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. 2019 Aug;61(2):209–218. doi: 10.1165/rcmb.2018-0301OC

Transforming Growth Factor-β1 Decreases β2-Agonist–induced Relaxation in Human Airway Smooth Muscle

Christie A Ojiaku 1,2, Elena Chung 2, Vishal Parikh 2, Jazmean K Williams 3, Anthony Schwab 2, Ana Lucia Fuentes 2, Maia L Corpuz 4, Victoria Lui 5, Sam Paek 5, Natalia M Bexiga 5,6, Shreya Narayan 5, Francisco J Nunez 4, Kwangmi Ahn 7, Rennolds S Ostrom 4, Steven S An 5,8,9, Reynold A Panettieri Jr 1,2,✉
PMCID: PMC6670035  PMID: 30742476

Abstract

Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-β1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-β1 affects the ability of HASM cells to relax in response to β2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-β1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-β1–treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-β1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-β1 decreases HASM cell β2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying β2-agonist hyporesponsiveness in asthma, and suggest TGF-β1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.

Keywords: human airway smooth muscle, TGF-β1, relaxation, severe asthma, β2-agonists

Clinical Relevance

In this study, we demonstrate a role for transforming growth factor (TGF)-β1 in mediating β2-agonist hyporesponsiveness in human airway smooth muscle. Collectively, our results suggest that therapeutically targeting TGF-β1 may decrease asthma exacerbations in severe and treatment-resistant asthma.

β2-agonist bronchodilators are a mainstay therapeutic used for acute and long-term control of asthma exacerbations. However, patients with severe asthma often respond poorly to β2-agonists, and increasing evidence demonstrates that frequent β2-agonist use leads to resistance and deterioration of asthma control (1, 2). Therefore, understanding the mechanisms mediating β2-agonist resistance is important for decreasing asthma-related morbidity and mortality.

Evidence suggests that a link exists between β2-adrenergic receptor (β2AR) hyporesponsiveness and airway hyperresponsiveness, where increased levels of bronchoconstriction can decrease bronchodilator responsiveness (2, 3). Unsurprisingly, several cytokines modulate hyperresponsiveness and β2-agonist resistance in human airway smooth muscle (HASM), the main regulator of bronchomotor tone (4, 5). We have previously reported that transforming growth factor (TGF)-β1—a profibrotic cytokine elevated in the airways of patients with asthma—augments agonist-induced contractile responses in HASM via a Smad3-dependent pathway (6). However, the role of TGF-β1 in modulating β2-agonist–induced relaxation responses in HASM remains unknown.

β2-agonists induce airway relaxation by binding to β2-adrenergic G protein–coupled receptors on HASM cells, stimulating adenylyl cyclase (AC) enzyme activity (7). AC activation by the β2AR Gs α subunit elevates intracellular cAMP levels, and increased cAMP leads to subsequent HASM cell relaxation by antagonizing HASM cell contractile pathways. HASM cell relaxation responses are also regulated by the action of prostaglandin E2 (PGE2), an arachidonic acid–derived mediator that exerts its effects via E-prostanoid (EP) receptors from the G protein–coupled receptor family (8). Stimulation of the Gs-coupled EP2 and EP4 receptor subtypes elevates intracellular cAMP levels via activation of AC, with EP4 receptor stimulation selectively leading to HASM cell relaxation (9, 10).

Intracellular cAMP levels in HASM cells are regulated by the balance between AC activation and cAMP-hydrolyzing phosphodiesterase (PDE) activity. Although HASM cells express multiple PDE isoforms (11), functional studies have established PDE3 and PDE4 as the major cAMP-hydrolyzing enzymes (12–14). PDE4, in particular, plays a pivotal role in HASM cell cAMP degradation, and is more widely studied as a therapeutic target in airway disease (15). Of the four PDE4 encoding genes (16), evidence supports a critical role for PDE4D in mediating HASM cell contractile and relaxation responses (17–20). Increased PDE4D activity and expression is associated with decreased β2-agonist–induced cAMP generation in HASM from subjects with asthma (20). Mice deficient in PDE4D also exhibit a loss of responsiveness to cholinergic stimulation (10), suggesting the therapeutic potential of PDE4D inhibitors in asthma.

Previous studies investigating the role of TGF-β1 in decreased β2AR responses have been purely biochemical in nature and largely limited to human tracheal smooth muscle cells and human lung embryonic fibroblasts (21, 22). As these studies were conducted in the presence of PDE inhibitors, neither study assessed the potential of TGF-β1 to modulate downstream components of the cAMP signaling pathway via PDE4. Therefore, we aimed to elucidate the mechanisms by which TGF-β1 modulates β2-agonist–induced relaxation responses in HASM cells.

Methods

HASM Cell Culture

Human lungs from otherwise healthy, aborted transplant donors were received from the International Institute for the Advancement of Medicine and the National Disease Research Interchange. HASM cells were isolated from the trachea and cultured as previously described (23).

Immunoblot Analysis

Confluent HASM cells were serum starved overnight before treatment and collected as previously described (24).

Magnetic Twisting Cytometry

Dynamic changes in cell stiffness were measured as an indicator of the single-cell contraction and/or relaxation of isolated HASM cells, as previously described (25, 26). Briefly, arginylglycylaspartic acid–coated ferrimagnetic microbeads bound to the cytoskeleton were magnetized horizontally and then twisted in a vertically aligned homogeneous magnetic field that varied sinusoidally in time (27). The ratio of specific torque to bead displacements is expressed here as the cell stiffness in units of Pascal per nanometer.

siRNA Transfection

In vitro siRNA knockdown was performed using a reverse transfection procedure as previously described (28). HASM cells were seeded onto cell culture plates for a final siRNA concentration of 10 μM.

Measurement of cAMP Levels

After stimulation, cAMP levels were measured in lysed HASM cells using the Applied Biosystems cAMP-Screen ELISA system according to manufacturer protocol. For kinetic measurement of cAMP production in live cells, HASM cells were infected with a recombinant BacMam virus expressing the cADDis cAMP sensor (Montana Molecular), as previously described (29). Cells were stimulated with agonist, and then fluorescence was measured at 30-second intervals for 30 minutes. Data were fit to a single-site decay model using GraphPad Prism 7.0 (GraphPad Software Inc.). Concentration–response curves were generated from each decay curve by multiplying the kinetic rate constant, k, with the plateau.

Quantitation of PDE Gene Expression

RNA was isolated from HASM cells using the RNeasy Mini Kit (Qiagen Sciences, Inc.). cDNA was generated using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific). Relative cDNA quantification was performed using TaqMan quantitative RT-PCR (Thermo Fisher Scientific) and the ΔΔCt method, and expression was normalized to β-actin control.

Statistical Analysis

Unless otherwise stated, statistical analysis was conducted using GraphPad Prism software, with significance evaluated at a P value of less than 0.05. Significance was determined using Fisher’s Least Significant Differences tests or multiple t tests with Holm-Sidak correction. For magnetic twisting cytometry experiments involving multiple lung donor cell responses, statistical analysis was conducted using mixed-effect models on SAS V.9.2 (SAS Institute Inc.) (30).

Materials

Compounds were purchased from Sigma-Aldrich (isoproterenol [ISO], PGE2, carbachol, perchloric acid), Selleck Chemicals (roflumilast), Cayman Chemicals (3-isobutyl-1-methylxanthine [IBMX]), and R&D Systems (TGF-β1; SB-431542). Immunoblot antibodies were purchased from Cell Signaling Technologies (phosphorylated myosin light chain [pMLC; 3674S]) and EMT Millipore (MLC; MABT180). siRNA was purchased from Thermo Fisher Scientific (Smad3; VHS41114), and Dharmacon (Smad2 [L-003561-00]; Nontargeting Pool [D-001810-10-05]).

Results

TGF-β1 Decreases β2-Agonist–induced Relaxation in HASM Cells

To determine the extent to which TGF-β1 mediates resistance to β2AR-induced relaxation in HASM cells, we investigated contractile outcomes in TGF-β1–pretreated HASM cells stimulated acutely with the β-agonist ISO (Figure 1). Single-cell relaxation responses were determined using magnetic twisting cytometry, a technique that measures changes in cell stiffness as a surrogate for agonist-induced force generation (26). TGF-β1 or vehicle pretreated cells were precontracted to carbachol and stimulated acutely with ISO. TGF-β1 significantly impaired ISO-induced single-cell relaxation in basal and carbachol-stimulated HASM cells as compared with vehicle control (Figure 1A). No significant changes in cell stiffness were observed in nonstimulated vehicle controls for the duration of our measurements (data not shown) (25, 26). To further confirm the effect of TGF-β1 on HASM cell contractile responses, we investigated the phosphorylation of MLC—an essential component of agonist-induced HASM cell contraction—after overnight TGF-β1 treatment. TGF-β1 augmented basal and agonist-induced MLC phosphorylation in a similar manner to previously published literature (6). After stimulation with ISO, MLC phosphorylation in TGF-β1–treated HASM cells remained significantly higher than that of vehicle control (Figure 1B). Notably, the addition of the contractile agonist, carbachol, to TGF-β1– and ISO-treated HASM cells significantly increased MLC phosphorylation to levels above that in TGF-β1– and ISO-treated HASM cells.

Figure 1.

Figure 1.

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Transforming growth factor-β1 (TGF-β1) decreases β2-agonist–induced relaxation in human airway smooth muscle (HASM) cells. (A) Single-cell relaxation of isoproterenol (ISO)-stimulated HASM cells in the presence or absence of TGF-β1 (10 ng/ml, 18 h) (n = 3 donors; ±SEM). HASM cells were contracted with carbachol (CCh) for 5 minutes and subsequently relaxed with ISO. CCh-stimulated stiffness was measured for the first 0–60 seconds, and changes in cell stiffness in response to ISO were measured continuously up to the indicated time (60–300 s). For each cell, stiffness was normalized to CCh-stimulated stiffness before ISO stimulation. (B) Phosphorylated myosin light chain (pMLC) after TGF-β1 (10 ng/ml, 18 h), CCh (20 μM; bottom left), and/or ISO (1 μM; bottom right) treatment (n = 4–7; ±SEM). Representative immunoblot of seven separate experiments. *P ≤ 0.05. ns = not significant.

TGF-β1 Blunts Agonist-induced cAMP Levels

To elucidate the mechanism by which TGF-β1 reduces HASM cell relaxation responses, total cAMP levels were measured in lysed TGF-β1–treated HASM cells. In TGF-β1–treated cells, ISO- and PGE2-induced cAMP levels were decreased versus that of the respective controls (Figure 2A). TGF-β1 treatment did not alter forskolin-stimulated cAMP levels (Figure 2B), suggesting that AC function was not negatively affected by TGF-β1; there were no significant differences in forskolin-evoked cAMP levels in vehicle control and TGF-β1–treated HASM cells.

Figure 2.

Figure 2.

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TGF-β1 blunts agonist-induced cAMP levels. (A) HASM cells were pretreated with TGF-β1 (10 ng/ml) overnight and acutely stimulated with ISO (1 μM, 5 min; n = 7 ±SEM; ISO 1 μM, 3,684.2 ± 1,170.0 pmol/well), prostaglandin E2 (PGE2) (100 nM, 5 min; n = 4 donors ±SEM; PGE2, 40,270.4 ± 25,537.2 pmol/well), or (B) forskolin (FSK; 10 μM, 15 min; n = 3 donors ±SEM; FSK 10 μM, 7,192.4 ± 3,244.3 pmol/well) before lysis for cAMP level determination. (C) Live HASM cells were pretreated with TGF-β1 (10 ng/ml) overnight then acutely stimulated with various concentrations of this indicated drug and cAMP levels monitored using cADDis. ISO (vehicle log half-maximal effective concentration (EC50) = −9.25 ± 0.258, maximum effect (Emax) = 0.0052 ± 0.00035; TGF-β1 logEC50 = −8.83 ± 0.433, Emax = 0.0031 ± 0.00038). (D) PGE2 (vehicle logEC50 = −9.08 ± 1.798, Emax = 0.0026 ± 0.00065; TGF-β1 logEC50 = −8.37 ± 0.647, Emax = 0.0021 ± 0.00048). (E) FSK (vehicle logEC50 = −5.40 ± 1.547, Emax = 0.010 ± 0.0012; TGF-β1 logEC50 = −5.44 ± 2.31, Emax = 0.0074 ± 0.0132). Data is expressed as mean (±SEM) of n = 5 donors. *P ≤ 0.05 and **P ≤ 0.01.

To further confirm these results, cAMP levels were monitored in live HASM cells pretreated with either vehicle or TGF-β1. In TGF-β1–treated cells, ISO-induced cAMP responses were 2.6-fold less potent and 1.7-fold less efficacious compared with the vehicle-treated control (Figures 2C and Figures E1A and E1B in the data supplement). TGF-β1 treatment appeared to decrease the potency of PGE2-stimulated cAMP responses, although this increase did not reach significance due to large variation of PGE2 responses between donors (Figures 2D and Figures E1C and E1D). Forskolin-stimulated cAMP responses were unaffected by TGF-β1 treatment in live HASM cells (Figures 2E and Figures E1E and E1F).

PDE Inhibition Rescues ISO-stimulated Responses in TGF-β1–treated HASM Cells

Intracellular cAMP levels are primarily reduced via hydrolysis—an effect mediated by the action of PDEs in HASM cells (31). To determine whether TGF-β1 mediates β2-agonist hyporesponsiveness by modulating PDE-mediated cAMP hydrolysis, MLC phosphorylation and cAMP levels were measured in TGF-β1– and ISO-treated HASM cells in the presence or absence of the pan-PDE inhibitor, IBMX (Figures 3 and E2A).

Figure 3.

Figure 3.

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Phosphodiesterase (PDE) inhibition rescues ISO-stimulated responses in TGF-β1–treated HASM cells. (A) MLC phosphorylation in HASM cells pretreated with vehicle or TGF-β1 (10 ng/ml; 18 h) and/or 3-isobutyl-1-methylxanthine (IBMX; 500 μM, 30 min) before stimulation with CCh (20 μM, 12 min) and/or ISO (1 μM, 10 min) (n = 4 ±SEM; maximum = 23.2-fold change over vehicle ± 9.4). (B) cAMP levels in TGF-β1 (10 ng/ml; 18 h)–treated HASM cells pretreated with vehicle (n = 7 ±SEM; ISO 1 μM, 3,684.2 ± 1,170.0 pmol/well) or IBMX (500 μM, 30 min) (n = 6 ±SEM; IBMX 1 μM ISO, 11,927.4 ± 1,599.3 pmol/well) before ISO (1 μM, 5 min) stimulation. n = 4 donors; ±SEM. *P ≤ 0.05.

MLC phosphorylation in HASM cells was increased after TGF-β1 treatment, and levels remained higher than vehicle control after ISO stimulation (Figure 3A, left). Treatment with IBMX, however, reduced MLC phosphorylation in TGF-β1–pretreated,ISO-stimulated HASM cells to a level similar to that of vehicle control (Figure 3A, left). In ISO-stimulated HASM cells, MLC phosphorylation levels were increased in TGF-β1– and carbachol-treated cells above those in TGF-β1–treated cells alone (Figure 3A, right). IBMX treatment decreased MLC phosphorylation in TGF-β1– and carbachol-treated cells to a level similar to that of vehicle control (Figure 3A, right).

We next investigated the role of PDE activity in TGF-β1–mediated decreases in ISO-induced cAMP (Figure 3B). Vehicle- or TGF-β1–treated HASM cells were pretreated with IBMX before ISO stimulation. IBMX pretreatment significantly elevated ISO-induced cAMP levels in TGF-β1–treated HASM cells (Figure 3B).

TGF-β1 Induces PDE4D Gene Expression in a Concentration-Dependent Manner

To determine the extent to which PDEs contribute to β2-agonist hyporesponsiveness in TGF-β1–treated HASM cells, we investigated the expression of HASM cell-specific PDEs in TGF-β1–treated HASM cells (Figure E3) (29). TGF-β1 selectively increased PDE4D gene expression in a concentration-dependent manner (Figures 4A and E3). Furthermore, inhibition of TβR-I receptor signaling with SB-431542 pretreatment blocked increased PDE4D gene expression evoked by TGF-β1.

Figure 4.

Figure 4.

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TGF-β1 induces PDE4D gene expression in a concentration-dependent manner. (A) PDE4D gene expression in TGF-β1–treated (10 ng/ml; 18 h) HASM cells in the presence or absence of SB-431542 (5 μM; 1 h pretreatment) (n = 3 donors ±SEM). (B) cAMP levels in ISO-stimulated HASM cells treated with TGF-β1 (10 ng/ml; 18 h) in the presence or absence of roflumilast (RF; 10 μM, 30 min) pretreatment (n = 6 ±SEM; ISO μM, 1,281.1 ± 406.6 pmol/well). (C) MLC phosphorylation in TGF-β1 (10 ng/ml; 18 h)–treated HASM cells in the presence of RF (10 μM, 30 min), CCh (20 μM, 12 min), and/or ISO (1 μM, 10 min) stimulation (n = 6 donors; ±SEM). (D) Single-cell relaxation of TGF-β1 (10 ng/ml, 18 h)–treated HASM cells in the presence or absence of RF (10 μM, 30 min) (n = 1 donor; n = 223 ±SEM). *P ≤ 0.05; relative to control unless otherwise shown.

To further determine the extent to which TGF-β1 modulates PDE4D to decrease β2-agonist–induced relaxation responses, cAMP accumulation, MLC phosphorylation, and cell stiffness were measured in HASM cells treated with the PDE4 inhibitor, roflumilast (Figures 4B–4D). Roflumilast pretreatment rescued blunted ISO-stimulated cAMP levels in TGF-β1–treated cells (Figure 4B). In the presence of roflumilast, TGF-β1–induced MLC phosphorylation in ISO-stimulated cells showed little increase over vehicle control (Figures 4C and Figure E2B). In addition, roflumilast pretreatment decreased augmented HASM cell stiffness in TGF-β1– and ISO-stimulated HASM cells (Figure 4D).

TGF-β1 Decreases β2-Agonist–induced Relaxation Responses in a Smad2/3-Dependent Manner

The canonical TGF-β1 signaling pathway involves the activation of Smad2/3–intracellular signaling proteins that mediate a variety of the effects of TGF-β1 on HASM cell signaling in asthma (32). To determine the role of Smad proteins in TGF-β1–mediated inhibition of HASM cell relaxation responses, we investigated TGF-β1’s modulation of ISO-induced cAMP levels in Smad2/3 siRNA-transfected cells (Figure 5). ISO-induced cAMP was significantly increased in Smad3 siRNA-transfected cells in the presence and absence of TGF-β1 treatment (Figure 5A). TGF-β1 blunted ISO-induced cAMP levels in HASM cells transfected with nontargeting and Smad2 siRNA, but had little effect on ISO-induced cAMP levels in Smad3 siRNA-transfected HASM cells.

Figure 5.

Figure 5.

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TGF-β1 decreases β2-agonist–induced relaxation responses in a Smad2/3-dependent manner. (A) Top: cAMP levels in nontargeting (NT) or Smad2/3 siRNA-transfected HASM cells pretreated with TGF-β1 (10 ng/ml, 18 h) and stimulated with CCh (20 μM; 10 min) and/or ISO (1 μM, 5 min) (n = 4 donors ±SEM; maximum, 15,397.2 ± 3,010.4 pmol/well). Bottom: representative immunoblot of total Smad3 (left, 16% of NT siRNA control ± 15%, n = 3) and total Smad2 (right, 10.7% of NT siRNA control ± 22.7%, n = 3) protein expression in Smad2/3 siRNA transfected HASM cells. (B) Top: PDE4D gene expression in NT- or Smad2/3 siRNA-transfected HASM cells pretreated with SB-431542 (5 μM, 30 min) before TGF-β1 (10 ng/ml) overnight treatment (n = 3–4 donors ±SEM). Bottom: representative immunoblot of total Smad3 (left, 20.3% of NT siRNA control ± 4.2%, n = 3) and total Smad2 (right, 38.1% of NT siRNA control ± 23.4%, n = 3) in Smad2/3 siRNA-transfected HASM cells. *P ≤ 0.05.

To determine the role of Smad signaling in TGF-β1–mediated induction of PDE4D gene expression, PDE4D gene expression was investigated in Smad2 or Smad3 siRNA-transfected HASM cells after overnight TGF-β1 treatment (Figure 5B). Smad2 and Smad3 knockdown reduced PDE4D gene expression induced by TGF-β1 treatment of HASM cells (Figure 5B).

Discussion

In the present study, we demonstrate that TGF-β1 attenuates β2-agonist–induced relaxation responses in HASM cells. To date, TGF-β1 has been shown to negatively modulate β-adrenergic responses in multiple cell types (21, 22, 33, 34). Here, we demonstrate that TGF-β1 treatment—in the presence or absence of the contractile agonist carbachol—significantly attenuates ISO-induced HASM cell relaxation via increased cell stiffness and MLC phosphorylation (Figure 1). Importantly, as β1 agonists have little bronchodilator effect in humans and HASM cell β receptors are solely of the β2 subtype, this study selectively demonstrates the effects of TGF-β1 and the β-agonist ISO- on β2AR-induced relaxation (35, 36). Although previous studies suggest that TGF-β1 modulates β2AR-mediated responses through a protein synthesis–dependent mechanism, the details by which this modulation occurs is not fully understood (21, 22). For the first time, we demonstrate that the effects of TGF-β1 on HASM cell relaxation responses occur via a Smad2/3 pathway that upregulates the expression of PDE4D. Collectively, our findings further establish TGF-β1 as a mediator of bronchodilator resistance via modulation of downstream cAMP pathway effects.

Previous studies suggest that TGF-β1 attenuates ISO-induced cAMP accumulation by negatively regulating β2AR number, protein, and gene expression (21, 22). However, our data suggest yet an additional mechanism for the attenuation of cAMP by TGF-β1. In our study, TGF-β1 blunted cAMP induced by both ISO and PGE2, a mediator that binds to the Gs/(Gi)-associated PG EP2 and EP4 G protein–coupled receptors to elevate intracellular cAMP levels (Figures 2A–2D) (37). Little is known regarding the effects of TGF-β1 on EP receptor expression in HASM, and it is unlikely that TGF-β1 blunts HASM cell cAMP by decreasing the expression of two independent Gs-coupled receptors.

Interestingly, other studies suggest a role for TGF-β1 in modulating G protein function. Treatment with pertussis toxin, an irreversible Gi inhibitor, blocked TGF-β1–induced PGE2 production in human lung fetal fibroblasts (38). In addition, a report demonstrating an augmentation of cholera- and pertussis toxin–induced ADP-ribosylation in TGF-β1–treated rat osteoblast-like cells suggests that TGF-β1 alters the abundance of both Gs and Gi proteins (39). TGF-β1 also modulates the expression of guanine nucleotide exchange factors—proteins that regulate the activity of small G proteins—in various cells (40, 41). A study in murine fibroblasts suggests that TGF-β1 increases GTPase activity via a pertussis-sensitive mechanism (42). Further studies will be needed to investigate whether TGF-β1 modulates G protein expression or activity in HASM cells, and whether this potential modulation further affects HASM cell relaxation responses. However, our present results suggest that TGF-β1—in addition to attenuating β2AR function—works downstream of the receptor level to impair ISO-stimulated cAMP levels.

We used forskolin—a direct activator of AC—as a tool to further investigate the downstream effects of TGF-β1 on the cAMP signaling pathway (43). In this study, TGF-β1 did not significantly alter forskolin-stimulated cAMP levels in HASM cells (Figures 2B and 2E). Current literature suggests an unclear role for cytokines in modulating AC activity. In previous reports using human and guinea pig airway smooth muscle, TGF-β1 treatment induced little or modest reductions in forskolin-stimulated cAMP accumulation (21, 34). Curiously, other reports demonstrate that chronic cytokine treatment sensitizes AC in HASM (44). In these studies, chronic incubation of HASM cells with the cytokine IL-1β or TNF-α caused a two- to threefold increase in forskolin-stimulated cAMP (44, 45). It is posited that AC sensitization may be a feedback response to upregulate relaxation pathways in the face of cytokine-induced airway hyperresponsiveness (45). Although TGF-β1 induces hyperresponsiveness in HASM cells (6), we did not find significant alteration of forskolin-stimulated cAMP in TGF-β1–treated HASM cells, (Figure 2B). Thus, further studies will be needed to determine the effect of TGF-β1 on AC activation.

As TGF-β1 did not negatively regulate AC function in HASM cells, we next investigated the role of cAMP-hydrolyzing PDE enzymes in TGF-β1’s attenuation of HASM cell relaxation responses. Previous reports suggest that TGF-β1 modulates PDE4 expression and activity. In human alveolar epithelial cells, TGF-β1 upregulated PDE4 mRNA, protein expression, and total cAMP-PDE activity (46). TGF-β1 has also been shown to mediate fibronectin, collagen I, and connective tissue growth factor induction in bronchial rings via a PDE4D-dependent mechanism (47). In human fetal lung fibroblasts, TGF-β1–mediated collagen gel contraction, fibronectin release, and fibroblast chemotaxis was inhibited in the presence of PDE4 pharmacological inhibitors (48). Therefore, we aimed to further investigate the role of PDE4 in the attenuation of ISO-induced cAMP by TGF-β1.

We demonstrate that TGF-β1 selectively induces PDE4D gene expression in HASM cells, and that PDE4D inhibition rescues attenuated ISO-induced cAMP levels in HASM cells (Figures 4A and 4B and Figure E3). Although roflumilast only modestly enhanced ISO-mediated decreases in TGF-β1–induced MLC phosphorylation (Figure 4C), roflumilast significantly enhanced ISO-induced, single-cell relaxation in TGF-β1–treated HASM cells (Figure 4D). Although discrepancies between biochemical and cell stiffness measurements in roflumilast-treated HASM cells are puzzling, studies suggest that both actomyosin cross-bridge cycling—regulated by MLC phosphorylation—and actin polymerization (49, 50) mediate HASM cell contractile responses. Reports demonstrate that TGF-β1 induces both MLC phosphorylation (6, 40) and actin polymerization (51, 52) in HASM cells. Although the individual contributions of these pathways to HASM cell shortening remain unclear, both pathways are modulated by cAMP signaling (31, 53). Evidence suggests that PDE enzymes regulate intracellular cAMP gradients in the cell, where subcellular PDE localization mediates variations in cAMP-stimulated responses (16, 29, 54). As both PDE3 and PDE4 hydrolyze cAMP in HASM, the observed discrepancy may result from the relative contribution of cAMP signaling to each pathway, driven by the spatially mediated effects of PDE isoforms.

To further determine the mechanism by which TGF-β1 attenuates ISO-induced responses, we investigated the role of the canonical TGF-β1 signaling pathway via Smad2/3 in HASM cells (Figure 5). In nontargeting and Smad2 siRNA-transfected cells, ISO-stimulated cAMP was decreased after TGF-β1 treatment (Figure 5A). In Smad3 siRNA-transfected cells, however, TGF-β1 had little effect on ISO-induced cAMP. Surprisingly, ISO stimulation induced significantly higher cAMP levels in Smad3 siRNA-transfected cells than those observed in nontargeting siRNA-transfected cells.

This increase in cAMP may indicate that Smad3 knockdown attenuates baseline TGF-β1 receptor activity after the release of biologically active TGF-β1 in HASM cells (55). Alternatively, it is possible that Smad3 knockdown augments basal cAMP levels through its association with HASM cell microtubules. Smad3 has been reported to bind directly to microtubules in the absence of TGF-β1 signaling (56), and TGF-β1 has been shown to induce microtubule stability in a variety of cell types (57, 58). Therefore, impaired TGF-β1 signaling via Smad3 knockdown may exert destabilizing effects on microtubule stability.

Microtubule destabilization has been correlated with impaired cAMP accumulation in multiple cell types. The microtubule assembly inhibitor, colchicine, has been shown to induce cAMP generation in human leukocytes in a concentration-dependent manner (59). In human leukocyte and S49 lymphoma cell studies, multiple microtubule assembly inhibitors enhanced β-adrenergic and PG-stimulated cAMP accumulation in a time- and concentration-dependent manner, potentially by acting on microtubules that inhibit AC activity (60, 61). However, further studies are needed to determine the significance of the interaction between Smad3 and microtubules in HASM cells, and how this interaction may affect microtubule stability and cAMP generation.

In addition to modulating HASM cell cAMP levels, Smad2/3 knockdown also decreased TGF-β1–stimulated PDE4D gene expression (Figure 5B). These findings were mirrored by a decrease in TGF-β1–stimulated PDE4D gene expression in HASM cells pretreated with the TβR-I receptor inhibitor, SB-431542 (Figure 4A). SB-431542 is a highly selective inhibitor of the TβR-I receptor, ALK5 (half-maximal inhibitory concentration = 94 nM), and, to a lesser extent, the activin type I receptor, ALK4, and the nodal type I receptor, ALK7, which share highly related kinase domains and Smad2/3 proteins as substrates (62). SB-431542 selectively inhibits TGF-β1 signaling in HASM at concentrations as high as 10 μM, and exerts little effect on more divergent ALK family members that recognize bone morphogenic proteins, suggesting it to be an effective and selective inhibitor of Smad2/3 signaling in HASM (6, 62, 63). Together, these experiments suggest that TGF-β1–induced PDE4D gene expression is Smad2/3 activation dependent.

In both Smad2 and Smad3 siRNA-transfected HASM cells, PDE4D gene expression in TGF-β1–treated cells was not significantly increased over vehicle control (Figure 5B). These results are surprising given that Smad2 and Smad3 exert differential effects on β2-agonist–induced cAMP in TGF-β1–treated cells (Figure 5A). However, these results support previous studies demonstrating that Smad2 and Smad3 can exert differential effects on cell function (6, 64, 65). It is possible that Smad3 selectively modulates PDE4D activity, whereas Smad2 and Smad3 mediate induction of PDE4D expression by TGF-β1. However, more studies will be needed to assess the potential role of Smad2/3 in PDE4D activation. Nonetheless, our collective findings demonstrate a role for TGF-β1 and Smad2/3 signaling in decreased HASM cell relaxation responses.

Due to the breadth and complexity of TGF-β1 signaling, there may be additional pathways by which TGF-β1 attenuates HASM cell cAMP levels that we did not investigate in this study. Other cytokines that attenuate HASM cell relaxation responses—such as IL-1β—attenuate ISO-induced cAMP via COX-2 induction and prostanoid release (66, 67). As TGF-β1 induces COX-2 expression in HASM cells (68), it is possible that prostanoid induction contributes to TGF-β1’s impairment of relaxation responses. Further studies will be needed to determine the contribution of potential TGF-β1 signaling pathways in HASM cell relaxation responses.

In conclusion, our study further establishes TGF-β1 as a mediator of bronchodilator resistance in asthma via a Smad3-dependent pathway (Figure 6). In light of our previous work on TGF-β1–induced hyperresponsiveness in HASM, these results further suggest TGF-β1 to be a promising therapeutic target to increase bronchodilator sensitivity and attenuate airway obstruction in asthma.

Figure 6.

Figure 6.

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Proposed role of TGF-β1 in HASM cell contractile responses in asthma. TGF-β1 signaling augments basal and HASM cell shortening through a Smad3, rho-associated protein kinase (ROCK)–dependent pathway, as previously described (6). In addition to modulating HASM cell contractile responses, Smad2/3 activation increases PDE4D gene expression, leading to increased cAMP hydrolysis and blunted HASM cell relaxation responses. GPCR = G protein–coupled receptor; MLC20 = 20-kD myosin light chain 20; MLCP = myosin light-chain phosphatase; RhoA = Ras homolog gene family, member A; TβR-I/II = TGF-β receptor I/II.

Supplementary Material

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Footnotes

Supported by National Institutes of Health (NIH) grant 3P01 HL114471-04S1 and T32 Training Grant T32GM008076. S.S.A. was supported by Discovery Award and Catalyst Award from the Johns Hopkins University, and the Patrick C. Walsh Prostate Cancer Research Fund. R.S.O. was supported by NIH grant GM107094.

Author Contributions: C.A.O., E.C., V.P., J.K.W., M.L.C., R.S.O., S.S.A., and R.A.P. contributed to the experimental concept and design; C.A.O., E.C., V.P., J.K.W., A.S., A.L.F., M.L.C., V.L., S.P., N.M.B., S.N., and F.J.N. performed the experiments; C.A.O., E.C., J.K.W., M.L.C., K.A., R.S.O., S.S.A., and R.A.P. contributed to the analysis and interpretation of the data; C.A.O. wrote the manuscript; C.A.O., R.S.O., S.S.A., and R.A.P. edited and reviewed the manuscript for important intellectual content.

This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2018-0301OC on February 11, 2019

Author disclosures are available with the text of this article at www.atsjournals.org.

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Author disclosures
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