were transfected with non-targeting
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siRNA or Orai1 siRNA as listed above and were allowed to grow for 72 hours on coverslips
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coated with collagen. At 4 °C, cells were washed with PBS and fixed with paraformaldehyde.
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Cells were then permeabilized for 5 minutes with PBS/0.2% Triton X100, followed by incubation
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in blocking buffer (PBS/0.1%Triton X100/0.5% fish gelatin) for 30min. The cells were allowed to
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incubate overnight in anti-Orai1 primary antibody diluted in blocking buffer at 1:25 (Alomone
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Labs) at 4 °C. The cells were rinsed and then treated with anti-rabbit secondary antibody
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diluted in blocking buffer at 1:300 for 1 hour. The coverslips were mounted in mounting media
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The sections were later imaged using
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containing DAPI and imaged using a wide-field microscope Zeiss Axio Observer Z1, at low
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magnification 40x. Images show intensity levels without modification. Non-permeabilized cells
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were treated for immunofluorescence using the same protocol in the absence of Triton X100.
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After treatment with secondary antibody, the cells were allowed to incubate in wheat germ
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agglutinin (WGA)-Alexa Fluor 594 at 4°C for 10 minutes at a concentration of 5µg/ml. The
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coverslips were mounted with a mounting media containing DAPI and imaged using
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LSM510Meta confocal microscope, using high magnification conditions and processed for
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visualization as described above.
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Statistical analysis
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Data were expressed as mean of the number of cells from several transfections ± SE. Each
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condition reflects results from several coverslips originating from at least 2 independent
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transfections. Statistical analysis, done with Origin software, was made using one-way factor
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ANOVA. Differences were considered (*) significant when P < 0.05.
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RESULTS
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Pharmacological characterization of PDGF-mediated Ca2+ entry
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We recently showed that store-operated Ca2+ entry (SOCE) in human umbilical vein and human
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pulmonary artery endothelial cells (HUVEC and HPAEC) and rat aortic VSMC is mediated by
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STIM1 and Orai1 independently of other Orai isoforms and TRPC proteins(1, 34). To test
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whether PDGF mediates Ca2+ entry in VSMC via store-dependent or store-independent
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channels, we used pharmacological inhibitors of SOCE and found that PDGF activates a Ca2+
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entry pathway that displays classical SOCE features similar to those observed in non-excitable
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cells such as HEK293 and RBL. In primary cultured VSMC cells (synthetic), PDGF-evoked Ca2+
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entry was essentially blocked with 5 µM Gd3+, 30 µM 2-APB and as recently demonstrated in
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HEK293 cells(41), by 50 µM ML-9 (Figure 1A), suggesting that STIM1 and Orai1 might be the
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molecular players of PDGF-activated Ca2+ entry pathway in VSMC. We have shown recently
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that SOCE in primary cultured VSMC displays a unique feature where concentrations of 2-APB
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as low as 5 µM are inhibitory (34). Additional experiments where cells were incubated with
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either Gd3+ (5 µM) or 2-APB (5 µM) before restoring extracellular Ca2+, showed that PDGF-
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activated Ca2+ entry was also sensitive to these concentrations of Gd3+ and 2-APB in a manner
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that is reminiscent of thapsigargin-activated SOCE in VSMC(34) (Figure 1B). Similar results to
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those described in Figure 1A, B were obtained with the VSMC cell line A7r5 (data not shown).
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Dose response analysis of Gd3+ inhibition showed that concentrations of Gd3+ as low as 0.5 µM
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substantially inhibited PDGF-activated Ca2+ entry (64.64%±3.98 inhibition, n=19) while 5 µM
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essentially inhibited PDGF-activated Ca2+ entry (88.18%±2.61 inhibition, n=19; Figure 1C) in a
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manner reminiscent of that seen with thapsigargin-activated SOCE in HEK293 cells(44).
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STIM1 and Orai1 mediate PDGF-activated Ca2+ entry in VSMC
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To determine the molecular identity of the plasma membrane Ca2+ channels activated by PDGF
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in primary cultured VSMC, we used silencing RNA (siRNA) against STIM1, Orai and TRPC
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isoforms. Knockdown of STIM1, Orai or TRPC isoforms in VSMC was achieved using 2 different
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siRNA sequences used individually (see(34) for siRNA sequences). SiRNA sequences induced
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a marked decrease in their target mRNA levels 72 hours post-transfection as measured by
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quantitative RT-PCR (Figure 2 A, B; decrease by 82.14%±1.34 for siOrai1#1; 57.38%±4.68 for
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siOrai2#1; 65.67%±3.55 for siOrai3#1; 90.18%±3.57 for siSTIM1#1; 68.79%±6.23 for
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siSTIM2#1; 87.6%±1.6 for siTRPC1#1; 90.82%±2.48 for siTRPC4#1; 66.1%±8.24 for
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siTRPC6#1; n=6). Extensive characterization of the same siRNA in VSMC by our group showed
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that they induce decrease in protein levels of their respective targets, as assessed by western
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blotting and published previously ((34); decrease by 74.7%±7.5 for STIM1#1; 58.3%±7.0 for
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Orai1#1; 58.8%±6.4 for Orai3#1; 61.8%±7.7 for TRPC1#1; 66.2%±12.1 for TRPC4#1 and
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85.4%±5.8 for TRPC6#1, n=3). Representative Western blots analysis of STIM1 (Figure 2D)
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and Orai1 (Figure 2E) from cells transfected with either scrambled control siRNA, siRNA
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against STIM1 or siRNA against Orai1 are shown. Interestingly, down-regulation of either
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STIM1 or Orai1 using siRNA significantly suppressed PDGF-activated Ca2+ entry in primary
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cultured VSMC while neither Orai2, Orai3, TRPC1, TRPC4 nor TRPC6 knockdown affected the
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amplitude of PDGF-induced Ca2+ entry in primary cultured VSMC (Figure 3). Figure 3 shows
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representative traces showing an average of several VSMC originating from single coverslips 72
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hours post transfection with either control scrambled siRNA or target siRNA and assayed on the
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same day. Knockdown of individual STIM, Orai or TRPC isoforms in VSMC had no significant
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effect on SR calcium release (data not shown). Figure 4A shows statistical analysis of Ca2+
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entry measured using Fura2 imaging after knockdown of different proteins in several individual
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cells obtained from different coverslips originating from at least 3 independent transfections. The
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inhibition of Ca2+ entry compared to control was 53.58%±8.36 for siSTIM1 (n=49) and
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54.17%±4.16 (n=50) for siOrai1, with no significant inhibition detected upon knockdown of other
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proteins (Figure 4A). Results are representative of two independent siRNA sequences per
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target gene and are normalized to control cells transfected with a scrambled siRNA (for
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sequence see(34)). Simultaneous knockdown of STIM1 and Orai1 had a similar effect
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(55.43%±5.08 inhibition; n=106) to that of knockdown of either STIM1 or Orai1 alone, strongly
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arguing that both proteins are involved in the same pathway. Figure 4B shows a representative
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trace with average cells from the same coverslip while Figure 4C represents statistical analysis
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on several cells (shown in bar graphs) from 3 independent experiments. Given the incomplete
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knockdown of Orai2 and Orai3, these proteins might have a relatively smaller contribution to
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SOCE in VSMC. We conducted a set of experiments using double knockdown of Orai1+Orai2
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or Orai1+Orai3 compared with single Orai1 knockdown. Figure 4D shows that the extent of
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SOCE inhibition is similar whether Orai1 is knocked down alone or in combination with Orai2
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and Orai3. This lack of additivity suggests that Orai2 and Orai3 do not participate in a significant
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manner to SOCE in VSMC. However, we cannot rule out completely a small contribution from
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Orai2 and/or Orai3 to SOCE in VSMC. Figure 4E and 4F shows statistical analysis on several
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cells (shown in bar graphs) from 3-4 independent experiments where the extent of Ca2+ release
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(Figure 4E) and Ca2+ entry (Figure 4F) were measured in cells transfected with control siRNA
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and siRNA against Orai isoforms.
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Protein knockdown of the PDGF receptor β (PDGFRβ) using 2 independent siRNA caused a
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substantial decrease in both Ca2+ release and Ca2+ entry in response to PDGF. Figure 4G
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shows representative traces from individual coverslips while Figure 4H and Figure 4I shows
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statistical analysis of Ca2+ release (82.24%±11.8 inhibition; n=88) and Ca2+ entry (89.29% ± 6.87
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inhibition; n=88) respectively from 4 independent experiments. The ability of siRNAs against
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PDGFRβ to induce substantial decrease in PDGFRβ mRNA was determined by quantitative
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PCR as shown in Figure 2C; 80.04%±1.12 decrease for siPDGFRβ#1 and 63.02%±3.3 for
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siPDGFRβ#2).
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STIM1, Orai1 and PDGFRβ are important for PDGF-induced VSMC migration
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Primary cultured VSMC transfected with either scrambled control siRNA or siRNA against
327
different STIM and Orai isoforms were used in a scratch wound assay to assess VSMC
328
migration in response to PDGF stimulation. The cultured rat aortic VSMC used in our lab have a
329
doubling time of 22-24 hours. To avoid contributions from VSMC proliferation, wound closures
330
were analyzed 12 hours post scratch at which time, VSMC migration is apparent while
331
proliferation is minimal. SiRNA against either STIM1 or Orai1 lead a substantial decrease in the
332
wound surface covered by VSMC in response to 100 ng/ml PDGF at 12 and 24 hours post-
333
wound (46.4%±10.7 at 12 hours for STIM1; 30.4%±6.9 at 12 hours for Orai1; Figure 5).
334
However, STIM2, Orai2 and Orai3 silencing had no significant effect on VSMC migration
335
(Figure 6). Figure 7 shows that knockdown of PDGFRβ in VSMC using siRNA caused a
336
substantial decrease in the wound surface covered by VSMC in response to 100 ng/ml PDGF at
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12 and 24 hours post-wound, compared to scrambled control-transfected cells (48.31%±6.56 at
338
12 hours; 60.46%±4.78 at 24 hours; Figure 7).
339
Recent studies have shown upregulation of STIM1 and Orai1 proteins in proliferative migratory
340
rat mesenteric arteries compared to quiescent freshly isolated VSMC from the same vascular
341
bed(8) and our group have recently published similar findings in rat aortic VSMC(34). The
342
involvement of STIM1 and Orai1 in PDGF-mediated VSMC migration in vitro, prompted us to
343
determine whether STIM and Orai proteins and PDGFRβ are upregulated in proliferative and
344
migratory VSMC in vivo in an animal model of vascular disease.
345
STIM1, Orai1 and PDGFRβ are upregulated in VSMC during vascular injury
346
While the role of Orai1 in neointima formation remain unknown, two recent reports described an
347
essential role for STIM1 in neointimal formation following arterial balloon injury in the rat(5, 16).
348
Therefore, we sought to analyze the potential upregulation of STIM1, Orai1 and PDGFRβ in
349
VSMC in vivo in a rat model of vascular injury. We performed balloon-injury on rat carotids from
350
male rats as described in the Methods section using sham-operated animals as controls. The
351
vascular injury model we used has been well described in numerous published studies over
352
many years(7, 25). Unlike diet-induced injuries, mechanical injuries are characterized
353
predominantly by VSMC proliferation and migration with only early and transient lymphocyte
354
and monocyte infiltration and very few detectable immune cells days after the injury(43). Figure
355
8 A, B shows representative sections from either sham control or injured carotid vessels 14
356
days post treatment stained with hematoxylin-eosin. Figure 8C shows that mRNA levels
357
encoding STIM1, Orai1 and PDGFRβ are significantly upregulated in injured carotid arteries 14
358
days post-injury compared to non-injured time matched sham-operated rats (12.03±3.33 fold for
359
STIM1; 15.13±3.02 fold for Orai1; and 6.33±0.44 fold for PDGFRβ). However, the mRNA levels
360
of STIM2, Orai2 and Orai3 were unaffected. As reported earlier(34), Figure 8D depicts
361
representative Western Blots showing that STIM1 and Orai1 upregulation also occurs in vitro in
14
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synthetic cultured VSMC, suggesting that contributions from infiltrating leukocytes to the
363
increase in STIM1 and Orai1 observed in vivo is unlikely.
364
Although we showed in Figure 2E that Orai1 antibody recognized a single band at the right
365
molecular weight that was decreased 72 hours post-transfection with specific siRNA against
366
Orai1, strongly arguing for the specificity of this antibody for Orai1, we performed additional
367
experiments in cultured VSMC to further validate its use in immunofluorescence studies in
368
vessel sections. We used a similar Orai1 knockdown approach followed by immunofluorescence
369
staining with anti-Orai1 in cultured synthetic VSMC as depicted in Figure 9A. In these
370
experiments, VSMC were processed for IF staining using standard protocols with Triton-X100
371
permeabilization and imaged using a wide-field microscope under low magnification conditions
372
as described in the Material & Methods. Figure 9A clearly shows that Orai1 staining is
373
dramatically decreased 72 hours after treatment with siRNA against Orai1. To address the
374
putative plasma membrane association of Orai1, we subjected VSMC cells to an IF protocol
375
under non-permebilized conditions followed by imaging using a confocal microscope at high
376
magnification. As shown in Figure 9B, Orai1 staining co-localizes with that of wheat germ
377
agglutinin (WGA), a well-known plasma membrane marker in non-permeabilized cells,
378
suggesting a plasma membrane localization of Orai1. Next, we performed immunofluorescence
379
staining in vessel sections of carotid arteries from sham-operated and injured rats probing for
380
STIM1 and Orai1 protein expression. Figure 10 shows increased protein expression of both
381
STIM1 and Orai1 in medial and neointimal VSMC from injured rat carotids (14 days post-injury)
382
compared to medial VSMC from non-injured vessels. In Figure 10, fluorescence images were
383
taken at similar imaging conditions at low magnification to provide an accurate representation of
384
differences in STIM1 and Orai1 fluorescence levels between the sham-operated and injured
385
carotid vessels. To further characterize the subcellular distribution of Orai1 in tissues from
386
control and injured carotid arteries, we collected vertical sections every 0.5 µm using confocal
387
microscopy (airy units =1) at high magnification (lens 63x and zoom =2). Image J volume viewer
15
Bisaillon et al.
388
plugin was used to generate a vertical cross-section of the tissue slice, which shows a
389
peripheral localization of Orai1, consistent with a plasma membrane association (Figure 11).
390
Orai1 staining appeared to be more polarized in cells from sections of injured carotids compared
391
to control vessels; additional studies are clearly needed to understand the contribution of this
392
polarized Orai1 staining to VSMC migration in vitro and in vivo.
393
16
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394
DISCUSSION
395
Agonist binding to G protein coupled receptors or receptor tyrosine kinase activate isoforms of
396
PLC to cause Ca2+ release from the SR and Ca2+ entry from the extracellular space. Receptor-
397
activated Ca2+ entry channels are of two major types: store-operated channels activated by the
398
fall of the Ca2+ concentration within the SR through the action of IP3 on its receptor, and store-
399
independent channels activated by diverse mechanisms involving among others second
400
messengers(31). Insights into the molecular identity of SOC channels and the signaling
401
mechanisms connecting internal Ca2+ store depletion to this Ca2+ entry have been recently
402
gained (for review see (35)) with the identification of a Ca2+-binding protein, Stromal Interaction
403
Molecule 1 (STIM1) as the long sought Ca2+ sensor in the ER/SR(24, 38) capable of
404
oligomerization and reorganization into punctuate structures in defined ER–plasma membrane
405
junctional areas upon store depletion to signal the activation of the SOC channel at the PM
406
composed of the membrane protein Orai1 (15, 48). The mechanisms of STIM1/Orai1 coupling
407
are beginning to emerge and appear to involve direct binding of a minimal, highly conserved
408
~107-amino acid region of STIM1 to the N- and C-termini of Orai1 to open the CRAC
409
channel(23, 29, 32, 51). However, a wide variety of store-dependent and store-independent
410
nonselective cation conductances activated downstream of PLC-coupled receptors have been
411
described by different groups in many cell types, including VSMC(3, 21, 31). Molecular
412
candidates for these cation channels include homomultimers and heteromultimers within Orai
413
and TRP family members as well as heteromutlimers of Orai/TRP isoforms(4, 12, 46).
414
In vitro and in vivo studies in mouse mutants have established PDGF as a major pro-
415
proliferative and pro-migratory growth factor for many cell types of mesenchymal origin,
416
including fibroblasts and smooth muscle cells(10). The importance of PDGF signaling during
417
cardiovascular diseases such as restenosis and atherosclerosis is clearly established, whereby
418
PDGF attracts PDGFRβ-expressing medial VSMC thus promoting their accumulation in the
419
neointima (37). PDGF receptors are receptor tyrosine kinase that couple to PLCγ activation and
17
Bisaillon et al.
420
cause increase in cytoplasmic Ca2+ via plasma membrane Ca2+ entry channels. However, the
421
molecular identity of PDGF-activated Ca2+ entry channels in VSMC remained unknown. In this
422
report we show that in cultured synthetic VSMC PDGF activates a Ca2+ entry pathway
423
pharmacologically reminiscent of classical SOCE, namely inhibition by low concentrations of
424
lanthanides (5µM Gd3+), 2-APB (5µM) and ML-9 (50µM). We also show that PDGF-induced
425
Ca2+ entry was substantially inhibited by knockdown of Orai1, STIM1 and PDGFRβ while
426
knockdown of Orai2, Orai3, TRPC1, TRPC4 and TRPC6 had no significant effect, establishing
427
STIM1/Orai1 as the Ca2+ entry route activated in response to PDGFRβ stimulation in VSMC.
428
The primary cultured VSMC used in this study (also known as synthetic) are dedifferentiated
429
VSMC that have lost their contractile markers and are highly proliferative and migratory
430
compared to the quiescent freshly isolated VSMC(19, 30). These synthetic VSMC cells
431
recapitulate most of the phenotype and gene expression patters of proliferative and migratory
432
VSMC found in vivo during vascular disease such as restenosis and atherosclerosis(19, 30).
433
We and others previously showed that these cultured VSMC have increased STIM1 and Orai1
434
protein expression and upregulated SOCE compared to their quiescent freshly isolated
435
counterparts(8, 34). Here we show that knockdown of either STIM1 or Orai1 inhibits VSMC
436
migration in vitro in response to PDGF, while STIM2, Orai2 and Orai3 silencing had no
437
significant effect suggesting that STIM1/Orai1 pathway is the mediator of PDGF action on
438
PDGFRβ in VSMC. Furthermore, we also show that during vascular injury, medial and
439
neointimal VSMC upregulate the mRNA and protein expressions of PDGFRβ, STIM1 and Orai1.
440
Two recent studies have demonstrated that in vivo silencing of STIM1 using viral particles
441
encoding STIM1-targeted shRNA in rat balloon-injured vessels inhibits neointima formation(5,
442
16). However, Orai1 upregulation or its contribution to VSMC migratory phenotype in vivo has
443
not been studied. Our results powerfully complement these studies by providing a plausible
444
mechanism for the role of STIM1/Orai1 in neointima formation. Clearly, additional studies are
18
Bisaillon et al.
445
needed to understand the roles of all STIM and Orai isoforms in mediating Ca2+ entry in
446
response to various growth factors/agonists so we might gain future insights into the use of
447
these proteins as targets for vascular disease therapy.
448
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449
ACKNOWLEDGMENTS
450
This work was supported by an NIH early career grant (K22ES014729) and supplements
451
(K22ES014729S1 and K22ES014729S2) allocated through the American Recovery and
452
Reinvestment Act (ARRA) mechanism to Mohamed Trebak. We are very grateful to Dr. David
453
W. Saffen (Fudan University, Shanghai, China) for his generous gifts of reagents and advice.
454
We also gratefully acknowledge Wendy Vienneau and Jo Anne Evans for administrative
455
support.
456
DISCLOSURES
457
None.
458
459
460
461
462
463
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Bisaillon et al.
464
LEGENDS TO FIGURES
465
Figure 1: A. In the absence of extracellular Ca2+, PDGF (50 ng/ml) activates Ca2+ release from
466
internal stores in primary cultured VSMC. Upon Ca2+ restoration to the extracellular milieu
467
(2mM), PDGF activates a Ca2+ entry pathway (control trace; average of n=18 cells), that is
468
sensitive to 5µM Gd3+ (n=8), 30µM 2-APB (n=29) and 50µM ML-9 (n=18). Drugs were added
469
where indicated by an arrow. B. Pre-incubation with either 5µM Gd3+ (n=10) or 5µM 2-APB
470
(n=18) essentially abrogated PDGF-activated Ca2+ entry; control trace (n=15). C. Dose
471
response inhibition of PDGF-mediated calcium entry by Gd3+ shows that concentrations as low
472
as 0.5µM Gd3+ cause a substantial inhibition of calcium entry (total number of cells are included
473
in the bar graphs). All Ca2+ imaging traces shown are average data from several cells analyzed
474
simultaneously. Data are representative of 3-4 independent experiments.
475
Figure 2: Quantitative RT-PCR assessing mRNA levels of STIM and Orai (A), TRPC (B)
476
isoforms and PDGF receptor β (C) after knockdown by transfection of 2 specific siRNA
477
sequences used independently or a scrambled siRNA sequence used as a control. After 72
478
hours, mRNA was isolated from cells, reverse transcribed and quantitative PCR was performed
479
using specific primers. Sequences for siRNA and primers for STIM, TRPC and Orai were
480
provided elsewhere(34). Sequences for primers and siRNA against PDGFRβ are provided in the
481
methods section. Data are representative of three independent transfections performed in
482
triplicates. Westerns blots showing STIM1 (D) and Orai1 (E) protein knockdown after siRNA
483
transfection as previously published(34). Data are representative of 4 independent experiments
484
and statistics on the STIM1/actin and Orai1/actin densitometry ratios of bands are included in
485
the text.
486
Figure 3: Representative traces showing average of PDGF-activated Ca2+ entry from several
487
cells in single coverslips from either scrambled control- or targeted siRNA-transfected VSMC
488
performed the same day. Representative Results obtained with targeted siRNA against STIM1
21
Bisaillon et al.
489
and Orai1 (A), Orai2 (B), Orai3 (C), TRPC1 (D), TRPC4 (E) and TRPC6 (F) are represented.
490
PDGF was used at 50 ng/ml for siOrai2 (B) and at 25 ng/ml for all other recordings. Data are
491
representative of 3-6 independent experiments per targeted siRNA. PDGF was first added in the
492
absence of extracellular calcium and exact times of calcium restoration to the extracellular
493
milieu for each trace are indicated with arrows.
494
Figure 4: A. PDGF-activated Ca2+ entry after VSMC transfection with either scrambled or
495
targeted siRNA (against STIM1, Orai1, Orai2, Orai3, TRPC1, TRPC4 and TRPC6) was
496
measured using Fura2 and the extent of Ca2+ entry was analyzed in several cells originating
497
from several independent experiments and is represented as the difference between the Fura2
498
signal before and after Ca2+ (2mM) restoration. Bar graphs represent average of cells originating
499
from 3-6 independent experiments per condition; data are represented as % of control. Total
500
numbers of cells for each condition are included (*P < 0.05). B. Representative traces depicting
501
the effect of STIM1 and Orai1 simultaneous silencing on PDGF-mediated calcium entry (n=15);
502
the condition with control scrambled siRNA-transfected cells (n=22) is also shown. Averages of
503
extent of calcium entry in many scrambled control- and siOrai1+siSTIM1-transfected cells from
504
3 independent experiments are represented in C; total number of cells in both conditions are
505
shown in bar graphs. D. Representative traces depicting the effect of Orai1 knockdown (n=15),
506
siOrai1+siOrai2 (n=18) and siOrai1+siOrai3 (n=14) simultaneous knockdown on PDGF-
507
mediated calcium entry as compared to control scrambled siRNA-transfected cells (n=20).
508
Averages of extent of calcium release (E) and calcium entry (F) in many scrambled control-,
509
siOrai1-, siOrai1+siOrai2-, and siOrai1+siOrai3-transfected cells from 3-4 independent
510
experiments are shown with total number of cells per condition shown in bar graphs. G.
511
Representative traces depicting the effect of PDGF receptor β (PDGFRβ) knockdown on PDGF-
512
mediated calcium release and entry (n=16); the control trace representing scrambled siRNA-
513
transfected cells is an average of 25 cells. Averages of the extent of calcium release (H) and
514
entry (I) in many scrambled control- and PDGFRβ-transfected cells from 4 independent
22
Bisaillon et al.
515
experiments are also shown with total number of cells indicated in the histogram. In all
516
experiments, PDGF was used at a concentration of 50ng/ml.
517
Figure 5: A, Bright field views of scratch wound migration assay performed in serum-free
518
media containing 100 ng/ml PDGF at different times post wound in primary cultured VSMC
519
either transfected with scrambled control siRNA or siRNA against either STIM1 or Orai1. Data
520
from 3 independent experiments (with 4 wells per condition) were quantified at 12 hours (B) and
521
24 hours (C) post wound and analyzed as described in methods and represented as % of
522
control (*P < 0.05).
523
Figure 6: A, Bright field views of scratch wound migration assay performed in serum-free
524
media containing 100 ng/ml PDGF at different times post wound in primary cultured VSMC
525
either transfected with scrambled control siRNA or siRNA against either STIM2, Orai2 or Orai3.
526
Data from 3 independent experiments (with 4 wells per condition) were quantified at 12 hours
527
(B) and 24 hours (C) post wound and analyzed as described in methods and represented as %
528
of control (*P < 0.05).
529
Figure 7: A, Bright field views of scratch wound migration assay performed in serum-free media
530
containing 100 ng/ml PDGF at different times post wound in primary cultured VSMC either
531
transfected with scrambled control siRNA or siRNA against PDGFRβ. Data from 2 independent
532
experiments (with 4 wells per condition) were quantified at 12 hours (B) and 24 hours (C) post
533
wound and analyzed as described in methods and represented as % of control (*P < 0.05). Data
534
shown were generated using PDGFRβ siRNA #1; similar results were obtained with PDGFRβ
535
siRNA #2 (not shown).
536
Figure 8: Hematoxylin-Eosin (HE) staining on rat carotid arteries from time matched (14 days)
537
non-injured sham control (A) and balloon-injured 14 days post-injury (B). C; Quantitative RT-
538
PCR were performed to assess the mRNA levels of STIM and Orai isoforms and PDGFRβ.
539
mRNAs were isolated from VSMC cells from sham control and injured rat carotids. Data are
540
represented as fold increase in mRNA in injured carotids compared to sham control. Data were
23
Bisaillon et al.
541
obtained from 3 control and 3 injured rats that were analyzed in 3-7 independent runs.
542
M=Media; NI=Neointima; L=Lumen. D; as demonstrated previously(34), synthetic (cultured
543
VSMC) express higher levels of STIM1 and Orai1 proteins compared to quiescent freshly
544
isolated VSMC. Rat basophilic leukemia (RBL) cells were used in parallel as a positive control.
545
Figure 9. A; VSMC cells were transfected with non-targeting siRNA (siControl) or with siRNA
546
targeting Orai1 (siOrai1), fixed and processed for immunofluorescence under permeabilized
547
conditions as described in the methods using anti-Orai1 and DAPI as a nuclei marker. Images
548
shown are representative of three independent experiments. A reduction in Orai1 expression is
549
clearly detected in the siOrai1 images. B; VSMC cells were fixed, processed for
550
immunofluorescence under non-permeabilized conditions (without Triton X100) using anti-Orai1
551
(Orai1), Wheat germ Agglutinin (WGA)-Alexa Fluor 594 (WGA) as a plasma membrane marker
552
and DAPI (DAPI) as a nuclei marker and images were collected using confocal microscopy.
553
Arrows indicate co-localization between Orai1 and WGA at the plasma membrane (Merge,
554
yellow staining). These images were modified in Adobe Photoshop to a higher level of contrast
555
for better visualization.
556
Figure 10. Immunofluorescence staining on carotid artery sections from rats subject to balloon
557
injury (C, F; 14 days post injury) and control non-injured vessels from sham-operated animals
558
(A, B, D, E) using anti-Orai1 (B, C) and anti-STIM1 (E, F) antibodies followed by a secondary
559
antibody coupled to FITC. A secondary antibody alone control was also performed (A, D). The
560
brackets indicate the neointima in injured vessels. Data are representative of results obtained
561
independently from sections originating from 3 control and 3 injured rats. M=Media;
562
NI=Neointima; L=Lumen
563
Figure 11. Carotid artery sections from rats subject to balloon injury (Panel B; 14 days post
564
injury) and control non-injured vessels from sham-operated animals (Panel A) were processed
565
for immunofluorescence staining using anti-Orai1 and DAPI. Vertical sections were collected
566
every 0.5µm using confocal microscopy at high magnification. A representative image is shown
24
Bisaillon et al.
567
in the right. The Image J Volume viewer plugin was used to generate a vertical cross-section
568
(yellow line) of the tissue slice. These images were modified in Adobe Photoshop to a higher
569
level of contrast for better visualization. Orai1 shows a peripheral subcellular localization, which
570
is consistent with a plasma membrane association.
571
572
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Figure 1
A
2.0
PDGF
2+
Ca
1.6
1.4
C
Cont.
1.2
1.0
2-APB
ML9
120
0.8
Gd
0.6
0
5
10
15
3+
20
25
Time, Min
B
2.0
Gd3+/ 2-APB
Ratio 340/380
1.8
PDGF
Ca2+
1.6
1.4
1.2
Control
1.0
2-APB
0.8
Gd3+
0.6
0
2
4
6
8
10 12 14 16 18 20
Time, Min
DRatio 340/380 % Control
Ratio 340/380
1.8
100
80
60
*
40
*
20
0
42
19
29
*
19
Control
0.5
1
5
mM Gd3+
Figure 2
125
100
75
50
*
*
* *
*
*
*
*
50
O
Control
D
100 __
75
__
*
25
si
Co
nt
ro
ra l
i
1si
1
O
ra
si i12
O
r
si ai2
O
ra 1
si i22
O
ra
si i3O
r 1
si ai3ST
2
si IM 1
S T -1
I
si M 1
S T -2
I
si M 2
S T -1
IM
22
25
* *
75
0
*
*
*
l
tro
100
-2
-1
-2
-2
-1
-1
C4
C4
C6
C1
C6
C1
n
P
P
P
P
P
P
o
C
TR
TR
TR
TR
TR
TR
si
si
si
si
si
si
E
STIM1
50 __
__
37
25 __
__
100
75 __
50 __
__
37
25 __
__
20
*
*
Control
100
0
C
B
mRNA expression (% of control)
mRNA expression (% of control)
125
75
50
*
25
0
*
Control
siOrai1
A
siSTIM1
mRNA expression (% of control)
125
__
100__
75 __
50
__
37
__
25
__
15
Orai1
__
Actin
100__
75
50 __
__
37
__
25
__
15
Actin
siPDGFRb-1
siPDGFRb-2
Figure 3
A
B
PDGF
2.0
2+
Ca
1.8
1.6
F340/380
F340/380
Control
siSTIM1
1.0
0.6
1.6
1.6
1.4
1.4
1.2
2
4
6
8
10
12
14
siOrai2
2
4
Time, min
D
6
8
10 12
Time, min
E
PDGF
2.0
14
1.4
1.4
0.6
1.2
F340/380
1.4
Control
1.0
siTRPC1
0.8
0
2
4
6
8
Time, min
10
12
6
8
10
Time, min
12
siTRPC4
0
2
4
6
Time, min
8
10
14
16
PDGF
2+
Ca
1.2
Control
1.0
0.8
0.6
4
1.8
1.6
1.0
2
2+
Ca
1.6
Control
0
2.0
1.6
1.2
siOrai3
F
1.8
F340/380
F340/340
0.6
18
PDGF
2+
1.8
0.8
16
2.0
Ca
Control
1.0
0
16
Ca 2+
1.2
Control
0.8
0
PDGF
1.8
Ca2+
1.0
siOrai1
0.8
2.0
1.8
1.4
1.2
PDGF
F340/380
2.0
C
siTRPC6
0.8
12
0.6
0
2
4
6
Time, min
8
10
B
1.8
*
32
40
49
1.0
0.6
PDGF
2
4
1.4
siOrai1
1.2
siOrai1 + siOrai2
1.0
0.8
0.6
siOrai1 + siOrai3
2
4
6
8
10
12
2+
Control
1.0
siPDGFRb
0.8
0.6
8
Time, min
10
12
DRatio 340/380 %Control
1.2
6
20
0
0.4
0.2
73
Control
siOrai1
siOrai1 +
siOrai2
54
1.0
Ca2+ Entry
0.8
*
*
0.6
*
0.4
0.2
0.0
siOrai1 +
siOrai3
145
73
63
45
Control
siOrai1
siOrai1 +
siOrai2
siOrai1 +
siOrai3
I
Ca2+ Release
100
80
60
40
*
20
0
106
siOrai1 + siSTIM1
1.2
0.6
58
103
Control
F
Ca2+ Release
31
*
40
14
Time, min
120
Ca
4
12
H
1.4
2
10
0.8
0.0
PDGF
1.6
8
1.0
Time (min)
G
6
60
D F340/380 (% of Control)
DF340/380 (% of Control)
2+
Control
0
0
80
1.2
Ca
0
siOrai1 + siSTIM1
E
1.6
Ratio 340/380
Control
D
1.8
1.8
1.2
0.8
siTRPC6
35
siTRPC4
36
siTRPC1
50
siOrai3
20
49
siOrai1
134
siSTIM1
40
*
siOrai2
60
Ca2+
1.4
100
D Ratio 340/380 % Control
80
0
Ratio 340/380
Ratio 340/380
100
0.4
PDGF
1.6
siCont
D F340/380, % of control
120
2.0
C
Figure 4
A
73
88
Control
siPDGFRb
120
DRatio 340/380 %Control
140
Ca2+ Entry
100
80
60
40
*
20
0
73
Control
88
siPDGFRb
Figure 5
B
125
A
Control
0 hr
12 hr
24 hr
Migration (% of control)
PDGF (100ng/ml)
12 hours PDGF
100
*
75
50
25
siOrai1
0
Control
siOrai1
siSTIM1
C
Migration (% of control)
125
siSTIM1
*
24 hours PDGF
100
75
*
*
siOrai1
siSTIM1
50
25
0
Control
Figure 6
A
0 hr
PDGF (100ng/ml)
12 hr
24 hr
B
12 hours PDGF
100
Migration (% of control)
siO
Orai2
Control
120
80
60
40
20
0
Cont.
siOrai2
siOrai3
siSTIM2
C
24 hours PDGF
Migration (% o
of control)
siiSTIM2
siOrai3
120
100
80
60
40
20
0
Cont.
siOrai2
siOrai3
siSTIM2
Figure 7
PDGF (100ng/ml)
A
12 hr
24 hr
si PDGFRb
Control
0 hr
C
B
12 hours PDGF
100
75
*
50
25
0
Control
siPDGFRb
125
Migration (% of control)
Migration (% of control)
125
24 hours PDGF
100
75
50
*
25
0
Control
siPDGFRb
B. 14 day Injured
A. 14 day Sham Control
Figure 8
L
NI
M
M
L
D
mRNA folld increase (Injured/Sham)
C
20
18
16
14
12
10
8
6
l
Cu
1.5
C
SM
V
t.
L
RB
i
Qu
es
M
VS
c.
C
ie
Qu
SM
.V
c
s
C
l
Cu
SM
t .V
C
STIM1
Orai1
Actin
Actin
1.0
0.5
0.0
STIM1 STIM2 Orai1 Orai2 Orai3 PDGFRE
A
Orai1
DAPI
Merge
Orai1
DAPI
Merge
siControl
siOrai1
40Pm
B
Orai1
WGA
Merge
Non-perm
Figure 9
10Pm
Figure 10
A
B
M
C
M
NI
M
L
L
Sham; 2nd Ab
(neg control)
(neg.
Sham;
Orai1
Injured;
Orai1
D
E
F
M
M
NI
L
L
M
Sham; 2nd Ab
(neg. control)
Sham;
STIM1
Injured;
STIM1
Figure 11
Sham
A
Orai1
Sham
DAPI
5Pm
Merge
g
Injured
B
Orai1
DAPI
5Pm
Merge
Injured