- Open Access
A key role for STIM1 in store operated calcium channel activation in airway smooth muscle
© Peel et al. 2006
Received: 20 June 2006
Accepted: 20 September 2006
Published: 20 September 2006
Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca2+ stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified.
To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay.
Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp.
Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.
Control of intracellular calcium is critical to regulation of smooth muscle function in many tissues. The relative contribution of SOC to the control of intracellular Ca2+ varies between different types of smooth muscle, with SOC being particularly prominent in airway myocytes. The contractile/relaxant state of the airway myocyte is a key determinant of airway calibre thus contributing to bronchoconstriction in diseases such as asthma. Previous studies have demonstrated that the contractile response of airway myocytes is dependent initially upon release of intracellular Ca2+ from the sarcoplasmic reticulum (reviewed in ) but that sustained contraction is dependent upon influx from extracellular sources. Two mechanisms have been proposed to account for this influx in airway myocytes involving either activation of SOC or ROC. In contrast to vascular smooth muscle, L type voltage dependent calcium channels (VDCCs) appear to play a negligible role in control of Ca2+ entry . In previous work we have demonstrated the expression of a number of TRP homologues including TRPC1, 3, 4 and 6 in cultured human airway myocytes and lung tissue and have suggested that TRPC6 may play an important role (probably together with other TRPC homologues including TRPC3 ) in contributing to agonist induced ROC activity .
SOC activation in many cell types including smooth muscle is known to involve depletion of the intracellular sarcoplasmic reticulum Ca2+ stores. Contractile agonists such as acetylcholine, histamine and bradykinin may vary in their ability to differentially activate ROC or SOC although all agonists are known to induce activation of phospholipase C with consequent IP3 mediated Ca2+ release from the intracellular stores. The mechanism underlying signalling for subsequent Ca2+ influx in response to store depletion, and hence refilling of the sarcoplasmic reticulum Ca2+ stores, remains unknown. In the current study we have set out to define the signals for SOC activation in human airway myocytes following both store depletion and agonist activation by spasmogens.
Using RNA interference techniques STIM (stromal interaction molecule) 1 has been shown to play a role in SOC induced calcium entry in Drosophilia S2 cells, Jurkat T cells  and Hela cells  with the latter study also implicating a role for STIM2. In particular, STIM1 appears to be a major activator of calcium release activated calcium channels (ICRAC) in T lymphocytes via a mechanism which has been proposed to involve translocation of STIM1 from endoplasmic reticulum like sites to the cell membrane . We therefore hypothesised that homologues of STIM may play a role in SOC activation in smooth muscle. To address this hypothesis we used specific siRNA sequences to suppress both STIM1 and STIM2.
Human bronchial tissue was obtained from patients without a history of asthma. Human airway smooth muscle (HASM) cells were isolated and cultured as previously described . Ethical approval for these studies was obtained from the Nottingham local ethical research committee. All subjects from whom tissue was obtained gave written consent. Primary human bronchial epithelial cells were obtained from Cambrex Bioscience (MD, USA) and grown in accordance with suppliers protocols. Cells at passage 4 were differentiated at an air-liquid interface on polyester tissue culture inserts (Corning, Costar) as described in a previous published method .
Transfection of siRNAs
siRNAs, including the scrambled siRNA control were purchased from Ambion (Huntingdon, Cambridge, UK). STIM1 siRNA (AAGGGAAGACCTCAATTACCA) was pre-designed from Ambion, STIM2 siRNA (AACTGAGAAGCAGTTGGTCTG) designed by Roos and colleagues . Cells were transfected with siRNA (1–50 nM) in serum free medium over a period of 6 h, the medium was then aspirated and replaced with serum containing medium for a further period of 42 h. The transfection reagent used was Lipofectamine 2000 (Invitrogen, Paisley, UK) at a final concentration of 2 μl/ml.
Total RNA extraction and reverse transcriptase PCR
Total RNA was isolated from pelleted cells using the RNeasy mini kit (Qiagen, West Sussex, UK) as per manufacturers' instructions. To examine for STIM1 and STIM2 expression, RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and random hexamers (Invitrogen). PCR was performed using specific primers against STIM1 (Forward; AGGCAGTCCGTAACATCCAC, Reverse; CTTCAGTCCGTAACATCCAC) and STIM2 (Forward; TCCCTGCATGTCACTGAGTC, Reverse; GGGAAGTGTCGTTCCTTTGA). Cycling was performed 35 times; 94°C, followed by 55°C (annealing temperature), then 72°C (all for 90 seconds) followed by 10 mins at 72°C. PCR products were visualized by ethidium bromide staining and confirmed by direct sequencing.
Real-Time PCR (Taqman)
siRNA targeted mRNA knockdown was measured using real time, quantitative PCR (Taqman). Gene specific primers and probes against STIM1 and STIM2 were designed using Primer Express™ software (Applied Biosystems, Foster City, CA) and using 18s RNA as the reference gene (Applied Biosystems). All probes were MGB probes, labeled with a 5'-reporter dye FAM and a non fluorescent quencher. Each sample was run in duplicate and mRNA knockdown was measured from mRNA obtained from 3 separate experiments. The relative expression of the target gene was calculated using the comparative method (2-ΔΔCt) .
STIM-1 forward primer: AAGGCTCTGGATACAGTGCTCTTT
reverse primer: AGCATGAAGTCCTTGAGGTGATTAT
STIM-2 forward primer: ACGACACTTCCCAGGATAGCA
reverse primer: GACTCCGGTCACTGATTTTCAAC
Measurement of [Ca2+]i
HASMs (passage 4–5) were plated in black walled, clear bottom 96 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in culture medium (DMEM) supplemented with 10% FCS and 2.5 mM probenecid (Sigma Chemical Co, Poole, Dorset, UK). Cells were then washed with Hanks' balanced saline solution containing 10 mM Hepes, 2.5 mM probenecid, 0.1 mM CaCl2 and 1 mM MgCl2. The fluorescence was continuously recorded at wavelengths of 485 nm excitation and 520 nm emission using a Flexstation (Molecular Devices, Wokingham, UK). Cells were treated with 10 μM cyclopiazonic acid (final concentration) for 4 minutes followed by the addition of 1.9 mM CaCl2 (2 mM final concentration). For agonist induced Ca2+ responses, cells were stimulated with bradykinin (1 μM) or histamine (100 μM final concentration) for 4 minutes in 0.1 mM CaCl2 buffer followed by the addition of 1.9 mM CaCl2. Data are presented as changes in fluorescence intensity (FI) compared with the baseline, the area under the curve was used as an estimation of changes in [Ca2+]i.
The conventional whole-cell patch-clamp technique  was employed to record store operated inward currents in single HASM cells with an EPC-10 double amplifier and Patchmaster version 2.10 software (HEKA, Lambrecht, Germany). The compositions of the internal and external solutions are as follows; Standard Internal Solution; 110 mM Cs-methanesulfonate, 25 mM CsCl, 2 mM MgCl2, 10 mM EGTA, 30 mM HEPES, 3.62 mM CaCl2. External Solution: 140 mM NaCl, 5 mM CsCl, 1 mM MgCl2, 10 mM D-Glucose, 10 mM HEPES, CaCl2 (as indicated). K+ was replaced by Cs+ in both external and internal solutions to block K+ currents and Cl- was replaced by an equal molar concentration of methanesulfonate to minimize Cl- currents. Nifedipine (5 μM) was included in the external solution. Pipettes were drawn from borosilicate glass and had resistances of 5–8 MΩ when filled with internal solution. HASM cells were placed directly into the cell chamber, allowed to settle and then were continuously perfused with external solution at a constant speed of 6 ml/min. Experimental drugs were delivered through a puffer pipette positioned 50 μm around the cells. Cells were held at a membrane potential of -60 mV and current-voltage relationships were analysed every 5s from voltage ramps from -100 to +100 mV at a rate of 0.5 Vs-1. Currents were filtered at 1 kHz and sampled at 4 KHz. Individual cell current densities were calculated by dividing peak current amplitude at maximum activation of inward current (at -100 mV) by cell capacitance.
HASMs grown on coverslips, transfected with either 20 nM scrambled siRNA or 20 nM STIM1 siRNA were fixed with 4% formaldehyde. Cells were permeabilized (0.5% TritonX-100) and blocked with 20% goat serum in PBS for 20 min. Cells were incubated with primary antibody (mouse, anti-human STIM1 mAb (1:100) (BD Biosciences, Pharmingen) overnight at 4°C followed by labeling with Alexa fluor 488 (Molecular probes). Cells were visualized on a Zeiss LS 510 confocal microscope (Hertfordshire, UK).
Averaged data are presented as mean ± sem. Where appropriate, statistical significance was assessed by unpaired Students T tests or one-way ANOVA followed by the Dunnets test for multiple group comparisons. Data were considered significant at *P < 0.05 or **P < 0.01.
Results and discussion
The mechanism whereby STIM1 is able to activate SOC in airway myocytes remains to be determined. The STIM genes encode type 1 transmembrane proteins that can potentially form hetero or homo-oligomers via coiled- coiled interactions [13, 14]. The NH2 terminus contains an EF-hand Ca2+ binding motif which is thought to be responsible for the detection of Ca2+ depletion in stores [6, 7, 12]. STIM1 is expressed in both plasma and intracellular membranes  and the EF hand is thought to be located outside the cell or in the lumen of intracellular stores. It is conceivable that STIM proteins may interact (possibly through coiled coil domains) between the two membranes providing the vital link between intracellular stores and the plasma membrane . Other models have been suggested including translocation of STIM1 from the endoplasmic reticulum to the plasma membrane  where STIM1 could directly activate SOC channels, or the involvement of STIM1 in the production of an unidentified Ca2+ influx factor .
The exact molecular identity of SOC in airway myocytes remains to be determined although potential candidates include a range of TRP homologues [3, 16, 17]. At present there are no specific tools to inhibit these channels directly and such approaches may be complicated by the formation of channels formed of heteromeric subunits.
Our data clearly implicates a role for STIM1 in SOC activation in airway myocytes providing for the first time molecular insight into this key signalling pathway in smooth muscle. Given the importance of control of intracellular Ca2+ to airway smooth muscle contraction STIM1 may provide a potential therapeutic target for diseases characterised by increased smooth muscle contractility such as asthma. However, one note of caution must be added in that STIM1 was initially identified as a candidate tumour suppressor gene  and the consequences therefore of long term inhibition of STIM1 expression need to be explored further.
Samantha Peel is in receipt of an MRC studentship.
- Hall IP: Second messengers, ion channels and pharmacology of airway smooth muscle. Eur Respir J 2000, 15:1120–1127.View ArticlePubMedGoogle Scholar
- Murray RK, Kotlikoff MI: Receptor-activated calcium influx in human airway smooth muscle cells. J Physiol 1991, 435:123–144.View ArticlePubMedPubMed CentralGoogle Scholar
- White TA, Xue A, Chini EN, Thompson M, Sieck GC, Wylam ME: Role of TRPC3 in Tumor Necrosis Factor-alpha Enhanced Calcium Influx in Human Airway Myocytes. Am J Respir Cell Mol Biol 2006.Google Scholar
- Corteling RL, Li S, Giddings J, Westwick J, Poll C, Hall IP: Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue. Am J Respir Cell Mol Biol 2004, 30:145–154.View ArticlePubMedGoogle Scholar
- Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Velicelebi G, Stauderman KA: STIM1, an essential and conserved component of store-operated Ca2+ channel function. J Cell Biol 2005, 169:435–445.View ArticlePubMedPubMed CentralGoogle Scholar
- Liou J, Kim ML, Heo WD, Jones JT, Myers JW, Ferrell JEJ, Meyer T: STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol 2005, 15:1235–1241.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, Stauderman KA, Cahalan MD: STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 2005, 437:902–905.View ArticlePubMedPubMed CentralGoogle Scholar
- Daykin K, Widdop S, Hall IP: Control of histamine induced inositol phospholipid hydrolysis in cultured human tracheal smooth muscle cells. Eur J Pharmacol 1993, 246:135–140.View ArticlePubMedGoogle Scholar
- Danahay H, Atherton H, Jones G, Bridges RJ, Poll CT: Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002, 282:L226–36.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402–408.View ArticlePubMedGoogle Scholar
- Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981, 391:85–100.View ArticlePubMedGoogle Scholar
- Spassova MA, Soboloff J, He LP, Xu W, Dziadek MA, Gill DL: STIM1 has a plasma membrane role in the activation of store-operated Ca(2+) channels. Proc Natl Acad Sci U S A 2006, 103:4040–4045.View ArticlePubMedPubMed CentralGoogle Scholar
- Manji SS, Parker NJ, Williams RT, van Stekelenburg L, Pearson RB, Dziadek M, Smith PJ: STIM1: a novel phosphoprotein located at the cell surface. Biochim Biophys Acta 2000, 1481:147–155.View ArticlePubMedGoogle Scholar
- Williams RT, Manji SS, Parker NJ, Hancock MS, Van Stekelenburg L, Eid JP, Senior PV, Kazenwadel JS, Shandala T, Saint R, Smith PJ, Dziadek MA: Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins. Biochem J 2001, 357:673–685.View ArticlePubMedPubMed CentralGoogle Scholar
- Putney JWJ: Capacitative calcium entry: sensing the calcium stores. J Cell Biol 2005, 169:381–382.View ArticlePubMedPubMed CentralGoogle Scholar
- Sweeney M, McDaniel SS, Platoshyn O, Zhang S, Yu Y, Lapp BR, Zhao Y, Thistlethwaite PA, Yuan JX: Role of capacitative Ca2+ entry in bronchial contraction and remodeling. J Appl Physiol 2002, 92:1594–1602.View ArticlePubMedGoogle Scholar
- Ong HL, Barritt GJ: Transient receptor potential and other ion channels as pharmaceutical targets in airway smooth muscle cells. Respirology 2004, 9:448–457.View ArticlePubMedGoogle Scholar
- Sabbioni S, Barbanti-Brodano G, Croce CM, Negrini M: GOK: a gene at 11p15 involved in rhabdomyosarcoma and rhabdoid tumor development. Cancer Res 1997, 57:4493–4497.PubMedGoogle Scholar
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