Canonical transient receptor potential 3 ... - The FASEB Journal

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Canonical transient receptor potential 3 channels activate NF-kB to mediate allergic airway disease via PKC-a/IkB-a and calcineurin/IkB-b pathways Tengyao Song,* Yun-Min Zheng,* Peter A. Vincent,* Dongsheng Cai,† Paul Rosenberg,‡ and Yong-Xiao Wang*,1 *Center for Cardiovascular Sciences, Albany Medical College, Albany, New York, USA; †Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA; and ‡Department of Medicine, Duke University School of Medicine, Durham, North Carolina, USA The purpose of this study was to determine the role of canonical transient receptor potential 3 (TRPC3) channel in allergen-induced airway disease (AIAD) and its underlying signaling mechanisms. The procedures included (1) intravenous injection of lentiviral TRPC3 channel or nonsilencing short hairpin ribonucleic acid (shRNA) to make the channel knockdown (KD) or control mice, (2) allergen sensitization/challenge to induce AIAD, (3) patch-clamp recording and Ca2+ imaging to examine the channel activity, and (4) gene manipulations and other methods to determine the underlying signaling mechanisms. The findings are that (1) intravenous or intranasal delivery of TRPC3 channel lentiviral shRNAs or blocker 1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino] phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid prevents AIAD in mice, (2) TRPC3 channel KD and overexpression, respectively, blocks and augments protein kinase C-a/nuclear factor of k light polypeptide gene enhancer in B-cell inhibitor-a (PKC-a/IkB-a)-mediated or calcineurin/IkB-b–dependent, NF-kB–dependent allergen-induced airway smooth muscle cell (ASMC) hyperproliferation and cyclin D1 (an important cell proliferation molecule) induction, and (3) the changes of the major molecules of the PKC-a/IkBa- and calcineurin/ IkB-b–dependent NF-kB signaling pathways are also observed in asthmatic human ASMCs. The conclusions are that TRPC3 channels plays an essential role in AIAD via the PKC-a/IkB-a– and calcineurin/IkB-b–dependent NF-kB signaling pathways, and lentiviral shRNA or inhibitor of TRPC3 channels may become novel and effective treatments for AIAD.—Song, T., Zheng, Y.-M., Vincent, P. A., Cai, D., Rosenberg, P., Wang, Y.-X. Canonical transient receptor potential 3 channels activate NF-kB to mediate allergic airway disease via PKC-a/IkB-a and calcineurin/IkB-b pathways. FASEB J. 30, 214–229 (2016). www.fasebj.org

ABSTRACT

Abbreviations: AEC, airway epithelial cell; AIAD, allergen induced airway disease; ASMC, airway smooth muscle cell; CAIP, calcineurin autoinhibitory peptide; CaMKII, Ca2+/ calmodulin-dependent protein kinase II; CNA, calcineurin A; CNACA, constitutively activated calcineurin A; CMV, cytomegalovirus; ds, double-stranded; GFP, green fluorescence protein; (continued on next page)

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Key Words: ion channel • signaling pathway • AIAD Ca2+ signaling, which is precisely generated and regulated by ion channels, plays an essential role in numerous diseases; however, its importance has not been well established in lung diseases such as asthma. Currently, the first-line treatment for asthma is the use of glucocorticoids, b-adrenergic agonists, and muscarinic antagonists. These drugs have been used for decades but do not always show therapeutic effect and may even produce severe side effects. The hallmark features of asthma are airway hyperresponsiveness and remodeling, which may result from increased Ca2+ signaling in airway smooth muscle cells (ASMCs). Thus, specific interventions for Ca2+ signaling may become novel and effective drugs for asthma (1–3). Recent in vitro studies from our laboratory and others indicate that canonical transient receptor potential 3 (TRPC3)-encoded channels show a predominant activity in ASMCs, and the channel expression and activity are increased in ASMCs from mice with allergen-induced airway disease (AIAD) and humans with asthma (2, 4, 5). In this study, we first addressed a fundamental question whether the in vivo knockdown (KD) or pharmacological inhibition of TRPC3 could prevent AIAD. Our data reveal that intravenous delivery of lentiviral short hairpin ribonucleic acid (shRNAs), a promising use in human gene therapy (6), to KD or intranasal administration of 1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (Pyr3) to inhibit TRPC3 channels block AIAD in mice, showing no airway hyperresponsiveness and remodeling. Subsequently, we conducted experiments to determine the molecular mechanisms for the role of TRPC3 channels. We found that TRPC3 channels control proliferation and cyclin D1 expression in AIAD ASMCs. TRPC3 channels can enhance NF-kB activity through 1

Correspondence: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA. E-mail: [email protected] doi: 10.1096/fj.15-274860 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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the PKC-a–dependent nuclear factor of k light polypeptide gene enhancer in IkB-a and calcineurindependent IkB-b signaling pathways. We have further discovered the similar changes in expression and activity of the aforementioned signaling molecules in asthmatic human ASMCs, suggesting a general fidelity of our findings to human biology.

Adenovirus production Adenovirus constructs were generated as reported previously (11). Full-length mouse TRPC3 and calcineurin A (CNA) cDNAs were cloned. Constitutively activated CNA (CNA CA ) was generated by deleting calmodulin binding and auto inhibitory sites. The targeted cDNAs were further cloned into Gateway (Life Technologies, Carlsbad, CA, USA) entry vector or pAD/CMV/V5 vector, and then infected in 293A cells to collect adenoviruses.

MATERIALS AND METHODS Production and titration of lentiviral shRNAs

Isolated cell preparation and culture

TRPC3 shRNAs and scrambled shRNAs with cytomegalovirus (CMV) promoter were purchased from ThermoScientific OpenBiosystems (Huntsville, AL, USA), and smooth muscle (SM) cell-specific SM22-a–driven TRPC3 and scrambled shRNAs from Biosettia (San Diego, CA, USA). Lentivirus packaging was performed, as we reported previously (7). Briefly, 293FT cells were grown in DMEM and then incubated with shRNA plasmid, pCMV-dR8.2 dvpr, and pCMV-VSV-G in CaCl2 and HEPES-buffered medium. After 72 h incubation, the medium was collected to obtain lentiviruses. Titration of lentiviruses was measured as described before (8).

Freshly isolated mouse ASMCs were prepared using the 2-step enzymatic digestion method, as described in detail in our previous report (5). In brief, ASM tissues were incubated in PSS containing 1 mg/ml papain (Sigma-Aldrich, St. Louis, MO, USA) for ;17 min, and then in PSS containing 1 mg/ml collagenase II (Sigma-Aldrich) for ;15 min. Single cells were harvested by gentle trituration. For primary culture, ASMCs were washed 3 times with DMEM including 4.5 g/L D-glucose, 10% fetal bovine serum, 100 U penicillin, 0.1 mg/ml streptomycin, and 2.5 mg/ml Fungizone (Life Technologies), and then cultured to 80% confluence used for experiments. Freshly isolated mouse small airway epithelial cells (AECs) were prepared following a previous publication (12). Small airways were incubated in PSS containing 1.5 mg/ml pronase (Roche, Basel, Switzerland) for ;18 h digestion. AECs were collected and cultured. ASMCs isolated from normal and asthmatic patients (passage 2) from Lonza (Hopkinton, MA, USA) were cultured up to 80% confluence used for experiments. Human donor information is shown in Table 1. All normal and asthmatic ASMCs were free of any other lung diseases.

Generation of global and SM-specific TRPC3 channel KD mice All animal experiments were performed according to an approved protocol by the Animal Care and Use Committee of Albany Medical College. Swiss Webster mice at 8 wk old were purchased from Taconic Biosciences (Germantown, NY, USA) and anesthetized by intraperitoneal injection of avertin (250 mg/kg). Lentiviruses (108 transforming unit/ml) encoding CMV- or SM22-a–driven TRPC3 or scrambled (nonsilence) shRNAs in saline were intravenously injected in mice via tail vein using a hydrodynamic intravenous injection method (9). Each mouse received the lentivirus injection 3 times, as shown in Supplemental Fig. 3E. Western blot analysis was conducted to confirm TRPC3 channel KD.

In vivo delivery of Pyr3 Pyr3 (Tocris Bioscience, Bristol, United Kingdom) was dissolved in PEG300, diluted in physiologic saline solution (PSS) at 3:1 ratio volume (10), and intranasally administered at 0.1 mg/g body weight in mice (Supplemental Fig. 3F).

(continued from previous page) HDM, house dust mite; KD, knockdown; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NS, nonsilencing; NSCC, nonselective cation channel; OE, overexpression; OVA, ovalbumin; Penh, enhanced pause; PSS, physiologic saline solution; Pyr3, 1-[4-[(2,3,3-trichloro-1-oxo-2propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4carboxylic acid; Rn, newtonian resistance; shRNA, short hairpin ribonucleic acid; SM, smooth muscle; sPASM, small pulmonary artery smooth muscle; TRPC, canonical transient receptor potential

ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

Preparation of cell cytoplasmic and nuclear extracts Cytosolic and nuclear extracts were prepared using the previously reported procedure (13) with modifications. Briefly, cells were incubated in cytoplasmic extraction buffer with a protease inhibitor cocktail (Roche) and centrifuged to gather extracted soluble (cytoplasmic) fractions. The pellet was placed in the nuclear extraction buffer and centrifuged to collect extracted soluble (nuclear) fractions.

Western blot analysis As we reported previously (5), isolated ASM tissues or cells were homogenized in RIPA buffer. The homogenate was sonicated and centrifuged. Collected proteins were transferred to a polyvinylidene difluoride membrane. The nonspecific binding sites were blocked by 5% nonfat milk. The membrane was incubated with specific TRPC3 channel antibodies (1:200 dilution; Alomone Laboratories, Jerusalem, Israel), PKC-a (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), calcineurin (1:200 dilution; Santa Cruz Biotechnology), IkB-b (1:300 dilution, Santa Cruz Biotechnology), IkB-a (1:300 dilution; Santa Cruz), cyclin D1 (1:300 dilution; Santa Cruz Biotechnology), lamin A (1:1000 dilution; Abcam, Cambridge, MA, USA), and GAPDH (1:1000 dilution; Santa Cruz Biotechnology), followed by a horseradish peroxidase–conjugated secondary antibody, and then developed using enhanced chemiluminescence reagents (Santa Cruz Biotechnology).

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TABLE 1. List of normal and asthmatic human subjects that were used to produce isolated ASMCs Parameter

Lot no. Age Gender Race

Normal

0000199524 11 Male Caucasian


Asthma

6f3647 39 Male Caucasian


0000208192 19 Female Hispanic

0f3070 41 Female Caucasian

Single-channel recordings

NF-kB activity assay

Single-channel activity was measured using the excised inside-out patch-clamp technique (5). Patch pipettes had resistances of ;7 MV. The holding potential was set at 250 mV. Single nonselective cation channel (NSCC) currents were recorded and analyzed using an Axon patch-clamp system (Sunnyvale, CA, USA). The channel open probability was calculated using the equation: channel open probability = total open time of all channel levels in the patch/sample recording.

NF-kB activity was measured using NF-kB luciferase reporter assay (18). ASMCs were transfected with NF-kB luciferase reporter plasmid that comprised 2 NF-kB binding sites with a truncated c-fos promoter/CAT reporter or control plasmid that contained 2 mutated kB binding sites.

Preparation of AIAD mice

As reported previously (19), isolated mouse lung lobes were washed with Tris-buffered saline, fixed with paraformaldehyde, sliced into sections of 4 mm thick, and then immunostained with anti-Ki67 antibody (Abcam) at 1:400 or 1:600. Antigen– antibody complexes were labeled by avidin-biotinylated enzyme complex (ABC kit) and 3,39-diaminobenzidine substrate kit, and then immunostained with anti–a-SM actin-alkaline phosphatase antibody. After overnight, red alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA, USA; 1:60) was applied for labeling a-SM actin, and hematoxylin for counter staining. Lung sections were visualized with Olympus (Tokyo, Japan) DP72 microscope. A relative area of SM layer was calculated, quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and expressed as the area of SM layer (pink) per square millimeter of bronchiole ring. A percentage of Ki67-positive cells was calculated by dividing the number of cell with Ki67-positive nucleus by the total number of cells with Ki67- and hematoxylin-positive nucleus.

The allergen ovalbumin (OVA)-induced airway disease mouse model was generated according to a modified protocol (5) (Supplemental Fig. 3C). Mice were sensitized by intraperitoneal injection of OVA/aluminum-hydroxide (600 mg/5 ml; SigmaAldrich) in PSS. On d 15–21, animals were challenged by intranasal instillation of OVA (150 mg). Control mice were treated with PSS alone. To produce the allergen house dust mite (HDM)induced airway disease (14) (Supplemental Fig. 3D), mice were sensitized by intranasal instillation of HDM (0.02 mg; Greer Laboratories, Lenoir, NC, USA) in PSS, followed by challenge via intranasal instillation HDM (0.2 mg) on d 7–11. Measurement of airway hyperresponsiveness After the last exposure to the allergen or saline, in vivo airway muscle contractile response to the muscarinic agonist methacholine was assessed by measuring enhanced pause (Penh) using an unrestricted whole-body plethysmography system (Buxco Research Systems, Wilmington, NC, USA) or newtonian resistance (Rn) using an FlexiVent (SCIREQ Scientific, Montreal, QC, Canada) system (5, 15). Muscle contraction in isolated airway (tracheal) rings was assessed using an organ bath technique. Contraction was induced by adding methacholine, and measured using a PowerLab/4SP recording system (AD Instruments, Colorado Springs, CO, USA), with a highly sensitive force transducer (Harvard Apparatus, Holliston, MA, USA). Muscle contraction in small airway was assessed in live lung slides as reported previously (16). Freshly sliced lung tissues were mounted in a bath chamber. Methacholine-induced contraction was measured using LSM510 laser scanning confocal microscope (Carl Zeiss GmbH, Jena, Germany).

Immunohistochemistry

Cell proliferation assay Cultured ASMCs were seeded at 7500 cells/well and cultured under a starvation condition (0.3% fetal bovine serum) for 18 h, followed in medium containing PDGF-BB (R&D Systems, Minneapolis, MN, USA) at 37°C for 24 h. After incubation with 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (20 ml, 5 mg/ml) for 3.5 h, the medium was removed, MTT solvent (150 ml) was added, and the plate was shaken at 4°C for 20 min. MTT absorbance was read at 590 nm. To directly count the cell number, ASMCs were seeded in 24-well plate at 20,000 cells/well, cultured in medium with 0.3% bovine serum albumin for 24 h, and then incubated with 1 ng/ml or 5 ng/ml PDGF for 3 d. Cell number was then counted.

Determination of [Ca2+]i Mouse gene expression microarrays [Ca2+]i was measured using a fluorescence imaging system (IonOptix, Westwood, MA, USA) (5, 17). ASMCs were incubated with 4 mM fura-2/AM. Fura-2 was excited at 340 and 380 nm wavelengths, and the emitted fluorescence at 510 nm was detected.

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RNA was obtained from mouse ASM tissues. The RNA quantity and quality were measured by NanoDrop ND-1000 (Wilmington, DE, USA), and RNA integrity assessed by standard denaturing agarose gel electrophoresis.

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The Mouse 12 3 135K Gene Expression Array was manufactured by Roche NimbleGen (Madison, WI, USA) to determine .44,170 genes. Double-stranded (ds)-cDNA was synthesized from total RNA using an SuperScript ds-cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with 100 pmol oligo dT primers. According to the NimbleGen Gene Expression Analysis protocol (Roche NimbleGene, Madison, WI, USA), ds-cDNA was incubated with 4 mg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. Cy3 labeling of cDNA was made using the NimbleGen One-Color DNA labeling kit. ds-cDNA (1 mg) was incubated for 10 min at 98°C with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added. The mix was incubated at 37°C for 2 h. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation. Microarrays were hybridized at 42°C during 16 to 20 h with 4 mg of Cy3-labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber. Following hybridization, washing was performed using the NimbleGen Wash Buffer kit. After being washed in an ozone-free environment, slides were scanned at 5 mm/pixel resolution using an Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA) piloted by GenePix Pro 6.0 software (Axon). The scanned images were imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average algorithm included in the NimbleScan software. All gene level files were imported into Agilent GeneSpring GX software (version 11.5.1; Agilent Technologies, Santa Clara, CA, USA) for further analysis. Differentially expressed genes with statistical significance between 2 groups were identified through volcano plot filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 11.5.1). Gene ontology analysis and Pathway analysis were performed using the standard enrichment computation method.

Statistical analysis Data are expressed as means 6 SD. Statistical analysis was performed using paired Student’s t test for comparisons before and after treatment in the same sample, unpaired (independent) Student’s t test for 2-sample comparisons, 1-way ANOVA with an appropriate post hoc test for multiple-sample comparisons, and 2-way ANOVA for comparisons of the means of populations that were classified in 2 different ways or the mean responses in an experiment with 2 factors. Values of P , 0.05 were considered statistically significant.

RESULTS Intravenous injection of lentiviral TRPC3 channel shRNAs abolishes airway hyperresponsiveness and remodeling in mice with AIAD Our previous in vitro studies have demonstrated that TRPC3 channels are increased in ASMCs from a mouse model of AIAD (5). An in vivo delivery of lentiviral shRNAs is a promising approach in human gene therapy (6). Thus, we sought to use this approach to test the effect of in vivo TRPC3 channel KD on AIAD. Lentiviruses containing ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

CMV promoter-driven, TRPC3 channel shRNAs with green fluorescence protein (GFP; an expression indicator) were injected in mice via tail vein using a hydrodynamic delivery technique (9). As shown in Fig. 1A, all isolated ASMCs from mice that received the lentivirus exhibited strong GFP-derived fluorescence, indicating the successful virus infection. TRPC3 channel protein expression was largely suppressed in ASMCs from mice that received TRPC3 channel shRNAs, but not nonsilencing (NS) shRNAs. An ion channel expression level is not always consistent with its functional activity, and the patch clamp recording is a standard approach in studies of ion channels. Thus, we made single-channel recordings and found that the cells from mice receiving TRPC3 channel shRNAs exhibited a significant decrease in the activity of NSCCs that have been shown to be primarily encoded by TRPC3 channel gene (5), relative to ASMCs from control mice (Supplemental Fig. 1B). To further determine the effect of TRPC3 channel KD, we examined whether the channel-associated extracellular Ca2+ influx was inhibited. The muscarinic agonist methacholine (25 mM) was applied to cells for 250 s to deplete intracellular Ca2+ store with nominally Ca2+-free and 0.5 mM EGTA PSS, followed by readmission of extracellular 1.8 mM Ca2+ in the continued presence of methacholine. As shown in Fig. 1C, Ca2+ entry following Ca2+ readmission was significantly decreased in cells from mice received TRPC3 channel, but not NS, shRNAs. These data demonstrate that in vivo delivery of lentiviral TRPC3 channel shRNAs can effectively KD the channel expression and activity in ASMCs, producing the channel KD mice. More excitingly, intravenous injection of lentiviral TRPC3 channel shRNAs abolished airway hyperresponsiveness in mice with OVA-evoked airway disease, evidenced by the absence of the enhanced methacholine-induced increase in airway contraction and resistance (airway lumen area change, Penh and Rn values) (Figs. 1D, E and 2A–C). Consistent with these genetic effects, in vivo administration of the TRPC3 channel blocker Pyr3 via intranasal inhalation diminished allergen-induced airway hyperresponsiveness in mice (Figs. 1F, G and 2B). OVA-treated mice also exhibited a large increase in ASM mass and cell proliferation, determined by a-SM cell actin and Ki67 immunohistochemistry staining. This wellcharacterized airway remodeling was eliminated by intravenous injection of lentiviral TRPC3 channel shRNAs, but not NS shRNAs (Fig. 2D). In vivo delivery of lentivirus encoding SM22-a–driven TRPC3 channel shRNAs also blocks AIAD in mice To specifically study the functional importance of TRPC3 channel in ASMCs, we made lentivirus encoding SM22-a promoter-driven TRPC3 and GFP (SM22-a-TRPC3) shRNAs as well as NS (control, SM22-a-NS) shRNAs. As indicated in Fig. 3A, B, after infection of lentiviral SM22a-TRPC3 and NS shRNAs, TRPC3 channel protein expression was increased in small (the third or smaller branch) ASM from mice with AIAD, but not in small pulmonary artery SM (sPASM) and small airway AECs. 217

Figure 1. In vivo genetic KD and pharmacological inhibition of TRPC3 channel blocks airway hyperresponsiveness in mice with AIAD. A) A fluorescence (top) and transmitted light image (bottom) of freshly isolated ASMCs from a mouse following intravenous injection of lentiviruses encoding specific TRPC3 shRNAs and GFP. TRPC3 channel expression was detected in freshly isolated ASMCs from mice that without virus infection (noninfected), received lentiviruses containing NS shRNA, or specific TRPC3 shRNAs (TRPC3 shRNA). The bar graph quantifies TRPC3 protein expression levels. B) TRPC3 channel activity in ASMCs isolated from noninfected mice and mice receiving intravenous injection of NS and TRPC3 shRNAs. The channel activity was recorded in excised membrane patches from ASMCs using the inside-out single-channel recording. C ) Methacholine (mAch)induced Ca2+ influx was decreased in TRPC3 shRNA-treated ASMCs. ASMCs were exposed to 25 mM mAch in Ca2+-free PSS, followed by 25 mM mAch in 1.8 mM Ca2+ PSS. D and E ) Penh to the muscarinic agonist mAch was assessed in nonAIAD (normal) mice, AIAD mice, and AIAD mice following intravenous injection of NS or TRPC3 shRNAs using a noninvasive unrestricted whole-body plethysmography system (left). In vitro airway muscle contractile responses to mAch were recorded in isolated airway (tracheal) rings from normal, asthmatic, and asthmatic treated with NS or TRPC3 shRNAs (right). F and G) Same measurement were performed on AIAD model treated with Pyr3 (TRPC3 channel blocker) or vehicle. n, number of mice examined. Numbers in parentheses indicated the numbers of airways/numbers of mice examined. *P , 0.05 compared with noninfected ASMCs or normal mice.

The increased channel expression in small ASM was blocked by TRPC3 channel KD. IL-5 and IL-13 expression were enhanced in bronchoalveolar lavage fluid from AIAD mice (Fig. 3C). The 218


enhanced IL-5 and IL-13 expression were unaffected by TRPC3 channel KD. Similarly, TRPC3 channel KD did not change the increased leukocyte number in BALF from AIAD mice (data not shown). These results indicate that


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Figure 2. In vivo genetic KD and pharmacological inhibition of TRPC3 channel blocks airway hyperresponsiveness and remodeling in mice with AIAD. A and B) In vivo airway muscle contractile responses to the muscarinic agonist mAch (Rn) were assessed in non-AIAD (normal) mice, AIAD mice, and AIAD mice following intravenous injection of NS or TRPC3 shRNAs using an invasive FlexiVent. C ) In vitro airway muscle contractile responses to mAch were recorded in freshly sliced lung tissue from normal, AIAD, and AIAD mice treated with NS or TRPC3 shRNAs. Graph indicated the quantification of lumen change. Scale bar, 25 mm. D) Immunohistochemistry costains of a-smooth muscle actin (pink) and Ki67 (brown) in airways in normal, asthmatic, asthmatic mice model treated with NS shRNA or TRPC3 shRNA. Green arrows indicate colocalization of a-smooth muscle actin and Ki67. Inserted picture was enlarged part of smooth muscle layer, which suggested colocalization of Ki67 and a-smooth muscle actin. Bar graph indicated the quantification of airway smooth muscle areas and summary of Ki67-positive cells in airway smooth muscle layers in each groups. Numbers in parentheses indicated the numbers of airways/numbers of mice examined. n, number of mice examined. *P , 0.05 compared with noninfected ASMCs or normal mice. # P , 0.05 compared with asthmatic mice treated with NS. Scale bar, 20 mm.

the lack of airway restriction in mice with functionally inhibited TRPC3 channels in ASMCs is not accompanied by a lack of inflammation. Intravenous injection of lentiviral SM22-a-TRPC3 shRNAs blocked airway hyperresponsiveness (augmented methacholine-evoked increase in airway resistance and SM cell contraction) in AIAD mice but not sPASM contraction (Fig. 3D, E and 4A–C). In contrast, lentiviral SM22-a-NS shRNAs had no effect. Likewise, lentiviral SM22-a-TRPC3 shRNAs eliminated ASM remodeling (increased thickness of ASM layer and the number of Ki67-positive cells) in mice ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

with allergic asthma (Fig. 4D). Taken together, lentiviral SM22-a–driven TRPC3 channel shRNAs can specifically KD the TRPC3 channel in ASMCs, thereby blocking allergic airway hyperresponsiveness and remodeling. TRPC3 channel-induced cyclin D1 expression mediates enhanced AIAD ASMC proliferation Lentiviral shRNA-mediated TRPC3 channel KD also blocked the increased proliferation of AIAD ASMCs in vitro 219

Figure 3. Lentiviral SM22-a– driven shRNA-mediated smooth muscle-specific TRPC3 channel KD mice have no asthmatic airway muscle hyperresponsiveness. A) Western blots detected and analyzed protein expression of TRPC3 channel in small ASM (sASM), small airway epithelial cells isolated from small airway AECs (sAEC), and sPASM. B) Bar graph quantifies blot intensity of (A). C ) Productions of IL13 and IL-5 in BALF were measured by ELISA. D and E ) Penh to the muscarinic agonist methacholine was assessed in nonasthmatic (normal) mice, asthmatic mice, and asthmatic mice following intravenous injection of lentivirus containing SM22-a-NS shRNA or TRPC3 shRNA using an unrestricted whole-body plethysmography system. In vitro airway muscle contractile responses to methacholine were recorded in isolated airway (tracheal) rings from normal, asthmatic, and asthmatic with SM22-a-NS shRNA or TRPC3 shRNA. n, number of mice examined. *P , 0.05 compared with normal group. #P , 0.05 compared with saline- or SM22-a-NS– injected groups.

but had no effect in normal ASMCs. Conversely, TRPC3 overexpression (OE) significantly increased both normal and AIAD ASM cell proliferation (Fig. 5A and Supplemental Fig. 1A). Cyclin D1 and other cyclins are involved in proliferation of ASMCs. As shown in Fig. 5B–D, TRPC3 channel KD or blocker (Pyr3) blocked the increased cyclin D1 expression in AIAD ASMCs, but did not have an effect in normal ASMCs. In contrast, TRPC3 channel OE increased cyclin D1 expression in AIAD and normal ASMCs (Fig. 5C and 220


Supplemental Fig. 1B). These findings further support the importance of TRPC3 channels in ASMC proliferation and remodeling. TRPC3 channels govern NF-kB activation to mediate AIAD ASM remodeling To study a mechanism by which TRPC3 channels mediate airway remodeling, we performed whole genome


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Figure 4. Lentiviral SM22a–driven shRNA-mediated smooth muscle-specific TRPC3 channel KD mice have no asthmatic airway muscle hyperresponsiveness and ASMC hyperproliferation. A) In vivo airway muscle contractile responses to the muscarinic agonist mAch (Rn) were assessed in non-AIAD (normal) mice, AIAD mice, and AIAD mice following i.v. injection of SM22a-NS or TRPC3 shRNAs using an invasive FlexiVent. B) In vitro sPASM contractile responses to norepinephrine (300 mM) were recorded in isolated sPASM from normal mice, asthmatic mice, and asthmatic mice with SM22a-NS shRNA or TRPC3 shRNA. C ) In vitro airway muscle contractile responses to mAch were recorded in freshly sliced lung tissue from normal, AIAD, and AIAD mice treated with SM22-a-NS or TRPC3 shRNAs. Graph indicated the quantification of lumen change. Scale bars, 25 mm. D) Immunohistochemistry costains of a-smooth muscle actin (pink) and Ki67 (brown) in airways in normal, AIAD, and AIAD mice model treated with SM22-a-NS shRNA or TRPC3 shRNA. Green arrows indicate colocalization of a-smooth muscle actin and Ki67. Bar graph indicates the quantification of airway smooth muscle areas and summary of Ki67-positive cells in airway smooth muscle layers in each groups. n, number of mice examined. Numbers in parentheses indicate the numbers of airways/numbers of mice examined. *P , 0.05 compared with normal group. # P , 0.05 compared with saline- or SM22-a-NS-injected groups. Scale bars, 20 mm.

microarray using freshly isolated mouse ASM tissues. As shown in Supplemental Fig. 2, NF-kB expression was upregulated in AIAD ASM tissues; these changes were blocked in TRPC3 channel KD mice. NF-kB signaling is involved in asthmatic airway SM cell proliferation and remodeling and can be activated by Ca2+ signaling in cancer cells. Thus, the up-regulated TRPC3 channels may increase NF-kB expression and activity to mediate AIAD. To test this possibility, we examined the effect of TRPC3 ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

channels KD and OE. ASMCs were infected with lentiviral TRPC3 channel shRNAs to make the channel KD or adenoviral TRPC3 channel cDNAs to make the channel OE. TRPC3 channel KD abolished the increased activity of NF-kB in AIAD ASMCs, but not in normal cells (Fig. 6A). On the other hand, TRPC3 channel OE increased NF-kB activity in normal and AIAD cells (Fig. 6B). TRPC3 channel KD could also eliminate the nuclear import of p65 in AIAD ASMCs, but had no effect in normal 221

Figure 5. TRPC3 channel induced cyclin D1 expression mediates enhanced AIAD ASMCs proliferation. A) Cell counting assay was performed on normal and AIAD ASMCs following TRPC3 silencing or TRPC3 OE. Lentiviruses encoding TRPC3 shRNA (TRPC3 shRNA) or NS shRNA were infected to primary cultured normal and AIAD ASMCs. Adenoviruses encoding TRPC3 cDNA (AD-TRPC3) or vector control (GFP) were also infected to normal and AIAD ASMCs. In TRPC3 silencing experiment, *P , 0.05 compared with normal ASMCs treated with NS shRNA. No statistical differences were observed among normal ASMCs with TRPC3 shRNA, normal ASMCs with NS shRNA, and AIAD ASMCs with TRPC3 shRNA. Experiment was repeated in three independent groups. In AD-TRPC3-treated experiment, *P , 0.05 compared with AIAD ASMCs treated with GFP; #P , 0.05 compared with normal ASMCs treated with GFP. B and C) Western blot detected and analyzed TRPC3 channel and cyclin D1 expression following TRPC3 shRNA (B) or AD-TRPC3 treatment (C ) (Supplemental Fig. 1A). Experiment was repeated in 3 independent groups. In TRPC3 shRNA-treated experiment, *P , 0.05 compared with normal or AIAD ASMCs treated with NS shRNA; # P , 0.05 compared with normal ASMCs treated with NS shRNA. In AD-TRPC3-treated experiment, *P , 0.05 compared with normal and AIAD ASMCs treated with GFP; #P , 0.05 compared with normal ASMCs treated with ADTRPC3. D) Western blot detected and analyzed protein expression of IkB-a and cyclin D1 in normal and AIAD ASMCs treated with Pyr3. *P , 0.05 compared with normal ASMCs treated with vehicle; #P , 0.05 compared with AIAD ASMCs treated with vehicle.

cells. TRPC3 channel OE increased p65 nuclear import in both AIAD and normal ASMCs (Fig. 6C, D). As the nuclear import of p65 represents the activation of NF-kB, the 222


results presented here further reveal that the up-regulated TRPC3 channels can activate NF-kB, playing an essential role in AIAD.


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Figure 6. TRPC3 channels govern NF-kB activation to mediate AIAD ASM remodeling. All the experiments in Fig. 4 were repeated in 3 independent groups. A and B) NF-kB activities were detected in normal and AIAD ASMCs treated with lentivirus encoding TRPC3 shRNA (TRPC3 shRNA) or adenovirus encoding TRPC3 cDNA (AD-TRPC3). NF-kB activity reporter gene (NF-kB-Luci) was transfected to TRPC3 shRNA(A) or AD-TRPC3–treated (B) normal and AIAD ASMCs. In TRPC3 shRNA-treated experiment, *P , 0.05 compared with normal ASMCs treated with NS shRNA; #P , 0.05 compared with AIAD ASMCs treated with NS shRNA. In AD-TRPC3-treated experiment, *P , 0.05 compared with normal ASMCs with the same treatment; #P , 0.05 compared with normal ASMCs treated with NS. C and D) Western blot detected and analyzed p65 expression in nuclear and cytosolic extraction from TRPC3 shRNA(C) or AD-TRPC3-treated (D) normal and AIAD ASMCs. In TRPC3 shRNA-treated experiment, *P , 0.05 compared with nuclear extraction from NS shRNAtreated AIAD ASMCs; #P , 0.05 compared with nuclear extraction from NS shRNA-treated normal ASMCs. In AD-TRPC3-treated experiment, *P , 0.05 compared with nuclear extraction from GFP-treated normal and AIAD ASMCs; #P , 0.05 compared with nuclear extraction from GFP-treated normal ASMCs.

TRPC3 channels cause NF-kB activation in AIAD ASMCs through the PKC-a–dependent IkB-a signaling Ca2+-dependent PKC-a can cause activation of NF-kB by inhibiting IkB-a; thus, we addressed an interesting question ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

of whether TRPC3 channels might activate NF-kB in AIAD ASMCs via PKC-a–mediated IkB-a inhibition pathway. The results presented in Fig. 7A shows that TRPC3 channel KD suppressed the increased PKC-a and cyclin D1 expression as well as the decreased IkB-a expression (NF-kB 223

activation) in AIAD ASMCs. However, no effect was detected in normal ASMCs. On the other hand, TRPC3 channel OE increased PKC-a and cyclin D1 expression as well as the decreased IkB-a expression in both normal and AIAD ASMCs (Fig. 7B and Supplemental Fig. 1C). Treatment with the novel PKC inhibitor Gö6976 to block PKC-a prevented the loss of IkB-a in AIAD, but had no effect in normal ASMCs (Supplemental Fig. 3B). Similarly, expression of dominant negative PKC-a to genetically inhibit PKC-a activity produced a similar effect. In contrast, OE of PKC-a by adenovirus infection decreased IkB-a expression in normal and AIAD ASMCs (Fig. 7E, F and Supplemental Fig. 1D). We have also shown that pharmacological and genetic inhibition of PKC-a diminished the increased NF-kB activity in AIAD ASMCs, but they did not exert an effect in normal ASMCs (Supplemental Fig. 3A and Fig. 7C). Oppositely, PKC-a activation increased NF-kB activity (Fig. 7D). Collectively, PKC-a is important for the role of TRPC3 channel-mediated NF-kB activation and associated cyclin D1 induction, thereby contributing to AIAD ASM cell hyperproliferation and remodeling. Calcineurin/IkB-b signaling pathway is also essential for the TRPC3 channel-mediated activation of NF-kB in AIAD ASMCs Since the activity of NF-kB is regulated by Ca2+-dependent phosphatase calcineurin-mediated IkB-b activation pathway, we wondered whether this signaling axis is also involved in the activation of NF-kB caused by TRPC3 channels in AIAD ASMCs. Our data reveal that TRPC3 channel KD blocked the increased calcineurin expression and decreased IkB-b expression in AIAD ASMCs (Fig. 8A). However, TRPC3 channel KD had no effect in normal ASMCs. TRPC3 channel OE increased calcineurin expression and decreased IkB-b expression in normal and AIAD ASMCs. Application of specific calcineurin auto inhibitory peptide (CAIP) to block calcineurin activity could inhibit the increased NF-kB activity in AIAD ASMCs, although no effect was observed in normal myocytes (Fig. 8B). Consistently, treatment with adenovirus encoding CNACA to increase calcineurin activity increased NF-kB activity in normal and AIAD ASMCs (Fig. 8C). CAIP could also inhibit the decreased IkB-b expression and increased cyclin D1 expression in AIAD ASMCs but did not exert an effect in normal ASMCs (Fig. 8D). Treatment with CNACA caused reduced IkB-b expression and increased cyclin D1 expression in normal and AIAD ASMCs. Altogether, these results indicate that TRPC3 channels can contribute to the development of AIAD via the calcineurin-dependent IkB-b–mediated NF-kB activation signaling axis. Both distinct PKC-a/IkB-a and calcineurin/IkB-b signaling pathways may also mediate the role of TRPC3 channels in activating NF-kB in asthmatic human ASMCs To prove that the overall findings in animal studies have a general fidelity to human biology, we first conducted 224


comparable studies to examine the activity and expression of TRPC3 channels in normal and asthmatic human as well as mouse ASMCs. Our single-channel recordings showed that the activity of TRPC3 channels (the NSCCs that could be blocked by specific TRPC3 channel antibodies) was significantly augmented in asthmatic human ASMCs, similar to that in AIAD mouse ASMCs (Fig. 9A). Additionally, TRPC3 channel protein expression was also increased in asthmatic human ASMCs, like that in AIAD mouse cells (Fig. 9B). Excitingly, all the tested signaling molecules such as PKC-a, calcineurin, IkB-a, IkB-b, and cyclin D1 had similar changes in asthmatic human and mouse ASMCs (Fig. 9C). We have also found that TRPC3 channels and associated signaling molecules all were significantly altered in ASMCs from mice with AIAD evoked by HDM, a major natural allergen in our living environment (Supplemental Fig. 4), further supporting their importance in AIAD. DISCUSSION Corticosteroids, b2-adrenergic agonists, and muscarinic receptor antagonists have been used as the first-line therapeutic drugs for treatment of asthma for several decades, However, these drugs may have significant adverse effects, such as lack of efficacy, exacerbation of asthma, and even death (1). Considering that ion channels play an essential role in ASMC contraction, scientists have made great efforts to develop novel and effective interventions targeting ion channels in the treatment of asthma; unfortunately, there has been no breakthrough so far (2, 3, 20). In this study, we demonstrate that intravenous injection of lentiviral TRPC3 channel shRNAs can sufficiently KD ASMC TRPC3 channel expression and activity in mice. These KD mice do not develop allergen-induced airway hyperresponsiveness. These results are consistent with the recent reports from our group and others, in which among all 7 members, TRPC3 channels exhibit a predominant functional activity in normal ASMCs and an increase in the activity and expression in AIAD ASMCs (2, 5). Our findings also provide further evidence that TRPC3 channels may play a vital role in AIAD. Besides hyperresponsiveness, airway remodeling is also a key contributor to the pathogenesis of asthma, which can be caused by the increased Ca2+ signaling (20). Our findings have shown that TRPC3 channel KD mice do not exhibit the AIAD ASMC proliferation and airway remodeling. Our in vitro experiments further unveil that TRPC3 channel KD inhibits the increased proliferation of isolated ASMCs from AIAD mice and its OE enhances normal and AIAD ASMC proliferation. We have also found that TRPC3 channel KD inhibits the increased protein expression of cyclin D1, an important cell proliferation molecule in ASMCs (20, 21). Furthermore, the channel OE promotes cyclin D1 expression in normal and AIAD ASMCs. Our previous study has revealed that TRPC3 channels show the increased expression levels and functional activity in AIAD mouse ASMCs (5). It has also been reported that TNF-a, an important asthmatic mediator, could significantly augment TRPC3 channel expression in cultured human ASMCs, and TRPC3 channel KD inhibits TNF-a-mediated enhancement of


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Figure 7. TRPC3-mediated NF-kB activation in AIAD ASMCs is required for the involvement of PKCa–dependent IkB-a signaling. All the experiments in Fig. 5 were repeated in 3 independent groups. A and B) Western blot detected and analyzed PKC-a and IkB-a expression following TRPC3 silencing (TRPC3 shRNA) (A) or TRPC3 OE (AD-TRPC3) in normal and AIAD ASMCs (B) (Supplemental Fig. 1C). In TRPC3 shRNA-treated experiment, *P , 0.05 compared with AIAD ASMCs treated with NS shRNA; #P , 0.05 compared with normal ASMCs treated with NS shRNA. In AD-TRPC3–treated experiment, *P , 0.05 compared with normal and AIAD ASMCs treated with GFP; #P , 0.05 compared with normal ASMCs treated with GFP. C and D) NF-kB activities were detected in normal and AIAD ASMCs with adenovirus encoding dominant negative PKC-a (PKC-aDN) or PKC-a cDNA (AD-PKC-a). NF-kB activity reporter gene (NF-kB-Luci) or reporter gene with mutant NF-kB binding sites (mNF-kB-Luci) was transfected to PKC-aDN– (C) or ADPKC-a2treated (D) normal and AIAD ASMCs. In PKC-aDN–treated experiment, *P , 0.05 compared with normal ASMCs treated with b-Gal; #P , 0.05 compared with AIAD ASMCs treated with b-Gal. In ADPKC-a–treated experiment, *P , 0.05 compared with normal ASMCs with the same treatment; # P , 0.05 compared with normal ASMCs treated with b-Gal. E and F) Western blot detected and analyzed IkB-a expression in normal and AIAD ASMCs treated with PKC-aDN (E) or ADPKC-a (F) (Supplemental Fig. 1D). In PKC-aDN–treated experiment, *P , 0.05 compared with AIAD ASMCs treated with b-Gal; #P , 0.05 compared with normal ASMCs treated with b-Gal. In AD-PKC-a–treated experiment, *P , 0.05 compared with normal and AIAD ASMCs treated with GFP; #P , 0.05 compared with normal ASMCs treated with GFP.

muscarinic increase in [Ca2+]i (22). Taken together, allergens and other asthmatic stimuli may increase TRPC3 channel expression and activity, which results in an ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

increase in [Ca2+]i and proliferation in ASMCs, leading to airway hyperresponsiveness, airway remodeling, and ultimately asthma. 225

Figure 8. Calcineurin/IkB-b signaling pathway is also essential for the TRPC3-mediated NFkB activation in AIAD ASMCs. All the experiments in Fig. 6 were repeated in 3 independent groups. A) Western blot detected and analyzed calcineurin (CaN) and IkB-b expression following TRPC3 silencing (TRPC3 shRNA) or TRPC3 OE (AD-TRPC3). *P , 0.05 compared with same cell type treated with control viruses (NS shRNA or GFP); #P , 0.05 compared with normal ASMCs treated with control viruses (NS shRNA or GFP). B and C) NF-kB activities were detected in normal and AIAD ASMCs treated with calcineurin inhibitory peptide (CAIP) or adenovirus encoding CNACA. NF-kB activity reporter gene (NF-kBLuci) or reporter gene with mutant NF-kB binding sites (mNF-kB-Luci) was transfected to CAIP- (B) or CNACA-treated (C) normal and AIAD ASMCs. In CAIP-treated experiment, *P , 0.05 compared with normal ASMCs treated with vehicle; #P , 0.05 compared with AIAD ASMCs treated with vehicle. In CNACA-treated experiment, *P , 0.05 compared with normal ASMCs with the same treatment; #P , 0.05 compared with normal ASMCs treated with GFP. D) Western blot detected and analyzed IkB-b and cyclin D1 expression in normal and AIAD ASMCs treated with CAIP or CNACA. *P , 0.05 compared with same type of cell treated with vehicle or GFP; #P , 0.05 compared with normal ASMCs treated with vehicle or GFP.

Lentivirus- and RNAi-mediated KD embodies a promising human gene therapy (10, 11). In vivo delivery of lentiviral TRPC3 shRNAs may become a promising new and effective therapeutic strategy in the clinical treatment of 226


asthma. Interestingly, treatment with the TRPC3 channel blocker Pyr3 in vivo produces a similar inhibitory effect on AIAD ASM hyperresponsiveness. This result is in complement of the genetic evidence for the important role of


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Figure 9. PKC-a–dependent IkB-a– and CaN-reliant IkB-b–mediated TRPC3NF-kB signaling pathway may be implicated in the development of asthma in humans. All the experiments in Fig. 7 were repeated in 3 independent groups. A) TRPC3 channel activity was recorded in normal and asthmatic human ASMCs. Original recording of single TRPC3 channel in an inside-out patch at 250 mV before and after applying specific TRPC3 antibody (1:200 dilution). Bar graph quantifies the open probability of TRPC3 channel in different conditions. B) Protein expression of each gene that participated in the proposed pathway was examined in normal and asthmatic ASMCs blots detected and analyzed the expression of each gene that participated in the proposed pathway. C) Bar graphs quantify Western blot band intensity. *P , 0.05 compared with normal mice or normal patients. D) Illustration shows the proposed signaling pathway.

TRPC3 channels and also suggests the use of pharmacological inhibitors of TRPC3 channels may also serve as a new and effective option in the clinical treatment of asthma and asthma attack. ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

PKC-a, a Ca2+-sensitive PKC isoform, may activate IkB-a and then NF-kB in cancer cells and pulmonary vascular SM cells (23) and induce cyclin D1 expression to promote proliferation in ASMCs (20, 21). As such, we sought to 227

answer an interesting question of whether TRPC3 channels may activate NF-kB via PKC-a because of Ca2+ influx. Our data, for the first time, reveal that PKC-a expression is increased in AIAD ASMCs; TRPC3 channel OE increases PKC-a expression in AIAD and normal ASMCs; and TRPC3 channel KD inhibits the increased PKC-a expression in AIAD ASMCs, but does not have an effect in normal cells. Similarly, PKC-a inhibition blocks, whereas PKC-a activation augments, NF-kB activity in AIAD ASMCs. Evidently, up-regulated TRPC3 channels induce the increased Ca2+ influx, which increases the activity of PKC-a, IkB-a, NF-kB, and cyclins, leading to airway hyperresponsiveness and airway remodeling, and ultimately AIAD. However, this PKC-a–dependent signaling pathway may not be functional in normal ASMCs under the conditions that TRPC3 channels are not up-regulated. TRPC3 channels can activate IkB-a and NF-kB in coronary artery endothelial cells via a Ca2+/calmodulindependent protein kinase II (CaMKII) pathway (24). Additionally, TRPC1 and TRPC4 channels are implicated in the activation of NF-kB through CaMKII (25). However, the expression and function of CaMKII has not been established in ASMCs, and TRPC1 and TRPC4 channel did not show a significant activity in ASMCs in this study. Perceptibly, the role of CaMKII in TRPC3 channel-dependent activation of NF-kB needs to be tested by further studies. In addition to IkB-a, IkB-b plays a crucial role in the control of NF-kB activation in C2C12 cells, which is dependent on the Ca2+-sensitive protein phosphatase calcineurin (26). Likewise, this calcineurin-reliant activation of IkB-b is also possibly involved in TRPC3 channel-mediated activation of NF-kB in AIAD ASMCs. In line with this hypothesis, our data reveal that calcineurin expression is increased in AIAD ASMCs, similar to previous reports (27–29); the increased calcineurin expression is blocked by TRPC3 channel KD, but augmented by TRPC3 channel OE. Moreover, specific calcineurin inhibition prevents, and its activation enhances, NF-kB activity. Consistent with our findings, previous studies have discovered that calcineurin plays an important role in TRPC3 channel-mediated activation of NF-kB in neurons and myocardiocytes (11, 30, 31). Thus, PKC-a–dependent IkB-a and calcineurin-reliant IkB-b, the 2 distinct signaling pathways, are critical for TRPC3 channel-mediated increased activity of NF-kB, which plays a vital role in the development of airway hyperresponsiveness and remodeling, eventually leading to AIAD. Very excitingly, a series of our comparative experiments have uncovered that TRPC3 channels are significantly upregulated in expression and activity in ASMCs from patients with asthma, similar to those from mice with AIAD. Furthermore, PKC-a, IkB-a, calcineurin, IkB-b, and NF-kB expression and/or activity are, in parallel, increased largely in asthmatic human ASMCs and AIAD mouse ASMCs. These results may prove that the overall doctrine outlined in animal studies has a general fidelity to human biology, which may facilitate future clinical studies. Moreover, similar changes in TRPC3 channels and related signaling molecules are observed in ASMCs from mice with the major natural allergen HDM-induced AIAD. Thus, our findings can also promote the potential use of pharmacological interventions and gene therapies specifically targeting at TRPC3 channels, PKC-a, IkB-a, calcineurin, 228


IkB-b, NF-kB, and cyclin D1 in the clinical treatment of asthma. This work was supported by the U.S. National Institutes of Health, National Heart, Lung, and Blood Institute Grants HL071000 and HL108232 (to Y.-X.W.), as well as American Heart Association Established Investigator Awards 0340160N (to Y.-X.W.) and 0630236N (to Y.-M.Z.). The authors thank Dennis Metzger, Ph.D., for assisting with experiments with the FlexiVent system and Qinghua Liu, M.D., Ph.D. for providing help with initial experiments.

REFERENCES 1. Barnes, P. J. (2012) New drugs for asthma. Semin. Respir. Crit. Care Med. 33, 685–694 2. Xiao, J.-H., Wang, Y.-X., and Zheng, Y.-M. (2014) Transient receptor potential and Orai channels in airway smooth muscle cells. In Calcium Signaling in Airway Smooth Muscle Cells (Wang, Y.-X., ed.), pp. 35–45, Springer International Publishing, Cham, Switzerland 3. Jansen, L. J. (2014) Novel mechanisms in calcium homeostasis and internal store refilling of airway smooth muscle. In Calcium Signaling in Airway Smooth Muscle Cells (Wang, Y.-X., ed.), pp. 195–209, Springer International Publishing, Cham, Switzerland 4. Wang, Y. X., and Zheng, Y. M. (2011) Molecular expression and functional role of canonical transient receptor potential channels in airway smooth muscle cells. Adv. Exp. Med. Biol. 704, 731–747 5. Xiao, J. H., Zheng, Y. M., Liao, B., and Wang, Y. X. (2010) Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 43, 17–25 6. Kumar, P., and Woon-Khiong, C. (2011) Optimization of lentiviral vectors generation for biomedical and clinical research purposes: contemporary trends in technology development and applications. Curr. Gene Ther. 11, 144–153 7. Herron, C. R., Lowery, A. M., Hollister, P. R., Reynolds, A. B., and Vincent, P. A. (2011) p120 regulates endothelial permeability independently of its NH2 terminus and Rho binding. Am. J. Physiol. Heart Circ. Physiol. 300, H36–H48 8. Tiscornia, G., Singer, O., and Verma, I. M. (2006) Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 9. Lewis, D. L., Hagstrom, J. E., Loomis, A. G., Wolff, J. A., and Herweijer, H. (2002) Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 32, 107–108 10. Kiyonaka, S., Kato, K., Nishida, M., Mio, K., Numaga, T., Sawaguchi, Y., Yoshida, T., Wakamori, M., Mori, E., Numata, T., Ishii, M., Takemoto, H., Ojida, A., Watanabe, K., Uemura, A., Kurose, H., Morii, T., Kobayashi, T., Sato, Y., Sato, C., Hamachi, I., and Mori, Y. (2009) Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc. Natl. Acad. Sci. USA 106, 5400–5405 11. Rosenberg, P., Hawkins, A., Stiber, J., Shelton, J. M., Hutcheson, K., Bassel-Duby, R., Shin, D. M., Yan, Z., and Williams, R. S. (2004) TRPC3 channels confer cellular memory of recent neuromuscular activity. Proc. Natl. Acad. Sci. USA 101, 9387–9392 12. Lam, H. C., Choi, A. M., and Ryter, S. W. (2011) Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface. J. Vis. Exp. (48):2513 13. Yu, Y., Keller, S. H., Remillard, C. V., Safrina, O., Nicholson, A., Zhang, S. L., Jiang, W., Vangala, N., Landsberg, J. W., Wang, J. Y., Thistlethwaite, P. A., Channick, R. N., Robbins, I. M., Loyd, J. E., Ghofrani, H. A., Grimminger, F., Schermuly, R. T., Cahalan, M. D., Rubin, L. J., and Yuan, J. X. (2009) A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension. Circulation 119, 2313–2322 14. Hammad, H., Plantinga, M., Deswarte, K., Pouliot, P., Willart, M. A., Kool, M., Muskens, F., and Lambrecht, B. N. (2010) Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J. Exp. Med. 207, 2097–2111 15. Furuya, Y., Roberts, S., Hurteau, G. J., Sanfilippo, A. M., Racine, R., and Metzger, D. W. (2014) Asthma increases susceptibility to heterologous but not homologous secondary influenza. J. Virol. 88, 9166–9181


SONG ET AL.

16. Bai, Y., and Sanderson, M. J. (2006) Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices. Am. J. Physiol. Lung Cell. Mol. Physiol. 291, L208–L221 17. Liu, Q. H., Zheng, Y. M., Korde, A. S., Yadav, V. R., Rathore, R., Wess, J., and Wang, Y. X. (2009) Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle. Proc. Natl. Acad. Sci. USA 106, 11418–11423 18. Grimm, S., Bauer, M. K., Baeuerle, P. A., and Schulze-Osthoff, K. (1996) Bcl-2 down-regulates the activity of transcription factor NFkappaB induced upon apoptosis. J. Cell Biol. 134, 13–23 19. Cho, J. Y., Miller, M., Baek, K. J., Han, J. W., Nayar, J., Lee, S. Y., McElwain, K., McElwain, S., Friedman, S., and Broide, D. H. (2004) Inhibition of airway remodeling in IL-5-deficient mice. J. Clin. Invest. 113, 551–560 20. Song, T., Zheng, Y.-M., Wang, Y.-X. (2014) Calcium signaling in airway smooth muscle remodeling. In Calcium Signaling in Airway Smooth Muscle Cells (Wang, Y.-X., ed.), pp. 393–407, Springer International Publishing, Cham, Switzerland 21. Du, C. L., Xu, Y. J., Liu, X. S., Xie, J. G., Xie, M., Zhang, Z. X., Zhang, J., and Qiao, L. F. (2010) Up-regulation of cyclin D1 expression in asthma serum-sensitized human airway smooth muscle promotes proliferation via protein kinase C alpha. Exp. Lung Res. 36, 201–210 22. White, T. A., Xue, A., Chini, E. N., Thompson, M., Sieck, G. C., and Wylam, M. E. (2006) Role of transient receptor potential C3 in TNFalpha-enhanced calcium influx in human airway myocytes. Am. J. Respir. Cell Mol. Biol. 35, 243–251 23. Vertegaal, A. C., Kuiperij, H. B., Yamaoka, S., Courtois, G., van der Eb, A. J., and Zantema, A. (2000) Protein kinase C-alpha is an upstream activator of the IkappaB kinase complex in the TPA signal transduction pathway to NF-kappaB in U2OS cells. Cell. Signal. 12, 759–768 24. Smedlund, K., Tano, J. Y., and Vazquez, G. (2010) The constitutive function of native TRPC3 channels modulates vascular cell adhesion

ROLE OF TRPC3 CHANNELS IN ALLERGIC AIRWAY DISEASE

25.

26.

27. 28. 29. 30. 31.

molecule-1 expression in coronary endothelial cells through nuclear factor kappaB signaling. Circ. Res. 106, 1479–1488 Bair, A. M., Thippegowda, P. B., Freichel, M., Cheng, N., Ye, R. D., Vogel, S. M., Yu, Y., Flockerzi, V., Malik, A. B., and Tiruppathi, C. (2009) Ca2+ entry via TRPC channels is necessary for thrombininduced NF-kappaB activation in endothelial cells through AMPactivated protein kinase and protein kinase Cdelta. J. Biol. Chem. 284, 563–574 Biswas, G., Anandatheerthavarada, H. K., Zaidi, M., and Avadhani, N. G. (2003) Mitochondria to nucleus stress signaling: a distinctive mechanism of NFkappaB/Rel activation through calcineurinmediated inactivation of IkappaBbeta. J. Cell Biol. 161, 507–519 Hai, C. M. (2007) Airway smooth muscle cell as therapeutic target of inflammation. Curr. Med. Chem. 14, 67–76 Walczak-Drzewiecka, A., Ratajewski, M., Wagner, W., and Dastych, J. (2008) HIF-1alpha is up-regulated in activated mast cells by a process that involves calcineurin and NFAT. J. Immunol. 181, 1665–1672 Said, S. I., Hamidi, S. A., and Gonzalez Bosc, L. (2010) Asthma and pulmonary arterial hypertension: do they share a key mechanism of pathogenesis? Eur. Respir. J. 35, 730–734 Nakayama, H., Wilkin, B. J., Bodi, I., and Molkentin, J. D. (2006) Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J. 20, 1660–1670 Poteser, M., Schleifer, H., Lichtenegger, M., Schernthaner, M., Stockner, T., Kappe, C. O., Glasnov, T. N., Romanin, C., and Groschner, K. (2011) PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. Proc. Natl. Acad. Sci. USA 108, 10556–10561 Received for publication April 28, 2015. Accepted for publication August 31, 2015.

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