All animal experiments were approved by the Animal Experimentation Ethics Committee of Shanghai Jiao Tong University School of Medicine (ethics protocol number: A-2015-010). KCa3.1 gene deletion (KCa3.1^-/-, KO) mice were obtained from the Jackson Laboratory. 8–10 week old wild type (KCa3.1^+/+, WT) and KO male mice were housed in a specific pathogen-free animal facility with free access to food and water.

The chronic asthmatic mice model was described previously (Yu et al., 2013b). Briefly, on Days 1 and 14, WT and KO mice were sensitized by an intraperitoneal (i.p.) injection of 20 mg ovalbumin (OVA, Grade V; Sigma–Aldrich) emulsified in 2.25 mg of aluminum hydroxide in phosphate buffered saline (PBS). Then the WT and KO mice were challenged with aerosolized 5% OVA on Days 21, three times per week for the following 8 weeks. These WT + OVA group mice (OVA-sensitized/challenged WT mice) and KO + OVA group mice (OVA-sensitized/challenged KO mice) were used as the model of chronic asthma. The control groups of WT and KO mice were sensitized with PBS plus aluminum hydroxide and challenged with PBS. After the final OVA challenge, airway isometric tension was assessed, and the lung samples were obtained for further analysis.

The mice were anesthetized with chloral hydrate and then the bronchus was removed as described previously (Xu et al., 2011). Briefly, bronchial rings were mounted in a modified Krebs solution (composition in mM: glucose 11, CaCl₂ 2.5; NaCl 118; KCl 4.7; NaHCO₃ 25; MgSO₄ 1.2; KH₂PO₄ 1.2; EDTA⋅Na₂ 0.5), maintained bubbled with a mixed gas of 95% O₂ and 5% CO₂ at 37°C. PowerLab 8sp Life Analysis System was used to record isometric tension (Ad Instruments Australia). A resting tension of 500 mg was used as the preparations connected vertically to a force-displacement transducer for 1 h. Preparations were washed every 15 min for 1 h until the cumulative concentration-response curves of methacholine were measured. The changes of the four groups (WT, WT + OVA, KO, KO + OVA) airway isometric tension were expressed as isometric force (mg) and EC₅₀, i.e., the concentration of methacholine causing 50% of the maximal force generated (EC₅₀). The EC₅₀ was calculated from the logarithmic regression of each concentration–response curve.

Airway inflammatory cell counts were scored on a five-point scale as described previously: 0 = no cells, 1 = a few cells, 2 = a ring of cells one cell layer deep, 3 = a ring of cells two to four cells deep, and 4 = a ring of cells of more than four cells deep (Yu et al., 2013b). Periodic acid-Schiff (PAS)-stained goblet cells of each bronchus epithelium were scored based on a 5-point scoring system: 0, <5% goblet cells, 1, 5–25%, 2, 25–50%, 3, 50–75%, and 4, >75% (Yu et al., 2013b). At least three different fields of each lung section were performed to score the inflammatory cells and mucus production. Mean scores were obtained from five animals. Image J software (Schneider et al., 2012) was used to quantify Masson’s trichrome staining intensity.

For immunofluorescence staining of lung sections and cultured asthmatic HBSM cells, the tissues and cells were blocked with 3% bovine serum albumin in PBS 1 h at room temperature. Sections and cells were incubated at 4°C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone labs), rabbit anti-TRPV4 (1:200; Alomone labs), mouse anti-α-SMA (1:400, Sigma, MO, United States). The sections and cells were then incubated with following secondary antibodies: Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen) for 1 h at room temperature. Then washed with PBS and mounted with DAPI. Immunofluorescence was visualized by a Leica TCS SP8 Confocal Laser Scanning Microscope (Leica, Germany).

Quantitative evaluation of sections stained with α-SMA antibody was performed using Leica LAS AF Lite software (Leica, Germany). All analyses were done blinded to the treatment groups. Results were shown as area of α-SMA labeling per micrometer length of bronchi’s basement membrane in internal diameter as described previously (Qin et al., 2012). At least 6 bronchi were counted on each slice.

Analysis of colocalization: For analysis of TRPV4 and KCa3.1 colocalization, images were processed and analyzed using Leica LAS AF Lite software (Leica, Germany). Pearson’s correlation was used to express the degree of colocalization as described early (Wu et al., 2012; Bradley et al., 2015). The colocalization of KCa3.1 and TRPV4 in the overlap of the two channels was assessed using the colocalization tool in Leica LAS AF Lite software. The Pearson correlation values range from -1 to +1. A correlation of 1 indicates complete colocalization between the two proteins. A correlation of -1 indicates a negative interaction, and a correlation of 0 indicates no colocalization between the two proteins (Wu et al., 2012).

Lung tissues of different groups mice were frozen in TRIzolsolution (Invitrogen, Carlsbad, CA, United States). According to the protocol of TRIzol, total RNA was isolated and transcribed to cDNA using the RevertAid^TM First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, United States). The primer sequences were as follows: for REST,

SYBR Green I was used to perform quantitative real-time PCR on an ABI 7500 sequence detector system (Applied Biosystems). Target gene expression was normalized to β-actin using the 2^-ΔΔCT method.

Bronchoalveolar lavage fluid (1 mM EDTA, 10% FBS, 0.01M PBS) was used to lavage the lung tissues through the tracheal tube. ELISA was performed using the kit for interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, and IL-13 (Rapidbio Labs, Langka Trade Co., Ltd., Shanghai, China).

Asthmatic HBSM cells (Lonza, Walkersville, MD, United States) were described previously (Yu et al., 2013b). The cells between Passages 4 and 8 were used. HBSM cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) media containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 1 × 10⁵ cells/cm². HBSM cells were serum-free for 24 h for a quiescent state, and were then treated with 20 ng/mL PDGF (ProSpec, ProSpec-Tany TechnoGene Ltd., Israel). In some cases, HBSM cells were pretreated with TRAM-34 or HC 067047 (Tocris Bioscience, United Kingdom) 1 h before PDGF was added.

The transfection of small interfering RNA (siRNA) was performed as described previously (Yu et al., 2013b). Briefly, HBSM cells were 70–80% confluent 24 h before transfection. HBSM cells were transfected with KCa3.1- and TRPV4-specific siRNA and with scrambled siRNA as a negative control (siCTL). TurboFect siRNA Transfection Reagent (Fermentas) was used to incubate the cells for 72 h after transfection.

Leica TCS SP8 Confocal Laser Scanning Microscope (Leica, Germany) was used to evaluate the relative changes in [Ca²⁺]_i by monitoring Fluo-4 fluorescence. 3 μM Fluo-4 AM (Sigma–Aldrich) was used to stain the asthmatic HBSM cells for 30 min at 37°C before measuring [Ca²⁺]_i. 200 μM 1-EBIO was added to the cells. Fluo-4 fluorescence was measured at 510 nm, with excitation at 488 nm. Confocal images were taken every 2 s for 360 s. Representative curves were used to show the fold changes in [Ca²⁺]_i relative to the unstimulated condition over 360 s.

The lysates of tissues or cells were homogenized in RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 4% protease inhibitor). The following primary antibodies were used: anti-TRPV4 (1:500; Alomone Labs), anti-KCa3.1 (1:500; Abcam), anti-α-SMA (1:1000; Sigma), anti-phospho-JNK/c-Jun antibodies (1:1000, Cell Signaling Technology, Danvers, MA, United States), anti-REST (1:500; Santa Cruz, Santa Cruz, CA, United States), and anti-β-actin (1:1000; Santa Cruz). HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (1:3000; Amersham Biosciences) were used for 1 h at room temperature. Bands were quantified by ImageJ software and normalized to the β-actin band as loading control (Schneider et al., 2012).

Cell proliferation assay was performed as reported previously (Cell Counting Kit-8, CCK-8, Dojindo Laboratories, Kumamoto, Japan) (Yu et al., 2013b). HBSM cells were serum-free for 24 h, and then the HBSM cells were pretreated with the blockers for 1 h before 10% FBS was added. All control experiments were performed in the presence of vehicle for the blockers.

All data are presented as means ± SEM. Statistical analyses were performed using Prism software (GraphPad Software, Inc., La Jolla, CA, United States). Data were tested for Gaussian distribution with the Kolmogorov–Smirnov normality test and then analyzed by one-way ANOVA and Dunnett’s post hoc tests. For AHR comparisons, two-way ANOVA with a Bonferroni post test was used. Non-parametric functional outcome scores were compared by the Kruskal–Wallis test with Dunn’s post hoc tests. Data were analyzed with unpaired, two-tailed Student’s t-test when comparing between two groups, or the non-parametric Mann–Whitney test was applied. Statistical significance was set at p < 0.05.

We previously reported that pharmacological blockade of KCa3.1 dose-dependently attenuated the airway AHR in a mouse model of chronic asthma (Yu et al., 2013b). Here we tested whether gene deletion of KCa3.1 exerted beneficial effect on airway AHR in the mouse model of chronic asthma. Figure 1 showed the responses of the WT, KO, WT + OVA, and KO + OVA groups’ bronchi to methacholine accumulating concentration. The methacholine concentration-response curve of bronchial preparations from the WT + OVA group mice was markedly shifted upward compared with that from the WT group mice (Figure 1, p < 0.01). Sensitivity to methacholine, however, was not affected (EC₅₀ 5.9 ± 0.3 mg and 5.7 ± 0.3 mg for WT group and WT + OVA group mice, respectively). In contrast, KO + OVA group significantly attenuated the up-regulation of contractility compared to the WT + OVA group (Figure 1, p < 0.01). The sensitivity to methacholine, was also not altered (KO + OVA group EC₅₀ 6.0 ± 0.4 mg).

Inflammatory cells recruitment in the airway is one of the characteristic features in the development of asthmatic pathophysiology. As shown in Figure 2, gene deletion of KCa3.1 exerted beneficial effects on airway inflammation in the mouse model of chronic asthma. Hematoxylin and eosin-stained lung tissues showed that gene deletion of KCa3.1 significantly reduced the inflammatory influx into the lung tissue in the KO + OVA group mice compared to the WT + OVA group mice (Figures 2A,B, p < 0.01).

It was well known that the aberrant production of the T helper cell type 2 (Th2) cytokines played an important role in allergic asthma. Th2 cells cytokines participated in airway inflammation via B cell activation, eosinophil proliferation, and secretion of inflammatory components. Th1 cytokines such as IFN-γ were well known to inhibit the production of Th2 cytokines, which participated in the process of asthma. The imbalance of Th1 and Th2 cytokines has been reported to be involved in the allergic asthma (Bui et al., 2017). Thus Th2 and Th1-type cytokines concentrations were measured in bronchoalveolar lavage fluid to investigate whether KCa3.1 was involved in OVA-induced dominant T cell-derived cytokines. The concentrations of Th2-type cytokines (IL-4, IL-5, and IL-13) appeared to be lower in WT group mice. As we expected, WT + OVA group mice showed up-regulation of Th2-type cytokines IL-4, IL-5, and IL-13 compared to WT group (Figure 2C). In contrast, WT + OVA group mice showed decreased concentration of the Th1-type cytokine IFN-γ compared to WT group (Figure 2C). Interestingly, gene deletion of KCa3.1 (KO + OVA group) significantly attenuated the up-regulation of IL-4, IL-5, and IL-13 and increased concentrations of IFN-γ, in comparison with WT + OVA group mice (Figure 2C, p < 0.01).

To study whether gene deletion of KCa3.1 channels can attenuate airway remodeling in the mouse model of chronic asthma, as measured by Masson trichrome staining, PAS staining and α-SMA expression, in comparison with WT + OVA mice. Firstly, images analyses were performed to measure the matrix density of lung sections from WT, KO, WT + OVA, and KO + OVA groups of mice. Matrix deposition was significantly increased in the WT + OVA group mice compared with WT group mice (Figures 3A,B, p < 0.01). While there was no significantly decreased of matrix deposition in the KO + OVA group mice compared with WT + OVA group mice (Figures 3A,B, p > 0.05). PAS staining was used to quantify mucus-secreting goblet. Gene deletion of KCa3.1 significantly decreased the number of mucus-secreting goblet cells in the KO + OVA group mice compared to the WT + OVA group mice (Figures 3C,D, p < 0.01).

The expressions of KCa3.1 and TRPV4 proteins were measured in WT and WT + OVA group mice bronchi, using Western blot analyses. The expressions of KCa3.1 and TRPV4 channels in WT + OVA group mice were approximately 2-fold and 1.5-fold higher than those in WT group mice, respectively (Figures 4A,B, p < 0.05).

Enhanced smooth muscle mass is also a pathogenic feature of asthmatic airway remodeling. We detected α-SMA expression of lung sections from WT, KO, WT + OVA, and KO + OVA group mice. As shown in Figure 4, OVA sensitization and challenge induced up-regulation of α-SMA protein staining in the airways of WT + OVA group mice compared to the WT group mice (Figures 4C,D, p < 0.01). KO + OVA group mice exhibited significantly less α-SMA staining (per μm of bronchi basement membrane) relative to WT + OVA group mice (Figures 4C,D, p < 0.05). However, the levels of airway α-SMA protein staining in the non-OVA challenged mice were similar in both the WT and KO group mice (Figures 4C,D). The absence of KCa3.1 in lung tissues was confirmed by examining KCa3.1 protein expression using Western blotting (Figure 4E). These data suggested that KCa3.1 and TRPV4 channels involved in airway remodeling in the process of chronic asthma.

It was previously reported that up-regulation of KCa3.1, whose expression was suppressed by REST and also regulated by nuclear transcription factor AP-1 (Fos/Jun) (Wisdom, 1999; Ghanshani et al., 2000; Cheong et al., 2005), was necessary for porcine coronary SMC migration in vitro (Tharp et al., 2006). The expressions of AP-1 (Fos/Jun) members and REST were determined, using real-time PCR analysis. As shown in Figure 5A, the expression of REST transcript was significantly lower found in WT + OVA group mice than that in the WT group bronchi (Figure 5A, p < 0.05). However, the expression of c-Jun transcript was significantly higher in WT + OVA group than that in the WT group bronchi (Figure 5A, p < 0.01). No significant differences were detected in the expressions of c-Fos, FosB, Fra-1, Fra-2, JunB, and JunD between these two groups (Figure 5A, p > 0.05).

c-Jun N-terminal kinases (JNK) were shown to bind and phosphorylate c-Jun within its transcriptional activation domain. JNK pathway was activated by different stressing factors, and played an important role in cell death, gene expression, and regulation of cellular senescence (Yarza et al., 2016). The expressions of JNK/c-Jun and REST proteins in the bronchi of WT, KO, WT + OVA, and KO + OVA group mice were determined by Western blot analysis. Anti-JNK, anti-c-Jun and anti-REST antibodies were used to detect JNK, c-Jun and REST proteins from the WT, KO, WT + OVA, and KO + OVA four groups mice. Western blot analysis revealed that JNK/c-Jun and REST expression levels were significantly higher and lower in WT+OVA group than those in WT group (Figures 5B,C). In the group of KO + OVA mice, gene deletion of KCa3.1 decreased the expression of JNK/c-Jun and increased the expression of REST expression compared to the WT + OVA group (Figures 5B,C).

It was demonstrated genetic deficiency of KCa3.1 protected against lung damage induced by activation of TRPV4 channel (Simonsen et al., 2017). TRPV4 and KCa3.1 also functionally couple as osmosensors in the paraventricular nucleus (Feetham et al., 2015). Our research had shown that KCa3.1 regulated Ca²⁺ influx via membrane hyperpolarization in the HBSM cells (Yu et al., 2013b).

To further study the molecular mechanism between TRPV4 and KCa3.1 in regulating asthmatic HBSM cells proliferation, we investigated the role of TRPV4 in KCa3.1-regulated Ca²⁺ influx. HBSM cells transfected with siTRPV4 or siKCa3.1 were used to confirm our hypothesis that there was functional coupling between TRPV4 and KCa3.1 channels. As mentioned above, the activation of KCa3.1 channels will hyperpolarize the membrane potential of HBSM cells and then enhance the calcium driving force. 1-EBIO, KCa3.1 specific agonist, induced [Ca²⁺]_i influx in the control HBSM cells which were abolished in Ca²⁺-free media (p < 0.05, Figures 6A,B). While in the HBSM cells transfected with siKCa3.1, the effect of 1-EBIO was reduced compared to control (p < 0.01, Figures 6C,D). We then investigated the role of TRPV4 in the process of Ca²⁺ influx induced by 1-EBIO-activated KCa3.1 channels. The HBSM cells transfected with siTRPV4 also showed a significant attenuation of Ca²⁺ influx induced by 1-EBIO (p < 0.01, Figures 6C,D). The basal Ca²⁺ concentrations of HBSM cells were decreased in both Ca²⁺-free media and transfected with siTRPV4 or siKCa3.1 (Supplementary Figure S1). It was also tested that mildly elevated K⁺ concentration (40 mM) counteracts the effects of 1-EBIO-induced Ca²⁺ influx (Supplementary Figure S2). Taken together, these data suggested that the HBSM cells membrane hyperpolarization-induced Ca²⁺ influx, due to activation of KCa3.1 channels, was via the TRPV4 channels.

Colocalization experiments were conducted using specific KCa3.1 and TRPV4 antibodies. Confocal analysis of double-labeled staining showed that KCa3.1 and TRPV4 channels colocalized in HBSM cells (Figures 7A,B). As shown in Figure 7C, histograms represent the ratio of the mean Pearson correlation coefficient using Leica LAS AF Lite software. The Pearson correlation values range from -1 to +1. A correlation of 1 indicates complete colocalization between the two proteins. A correlation of -1 indicates a negative interaction, and a correlation of 0 indicates no colocalization between the two proteins. When double-labeled staining of KCa3.1 and TRPV4 channels, colocalization was observed in HBSM cells (Figures 7A,B). Pearson correlation coefficient is 0.59, confirming colocalization between KCa3.1 and TRPV4 (Figure 7C). Taken together, our results demonstrate that KCa3.1 and TRPV4 were colocalized in HBSM cells where they functional coupled and interacted in regulating Ca²⁺ entry.

Although proliferation of HBSM cells has been known to be a major characteristic feature of asthmatic airway remodeling, the mechanisms remain poorly understood. We have demonstrated that KCa3.1 was involve in cell cycle progression of asthmatic HBSM cells via membrane hyperpolarization and regulating Ca²⁺ influx (Yu et al., 2013b). The asthmatic HBSM cells proliferation can also be dependent on Ca²⁺ influx through TRPV4 channels (Dietrich et al., 2006). In this study, we investigated whether KCa3.1 and TRPV4 channels used a common pathway in regulating HBSM cells proliferation.

HBSM cells were transfected with KCa3.1-specific siRNA (siKCa3.1) and TRPV4-specific siRNA (siTRPV4) and with scrambled siRNA as a negative control (siCTL). The efficacy of siKCa3.1 and siTRPV4 was firstly measured. The proteins expressions of TRPV4 and KCa3.1 were reduced following 72 h treatment with siKCa3.1 and siTRPV4, respectively (Figures 8A,B). Additionally, siTRPV4 did not affect the expression of KCa3.1 (Figure 8A) and neither did siKCa3.1 for the expression of TRPV4 (Figure 8B). We then measured the effect of KCa3.1 and TRPV4 silencing on the proliferation of HBSM cells using a CCK-8 assay. The proliferation of HBSM cells was significantly attenuated in the cells of transfected with either siTRPV4 (Figure 8C, p < 0.01) or siKCa3.1 (Figure 8C, p < 0.01) compared to siCTL. No synergistic effects were observed in HBSM cells transfected with both siTRPV4 and siKCa3.1 compared to the effects obtained with either siTRPV4 or siKCa3.1, respectively (Figure 8C, p < 0.01).

The expressions of JNK/c-Jun and REST proteins in the HBSM cells with or without the stimulation of PDGF were determined by Western blot analysis. As shown in Figures 9A,B, anti-JNK, anti-c-Jun and anti-REST antibodies were used to detect JNK, c-Jun and REST proteins from the 1h PDGF-stimulated HBSM cells with or without the pretreatment of TRAM-34 (1 μM) or HC 067047 (10 μM). Western blot analysis revealed that the expressions of JNK/c-Jun and REST were significantly higher and lower in PDGF stimulation than those in control group (Figure 9B, p < 0.05). In the pretreatment of TRAM-34 or HC 067047, blockade of KCa3.1 or TRPV4 decreased the expression of JNK/c-Jun and increased the expression of REST expression compared to the PDGF group (Figures 9A,B). We then measured the pharmacological blockade of KCa3.1 or TRPV4 channels, and inhibition of MAPK/JNK/c-Jun signal pathway on the effect of HBSM cells proliferation using a CCK-8 assay. The proliferation rate of HBSM cells was significantly attenuated in the cells pretreated with either TRAM-34 (0.1, 1, 5 μM) or HC 067047 (1, 5, 10 μM) compared to control group (Figure 9C). With the pretreatment of MAPK/JNK/c-Jun inhibitor SP600125 (0.1, 1, 5, 10 μM), the proliferation rate of HBSM cells was also significantly attenuated (Figure 9C, p < 0.05).

The simple scheme of positive feedback, whereby KCa3.1-induced hyperpolarization led to an increase in Ca²⁺ influx via TRPV4, and the gene expressions of REST and AP-1 were correlated negatively or positively with the expression of KCa3.1 channels (Supplementary Figure S3).