Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle

Abstract

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca²⁺]_i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca²⁺]_i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca²⁺]_i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.

NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

INTRODUCTION

Asthma, a significant global public health issue, affects ∼339 million people of all age groups worldwide (1). It is a leading cause of work and school absenteeism, resulting in substantial economic, social, and medical burdens (2). Clinically, asthma is characterized by breathlessness, wheezing, and airflow obstruction (3). Furthermore, airway inflammation with airway remodeling and hyperresponsiveness are the hallmarks of asthma (4). As asthma is an inflammatory disease, various immune cells contribute to its disease pathogenesis (2–4). Indeed, major treatments for asthma involve anti-inflammatories or bronchodilators to reduce inflammation and relax airways (5). Airway smooth muscle cells (ASMs) in particular have gained attention as they substantially contribute to asthma pathophysiology (6–8). Especially, ASM is well known for its involvement in acute bronchoconstriction (9), where contraction reduces the airway lumen leading to shortness of breath and wheezing (10). Intracellular calcium ([Ca²⁺]_i) regulation in ASM is a key regulatory mechanism in airway contraction (11, 12), proliferation (8, 13), and production of extracellular matrix (14–16), contributing to asthma pathophysiology.

Despite substantial research into the pathophysiology of asthma and its treatment, a sizeable portion of patients with asthma still experience poor control, especially concerning the alleviation of airway inflammation, hypercontractility, and remodeling (17, 18). Essentially, these aspects remainunaddressed by existing therapies, emphasizing the need to identify unexplored mechanisms of asthma and develop new treatment strategies. One potential avenue for exploration is protein phosphatase 2 A (PP2A), a major serine/threonine phosphatase found in cells (19). PP2As play a significant role in eukaryotic cells by contributing to a large fraction of phosphatase activity. It exists as a diverse collection of oligomeric enzymes with a shared catalytic subunit (20). This enzyme complex consists of a catalytic subunit (C), a structural subunit (A), and a regulatory/variable B-type subunit (19). Furthermore, C subunits have two different encoding genes, Cα and Cβ. The A subunit also has two encoding genes Aα and Aβ, which share 87% homology, however, the abundance of Aα is 90% of PP2A holoenzymes while Aβ has only 10%. The subunit B is more diverse and responsible for at least 15 regulatory subunits of PP2A holoenzyme (19, 21). PP2A regulates cellular contractile mechanisms by dephosphorylating ion channels and respective regulatory proteins, such as L-type calcium channel, ryanodine receptor 2, and Na⁺/K⁺ ATPase (22). PP2A plays a central role in [Ca²⁺]_i handling in cardiomyocytes regulating heart contraction (23–25).

Furthermore, several studies have highlighted the beneficial role of PP2A in various inflammatory diseases, including cancer (26, 27), Alzheimer’s disease (28, 29), and multiple sclerosis (30, 31). However, its role in respiratory diseases involving inflammation, particularly asthma, remains unexplored (32–34). Concerning asthma, one study reported that, defects in PP2A in human monocytes contribute to corticosteroid insensitivity in asthma (35). Subsequent studies have shown the anti-inflammatory effects of PP2A activation in lung cells (32, 34). As a result, it is conceivable to hypothesize that PP2A represents a promising target for addressing airway inflammation and asthma.

Although PP2A’s role in normal airways is not well-defined, it is even less understood in asthmatic airways. Considering the importance of ASM cells in asthma pathophysiology, investigating PP2A’s expression and function in ASM, particularly in the setting of inflammation and asthma, is crucial. Therefore, the current study seeks to establish PP2A signaling in ASM and asthma pathophysiology, with the ultimate goal of developing novel therapeutics to alleviate airway hyperresponsiveness, an unmet therapeutic need in asthma.

MATERIALS AND METHODS

Chemicals, Drugs, and Antibodies

PPP2R1A antibody (PA5-27643), fluorescent secondary antibodies (Alexa Fluor), DMEM/F12, and AbAm were obtained from Thermo Scientific (Waltham, MA). IRDye goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Li-Cor Biosciences (Lincoln, NE). β-Actin antibody (sc-47778) and PP2A agonist (FTY720, sc-202161) were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). Phospho-myosin light chain (pMLC:ser19/Thr18; 3674S) antibody was procured from Cell Signaling Technologies (Danvers, MA). Alpha-smooth muscle actin (α-SMA) antibody was procured from Millipore Sigma (Burlington, MA, A2547). Tumor necrosis factor (TNFα) and interleukin-13 (IL-13) were obtained from R&D systems (Minneapolis, MN). All other chemicals or antibodies were obtained from Sigma-Aldrich unless otherwise specified.

Human Tissue and ASM Cells

Human lung tissues and primary human ASM cells were isolated from healthy individual and patients with asthma (mild to moderate) undergoing thoracic surgery at Mayo Clinic, Saint Mary’s campus, Rochester (approved by Mayo Clinic IRB for lung tissue samples of focal noninfectious area and with patient written informed consent) as previously described (36, 37). For immunofluorescence studies, formalin-fixed paraffin-embedded (FFPE) human lung tissue sections were used. To generate ASM cells, epithelium-denuded airway samples were enzymatically dissociated following the manufacturer’s instructions (Worthington Biochemical). Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AbAm) and maintained under standard conditions (37°C temperature, 5% CO₂, and 95% air). Studies were performed on primary ASM cells with a maximum passage of 5. The human ASM cell phenotype was validated periodically by determining the expression of ASM-specific markers (α-SMA and Caldesmon) using immunoblotting. Furthermore, cells were periodically analyzed to ensure the absence of mycoplasma using PCR (Abm goods #G238). Human ASM cells were negative for mycoplasma. Donor ASM samples background information has been provided in the supplement (Supplemental Table S1).

Treatments

To determine the effect of inflammation on PP2Aα expression, human ASM cells were serum-deprived for 24 h and treated with proinflammatory cytokines TNFα (20 ng/mL) or IL-13 (50 ng/mL) for 24 h as previously described (38). For mechanistic studies, cells were serum-deprived for 24 h and pretreated with PP2Aα agonist FTY720 (2.5 µM) for 6 h (32), followed by incubation with IL-13 for 24 h (for calcium imaging) or 3 h (for phosphorylation studies).

RNA-Seq Transcriptome Profiling of Human ASM Cells

The RNA-Seq data analysis was performed as previously described (39). Briefly, the RNA-Seq data from nonasthmatic and asthmatic human ASM cells (6 independent samples) were processed and analyzed at NDSU using the Center for computationally assisted science and technology (CCAST) resources. Furthermore, FastQC v0.11.8 (40) and MultiQC v1.9 (41) were used to perform data quality control (QC). Subsequently, the reads mapping was done using human genome reference (hg38) and gene annotation (Homo_sapiens.GRCh38.gtf) from the Ensembl database using STAR aligner v2.7.5a (42). Furthermore, the read was quantified to obtain the raw counts per gene using quantMode gene counts flag. Read counts were transformed to counts per million (CPM) using edgeR (43). Genes with CPM < 0.5 in 50% of the samples were considered with low count (expression) and filtered out. Furthermore, postmapping QC was performed using MultiQC and edgeR.

Immunofluorescence

Immunofluorescence of nonasthmatic and asthmatic human airway tissue and ASM cells was performed following the established protocol (44). For tissues, 6-μm thick human lung sections on slides were baked for 2 h at 56°C, deparaffinized using xylene, and hydrated with decreasing grades of ethanol (100% to 70%). Antigen retrieval was performed using citrate buffer (pH 6.0) and the retrieved sections were permeabilized with 0.1% Triton X-100. Furthermore, sections were blocked using 10% goat serum and incubated overnight with α-SMA (1:100 dilution) in the presence or absence (for primary antibody control) of PP2Aα antibody (1:100 dilution) at 4°C. Next day, sections were washed with phosphate buffer saline (PBS) with 0.1% Tween and fluorescence labeled secondary antibodies, Alexa Fluor 647 (1:200 dilution) for PP2Aα and Alexa Fluor 488 (1:200 dilution) for α-SMA were probed for 2 h. Subsequently, sections were washed, counter-stained with DAPI, and visualized under Zeiss-LSM900 with Airyscan2 confocal microscope.

Separately, human ASM cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.05% Triton X-100, and blocked using 10% goat serum. Cells were incubated overnight in the presence or absence (for primary antibody control) of PP2Aα antibodies (1:100 dilution) at 4°C. Next day, cells were washed with PBS with 0.1% Tween and Alexa Fluor 647 (1:200 dilution) with Phalloidin (smooth muscle marker) was probed for 1 h. Subsequently, cells were washed, counter-stained with DAPI, and visualized under Zeiss-LSM900 with Airyscan2 confocal microscope. Fluorescence images were quantified using Zeiss Zen 3.0 software. Images for control and treatment groups were recorded and collected at the same time under the same conditions.

Immunoblotting

Immunoblotting was performed as per the previously established standard protocol (14). Cells were washed using PBS, harvested, and centrifuged to get cell pellet. Pellets were lysed in cell lysis buffer (Cell Signaling Technology) containing 1% protease and phosphatase inhibitors (PPIs) cocktail and centrifuged to get cell lysates. Furthermore, protein was quantified using DC Protein Assay Kit (Bio-Rad, CA) and 40 µg of samples were loaded on 4–15% gradient gels (Criterion Gel System; Bio-Rad). Subsequently, the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (0.22-μm) using a rapid transfer system (Trans-Blot Turbo; Bio-Rad). The membrane was dried and following postrenaturation with methanol, the blots were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Furthermore, the membrane was probed with specific primary antibodies (PP2Aα, 1:1,000; pMLC, 1:1,000; β-actin, 1:2,000 dilutions) overnight at 4°C. Next day, primary antibodies were retrieved, membranes were washed with Tris-buffered saline with 0.1% Tween, and probed with Li-Cor near-red-conjugated secondary antibodies (1:10,000 dilution). Finally, membranes were washed with Tris-buffered saline with 0.1% Tween and scanned using Li-Cor Odyssey XL System. The densitometry analysis was performed using Image Studio v.5.2 software. The intensity of protein of interest was normalized by β-actin and presented as relative protein expression. The representative blot for each protein showing the entire lane has been provided in the supplement (Supplemental Figs. S1 and S2).

To determine pMLC expression, cell lysates were prepared differently following the previous protocol (45). Following treatments, cells were exposed to histamine (1 µM) for 5 min, washed with Hanks’ balanced salt solution (HBSS), and treated with stop buffer (0.5 N perchloric acid) for 5 min. Subsequently, cells were washed with ice-cold PBS three times and lysed in cell lysis buffer containing 1% PPI cocktail. Lysates were sonicated and centrifuged to remove debris. The remaining immunoblotting procedures were the same as described earlier.

Real-Time Intracellular Calcium Imaging

The intracellular calcium ([Ca²⁺]_i) imaging was performed as previously described (46, 47). ASM cells were grown on Lab-tek eight-well plate, treated as described in Treatments, and serum-deprived for 24 h. Subsequently, cells were washed with HBSS buffer (containing 2.5 mM calcium and 1 mM magnesium) and incubated with Fura-2 AM (4 μM) for 1 h at room temperature. Subsequently, dye was aspirated, cells were washed, and imaging was done with ratiometric Fura-2 filters (alternatively excited at 340 and 380 nm), and emissions at 510 nm using fluorescence microscope (Olympus, Fluoview FV300) equipped with Hamamatsu camera and Cool-LED lamp. Cells were perfused with HBSS using Gilson minipump and vacuum system. [Ca²⁺]_i responses to 10 μM histamine were recorded and calculated as previously described (46).

Multiwire Myography

Age-matched C57BL/6J mice (in-house breed) of either sex were used in the study. The mice breeding and experiment protocols were approved by Institutional Animal Care and Use Committee (IACUC) at North Dakota State University. The experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (48). Animals were maintained in standard housing conditions with 12:12-h light and dark cycles. The food and water were provided ad libitum. The force study was performed using multiwire myograph system (DMT, MI) following the previously reported procedure (49). The mice were euthanized under CO₂. The bronchi were isolated, connective tissues were removed, and rings of equal size were made. The rings were mounted between two pins in a chamber filled with Krebs-Henseleit (KH) buffer. The composition of KH buffer was (in mM): 115 NaCl, 2.5 KCl, 1.91 CaCl₂, 2.46 MgSO₄, 1.38 NaH₂PO₄, 25 NaHCO₃, and 5.56 d-glucose. The pH of 7.4 was ensured and the buffer was continuously bubbled with 95% O₂-5% CO₂ and maintained at 37°C. The rings were stretched up to 5 mN force and normalized. After equilibration of 30 min with a buffer exchange every 10 min, the rings were contracted using 60 mM KCl. Subsequently, rings were washed by exchanging buffer three times with 10 min each wash. For contraction study, rings were pretreated with PP2Aα activator (FTY720: 2.5 µM) for 45 min and then the rings were contracted using increasing log dose (10⁻⁶ to 10⁻³ M) of acetylcholine (Ach) or methacholine (Mch). Furthermore, for relaxation study, the rings were contracted using single dose of Ach (10⁻⁶ M) and then increasing log dose (10⁻⁹ to 10⁻⁶ M) of FTY720, isoproterenol, and combination of both were added to induce relaxation. The force was recorded using LabChart 5 software (ADI Instrument).

Statistical Analyses

Statistical analysis was performed using Prism version 10.1.1 (GraphPad, CA). Normal distribution was tested using the Shapiro-Wilk normality test. Furthermore, parametric unpaired Student’s t test (for two groups) or one-way ANOVA (for multiple groups) followed by a Tukey post hoc test was performed. P values less than 0.05 were considered statistically significant. All values are expressed as means ± SD.

RESULTS

PP2Aα Expression in Airway Smooth Muscle

We have analyzed the RNA sequencing data of healthy and asthmatic human ASM cells (obtained from our original study deposited at NCBI GEO GSE119579) to screen for PP2A genes in ASM cells. The transcriptome profile demonstrated notably high gene expression of PPP2R1A (PP2Aα) compared with all other isoforms in healthy and asthmatic human ASM cells (Fig. 1, A and B). Furthermore, we validated the PP2Aα expression in isolated nonasthmatic and asthmatic ASM cells using immunoblotting and immunofluorescence analysis. We observed significant downregulation of PP2Aα expression in asthmatic ASM cells (Fig. 1, C–F) compared with nonasthmatic (Fig. 1, C–F). Separately, we exposed nonasthmatic ASM cells to proinflammatory cytokines TNFα or IL-13 and analyzed PP2Aα expression. We found significantly reduced expression of PP2Aα in IL-13-encountered nonasthmatic cells compared with vehicle (Fig. 1G). However, TNFα-exposed cells had reduced PP2Aα expression but was not significant. Overall, these results indicated PP2Aα is downregulated during asthma and/or inflammation.

Figure 1.

Heatmap (A) and bar graph (B) of RNA Seq showing normalized read counts of PP2A isoforms expression in nonasthmatic and asthmatic human ASM cells; data represented as box plots showing minimum, maximum, and median, with 25th to 75th percentile range. C: PP2Aα protein expression in nonasthmatic- versus asthmatic-isolated ASM cells. Immunofluorescence showing PP2Aα expression in nonasthmatic (D) and asthmatic (E) ASM cells, with image quantification (F). G: PP2Aα expression in nonasthmatic ASM cells during proinflammatory cytokine exposure (24 h). Images were quantified using Zeiss Zen 3.0 software. Data were analyzed using unpaired Student’s t test or one-way ANOVA followed by Tukey’s post hoc test. Data represented as means ± SD (n = 6 each, nonasthmatic 3 M/3 F, asthmatic 2 M/4 F, independent donor samples, biological replicates); *vs. nonasthmatic or vehicle, *P < 0.05, ***P < 0.001. ASM, airway smooth muscle; PP2A, protein phosphatase 2A.

PP2Aα Expression in Human Lung Section

After confirming PP2Aα expression in human ASM cells, we verified its expression in human lung tissue sections. The immunofluorescence of PP2Aα was performed in nonasthmatic and asthmatic human lung sections. We probed the ASM using an α-SMA (marker for ASM) antibody to show the localization of PP2Aα. Interestingly, following the pattern of asthmatic ASM cells, asthmatic lung section also depicted the significant downregulation of PP2Aα expression in the ASM region (Fig. 2, B and C) compared with nonasthmatic airway (Fig. 2, A and C).

Figure 2.

Nonasthmatic (A) and asthmatic (B) human lung sections immunostained for PP2Aα (AF-647) and DAPI (AF-408). C: graphical representation of ASM PP2Aα. α-SMA (AF-488) shows smooth muscle-specific colocalization. Scale bar: 10 μm. Inset: 2 μm. Images were quantified using Zeiss Zen 3.0 software. Data were analyzed using unpaired Student’s t test. Data represented as means ± SD (n = 4 each, nonasthmatic 2 M/2 F, asthmatic 2 M/2 F, independent donor samples, biological replicates); *vs. nonasthmatic, **P < 0.01. Arrows pointing Epi or ASM. ASM, airway smooth muscle; Epi, epithelium.

PP2Aα and Airway Contractility

To examine the functional importance of ASM PP2Aα, we examined the role of PP2Aα on mouse bronchial contraction and relaxation using DMT myograph. We pretreated the bronchial ring with FTY720, a PP2A agonist, for 45 min and then added an increasing log dose of Ach or Mch (10⁻⁶ to 10⁻³ M). Interestingly, FTY720 pretreatment significantly inhibited Ach-induced bronchial contraction (Fig. 3, A and B). Conversely, Mch-induced bronchial contraction was not significantly affected by FTY720 (Fig. 3, C and D). Furthermore, we investigated the effect of PP2Aα on bronchial relaxation. We induced bronchoconstriction using a single dose of Ach (10⁻⁶ M) and then added an increasing log dose (10⁻⁹ to 10⁻⁶ M) of FTY720, isoproterenol, and a combination of both. FTY720 alone failed to induce relaxation on Ach-induced precontracted bronchial rings. However, FTY720 in combination with isoproterenol tends to show augmented relaxation compared with isoproterenol alone (Fig. 3E).

Figure 3.

FTY720 (PP2Aα activator) inhibits acetylcholine- (A and B) or methacholine- (C and D) induced bronchoconstriction. FTY720 potentiates the isoproterenol-induced bronchial relaxation (E). FTY720 inhibits IL13-induced calcium response to histamine (F). FTY720 inhibits histamine-induced myosin light chain (MLC) phosphorylation in the presence of IL13 (G). Data were analyzed using unpaired Student’s t test or one-way ANOVA followed by Tukey’s post hoc test. Data represented as means ± SD (n = 4–6 mice or nonasthmatic donor samples 3 M/3 F, biological replicates); *vs. acetylcholine or vehicle, #vs. IL13, $vs. -Histamine vehicle, *P < 0.05, **P < 0.01, ***###$$$P < 0.001.

To identify potential mechanisms of PP2Aα-induced inhibition of airway contraction, we determined the effect of PP2Aα activation on [Ca²⁺]_i and MLC phosphorylation using nonasthmatic human ASM cells. Exposure of histamine to IL-13-incubated nonasthmatic ASM cells demonstrated significantly higher [Ca²⁺]_i response than untreated ASM cells. Interestingly, FTY720 pretreatment in IL-13-incubated ASM cells showed substantial inhibition of IL-13-induced calcium response to histamine (Fig. 3F). Furthermore, for MLC phosphorylation study, nonasthmatic ASM cells were treated with FTY720 for 6 h followed by incubation with IL-13 for 3 h. Subsequently, cells were stimulated with histamine for 5 min. We observed increased phosphorylation of MLC in all histamine-treated groups compared with untreated group. Furthermore, IL-13-incubated ASM cells produced significantly higher histamine-induced MLC phosphorylation than vehicle. FTY720 pretreatment significantly inhibited the histamine-induced MLC phosphorylation in the presence of IL-13 (Fig. 3G).

DISCUSSION

Considering that asthma is a chronic inflammatory disease, the majority of studies have centered around inflammatory cells (2–4). However, this trend is changing as recent studies have deciphered the importance of airway resident cells in asthma pathophysiology, such as epithelial (50) and ASM cells (10). Notably, ASM cells are involved in airway hyperresponsiveness, one of the paramount hallmarks of asthma through impaired ASM relaxation (7, 51) and bronchoconstriction (11, 12). As ASM plays a significant role in airway contractility, it is an attractive target for asthma therapeutics. PP2A is a serine/threonine phosphatase that plays a significant role in eukaryotic cells by regulating a large fraction of phosphatase activity (20). It regulates cellular contractile behavior by dephosphorylating ion channels and related regulatory proteins, such as L-type calcium channel, ryanodine receptor 2, and Na⁺/K⁺ ATPase (22). Based on these mechanisms, PP2A has been widely explored in cardiac contraction (23–25), which implicated PP2A as a potential target to elucidate in the context of airway contraction.

Considering the importance of ASM cells in airway contraction, exploration of PP2A in ASM becomes indispensable. However, based on our literature survey, the expression of PP2As in ASM and their functional role are not known. Considering the several isoforms of PP2A (19, 33), we screened abundantly expressed isoforms in the healthy and asthmatic human ASM cells using RNA-seq data (NCBI GEO GSE119579). PPP2R1A (PP2Aα) was predominantly expressed compared with all other isoforms, suggesting PP2Aα signaling is a plausible target to unveil in airway biology. These results are consistent with previous findings showing PP2Aα accounts for up to 90% of PP2A scaffold (21). Thereby, we selected PP2Aα as our protein of interest for our initial investigation.

We found robust PP2Aα protein expression in the isolated ASM cells and lung tissue from healthy and patients with asthma. Notably, asthmatic ASM cells and tissue had lower PP2Aα, suggesting possible intrinsic changes in PP2Aα signaling in asthma. Here, reinstating PP2Aα signaling may mitigate bronchoconstriction and thereby airway hyperresponsiveness in asthma, considering its phosphatase nature (19). Notably, we found a change in PP2Aα protein with no change in its transcripts level (RNA-Seq data), suggesting a posttranscriptional or -translational regulation may account for the diminished PP2Aα protein expression in asthmatic ASM cells.

Asthmatic airway is recognized to have immune response driven by Th1, Th2, and Th17 CD4+ helper T cells that secrete effector cytokines that promote ASM hypercontractility (52). Th1 cells produces TNFα and interferon-γ (IFNγ) while Th2 cells produce IL-13 and IL-4 (53). Thus, we examined the effect of TNFα and IL-13 on PP2Aα expression. Our results demonstrated significantly reduced expression of PP2Aα in IL-13-exposed cells but not in TNFα, suggesting Th2 inflammation can alter PP2Aα expression. This is an interesting finding as asthma is more influenced by Th2, thereby signifying the importance of PP2Aα in asthma. Moreover, how/why PP2Aα is downregulated in humans with asthma and IL13-exposed ASM cells was not elucidated. As IL13 transcriptionally regulates genes through signal transducer and activator of transcription 6 (STAT6) (54), STAT6 can be modulated using pharmacological or molecular tool to check its effect on PP2Aα expression in human ASM. Considering the phosphatase activity of PP2Aα and its involvement in cellular contraction and relaxation (23–25), we delineated its functional importance in ASM (9) by investigating its role on airway contraction. As asthma primarily affects lower airways (3), we performed force studies in isolated bronchial rings from mice using multiwire myograph system. Interestingly, FTY720 significantly inhibited Ach-induced bronchial contraction and further potentiated isoproterenol-induced bronchial relaxation. Interestingly, Kirchhefer et al. (23) revealed that enhancing PP2A activity in cardiomyocyte mice blunts isoproterenol-induced contractility of isolated hearts, which supports our findings in the airway. At the same time, FTY720 failed to significantly inhibit Mch-induced bronchial contraction in mice. This could be due to the long-lasting effect produced by Mch as its rate of hydrolysis by acetylcholinesterase is significantly less compared with Ach (55). The FTY720 dose and/or time of treatment used in the current study may not be enough to blunt the Mch effect significantly. These important aspects should be considered in the future investigations.

As [Ca²⁺]_i in ASM cells is one of the most important factors for airway constriction (11, 12) and PP2Aα has been reported to regulate [Ca²⁺]_i in other cell types (25, 56), we investigated PP2Aα role on [Ca²⁺]_i in ASM cells in the presence of IL-13. FTY720 inhibited IL-13-induced [Ca²⁺]_i response to histamine in ASM cells, suggesting a potential mechanism of action. Previous studies reported that enhanced [Ca²⁺]_i promotes phosphorylation of MLC in ASM cells, thereby inducing contraction (57). As speculated, FTY720 inhibited the histamine-induced MLC phosphorylation in the presence of IL-13, which further proves strong phosphatase activity of ASM PP2Aα. This study was further supported by previous study where Kitazawa et al. (58) unveiled that PP2A dephosphorylates MLC kinase in smooth muscles. As a limitation, it should be noted that there are currently no specific PP2Aα activators available. Accordingly, we used nonspecific PP2A activator FTY720 in this present study. Therefore, future studies aimed to understand PP2Aα’s mechanistic and functional roles should be elucidated using more specific methods, notably PP2Aα overexpression and/or silencing experiments.

Overall, we for the first time demonstrated robust expression of PP2Aα in human ASM. In addition, we observed a significant downregulation of ASM PP2Aα in asthma or inflammation, suggesting reduced PP2Aα activity and signaling in asthma and inflammation. Interestingly, PP2Aα activation inhibited Ach-induced bronchial contraction in mice and further potentiated isoproterenol-induced bronchial relaxation. Furthermore, PP2Aα activation inhibited [Ca²⁺]_i response to histamine and histamine-induced MLC phosphorylation in the presence of IL-13. Collectively, our data established the importance of PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2 and Supplemental Table S1: 10.6084/m9.figshare.25421125.

GRANTS

This study was supported by NIH grants: R01-HL146705 (to V.S.), R01-HL142061 (to C.M.P. and Y.S.P.), R01-HL088029 (to Y.S.P.), and R01-HL155095 (to R.D.B.). We also acknowledge the DaCCoTA-CTR confocal Microscopy Core Facility supported by the National Institute of General Medical Sciences (NIGMS)-U54GM128729 grant.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.I.R., R.D.B., and V.S. conceived and designed research; M.I.R. and A.K. performed experiments; M.I.R. and A.K. analyzed data; M.I.R. and V.S. interpreted results of experiments; M.I.R. prepared figures; M.I.R. drafted manuscript; M.I.R., A.K., C.M.P., R.D.B., Y.S.P., and V.S. edited and revised manuscript; M.I.R., A.K., C.M.P., R.D.B., Y.S.P., and V.S. approved final version of manuscript.