Introduction
Obesity contributes to asthma diathesis by enhancing airway hyperresponsiveness (AHR) and attenuating the response to standard asthma therapy [
1‐
3]. Human airway smooth muscle (HASM) cells play pivotal roles in asthma through its contractile, immunomodulatory and remodeling functions [
4,
5]. We previously reported that HASM cells from obese lung donors show amplified cell shortening in response to contractile agonists [
6]. Contractile agonists elevate intracellular calcium ([Ca
2+]
i) levels, activate myosin light chain kinase (MLCK), and increase the phosphorylation of myosin light chain (MLC), promoting cross-bridge formation and ASM cell shortening [
7]. In a parallel mechanism, myosin light chain phosphatase (MLCP) is inhibited through RhoA-activated Rho-associated kinase (ROCK), maintaining increased MLC phosphorylation (pMLC) levels in HASM cells [
8,
9]. Additionally, RhoA can activate actin polymerization and focal adhesion protein paxillin phosphorylation and, thereby, reinforcing mechanotransduction through the cell surface integrin receptors and cortical tension development [
10]. How obesity affects these surrogate measures (i.e. [Ca
2+]
i, pMLC, and paxillin phosphorylation) of excitation–contraction (E–C) coupling in HASM shortening is unclear. It is equally unclear the structure–function relationship between an altered E–C coupling and obesity-associated AHR.
Lipid mediators play pivotal roles in various aspects of asthma pathogenesis. Cysteinyl leukotrienes, generated from arachidonic acid metabolism, modulate AHR and leukotriene inhibitors are used as therapeutics in asthma patients [
11,
12]. There is a renewed interest in other lipids and their mechanisms of action on airway structural cells [
13]. Fatty acid receptors are expressed in a variety of tissues and modulate various cellular functions. Free fatty acid receptor 1 (FFAR1), also known as GPR40, is a de-orphaned G protein-coupled receptor (GPCR) expressed in a variety of tissues [
14]. FFAR1, activated by long-chain fatty acid linoleic acid, is reportedly linked to Gα
q [
14]. In guinea pig bronchial rings, activation of FFAR1 has been shown to potentiate acetylcholine-induced contraction [
15].
Serum levels of free fatty acids are elevated in obesity and modulate systemic inflammation associated with the metabolic syndrome (metabolic inflammation) [
16]. These fatty acids, in addition to acting as biomarkers of dyslipidemia in obesity, play (patho)physiological roles in several organ systems in obese subjects. Supporting the negative impact of elevated plasma free fatty acids, studies found that high-fat diet acutely increases airway inflammation and attenuates bronchodilator response in asthma patients [
17]. However, it appears that not all free fatty acids negatively impact lung functions. A recent study found that FFAR4 (GPR120), a long chain free fatty acid receptor, mediates ASM relaxation [
18]. Accordingly, we posit that free fatty acid receptors (FFAR1 and FFAR4) can modulate E–C coupling in HASM cells and play a role in obesity-associated AHR. Unexpectedly, our findings show that TAK875, a pharmacological agonist for FFAR1, but not GW9508 that targets both FFAR1 and FFAR4, attenuated histamine-evoked cortical tension development in HASM cells. Inhibition of tension was associated with decreases in MLC phosphorylation, but not calcium flux, phosphorylation of AKT or MYPT1. Of note, FFAR1 agonist-mediated inhibition of MLC phosphorylation was operational with carbachol stimulation, maintained in β
2AR-desensitized HASM cells, and across obese and non-obese donor-derived HASM cells. These findings warrant further investigation on FFAR1 agonists as novel bronchoprotective agents.
Material and methods
Reagents
HAM’s F-12 medium, PBS, FBS, 0.05% Trypsin and EDTA, and PAGE/western blotting were purchased from Life Technologies (Carlsbad, CA). Antibodies for pMLC (pS18/T19-MLC), total MLC, pS507MYPT1, total MYPT1, pAKT, total AKT and tubulin were purchased from Cell Signaling Technology (Danvers, MA). Fluo-8 calcium flux assay kit was purchased from Abcam (Cambridge, MA). GW9508 and TAK875 were purchased from Cayman Chemical Company (Ann Arbor, MI). Cyclic AMP-Screen assay kit was purchased from Applied Biosystems (Bedford, MA).
Culture of HASM cells
Primary HASM cells were harvested, characterized and grown in culture as described by us in detail previously [
19]. For all experiments, cells were used within the first 4 passages to ensure proper smooth muscle phenotype. HASM cells were serum-deprived 48 h prior to experimental exposures.
Exposure to testing compounds
HASM cells were exposed to compounds in F-12 culture medium without serum. GW9508 and TAK875 were dissolved in DMSO. All subsequent dilutions were made in serum-free F-12 medium. GW9508 or TAK875 were initially used at 0.1–10 μM concentrations for 10 and 30 min to detect the effect on pMLC level. In subsequent experiments, both compounds were used at 10 μM for 30 min. The DMSO concentration in the vehicle control was 0.05–0.1%. To determine MLC, MYPT1 or AKT phosphorylation, HASM cells were exposed to 25 μM carbachol (CCh) or 2.5 μM histamine for 10 min. Cell lysates were collected in 0.6 M HClO3 to precipitate proteins.
Magnetic twisting cytometry
We used magnetic twisting cytometry (MTC) to measure dynamic changes in the cytoskeletal stiffness as a surrogate for agonist-induced force generation at the single-cell level. An RGD-coated ferrimagnetic microbead functionalized to the cytoskeleton through cell surface integrin receptors was magnetized, twisted by an external magnetic field that varied sinusoidally in time, and forced bead motions were detected as previously described [
20]. Cell stiffness is expressed as Pascal per nm. We applied mixed effect model using SAS V.9.2. and report estimated mean ± SEM.
Measurement of [Ca2+]i in HASM cells
Agonist-induced [Ca
2+]
i in HASM cells was determined as previously described with some modifications [
21]. Briefly, HASM cells grown to confluence in a 48-well plate were loaded with fluo-8 Ca
2+-binding dye. Carbachol (25 μM) or histamine (2.5 μM) were used to elicit Ca
2+ response in HASM cells. Fluorescence intensity was monitored for up to 100 s following agonist injection. Area under the curve (AUC) of the time-dependent fluorescence (relative fluorescence units-RFU) was calculated from the response curve.
Measurement of cyclic adenosine monophosphate (cAMP) in HASM cells
HASM cells were seeded and grown in a 24‐well plate until about 80% confluent before serum-withdrawal. Cells were stimulated, lysed and analyzed by cAMP screen ELISA system by Applied Biosystems (Bedford, MA) following manufacturer’s instructions.
Data analysis
HASM cells from at least 5 donors (Additional file
1: Table S1) were used in the experiments. When applicable, the experimental readouts were first normalized to vehicle control in each donor to obtain the fold change. The fold changes from individual donors were used to obtain group mean graphs. The data are expressed as mean ± SEM. Unless otherwise noted, GraphPad Prism 5.0 was used for statistical analysis using One-Way ANOVA with Dunnett’s multiple comparison test and the means were considered significantly different when p ≤ 0.05.
Discussion
Elevated serum free fatty acid levels are associated with increased adiposity, insulin resistance and cardiovascular disease risk [
24]. Evidence suggests that agonists of free fatty acid receptors FFAR1 and FFAR4 increased cytosolic Ca
2+ and amplified ASM shortening [
15]. These observations prompted us to explore whether FFAR1 amplifies E–C coupling in obesity [
6]. Surprisingly, our results show that FFAR1 agonists GW9508 and TAK975 attenuated agonist-induced MLC phosphorylation while TAK875 decreased agonist-induced cell stiffness in HASM cells. These FFAR1 agonists, however, had little effect on cytosolic Ca
2+ or MYPT1 phosphorylation.
The concentrations of TAK875 and GW9508 used in this study were based our preliminary experiments and those of others [
15,
25]. TAK875 was developed as an anti-diabetic drug (Fasiglifam, Takeda Pharmaceutical Company Ltd., Kanagawa, Japan) and underwent extensive pharmacokinetic profiling. In healthy human subjects, maximal plasma concentrations (C
max) of TAK875 reached 41.8 μM and 76.7 μM, following single daily oral dosing with 400 mg and 800 mg fasiglifam, respectively [
26]. Therefore, the TAK875 concentration of 10 μM in our experiments appears physiologically relevant and pharmacologically achievable.
Intracellular elevation of Ca
2+ is a key step in E–C coupling in HASM cells. Some allergens and inflammatory cytokines promote AHR by enhancing Ca
2+ mobilization in ASM cells [
27‐
29]. Therefore, we tested the hypothesis that FFAR1 agonists attenuate E–C coupling and cell stiffness by abrogating cytosolic Ca
2+ levels. Since the broncho-protective effects of FFAR1 was independent of Ca
2+ flux, we focused on two other pathways that could be modulated by FFAR1 receptors. We previously reported that muscarinic cholinergic receptor activation in HASM cells elicit cell shortening through G
α12-coupled PI3K activation and Ca
2+ sensitization [
22]. This mechanism is unlikely to be the target of FFAR1 agonists since pAKT and pMYPT1 levels are unaltered by FFAR1 agonists. Others reported that activation of FFAR1 increases actin polymerization in ASM cells [
15]. It remains to be seen whether these FFAR1 agonists, particularly TAK875, modulate actin polymerization in HASM cells to elicit bronchoprotective effects. Evidence suggests that FFAR1 activation enhanced HASM cell proliferation in a MEK/ERK- and PI3K/Akt-dependent manner [
25]. While not measuring HASM cell proliferation, we showed unaltered Akt phosphorylation by FFAR1 agonists that suggests these agents are not mitogenic. We and others have shown that PI3K activation is necessary for HASM cell growth [
22]. Generally, pro-contractile signaling overlaps and shares signaling entities with proliferative signaling in many cell types. Theoretically, the bronchoprotective effects of FFAR1 agonists should not induce HASM cell proliferation. Furthermore, TAK875 and GW9508 acting through FFAR1, elicited potent anti-proliferative effects in multiple types of human melanoma cells lines, suggesting that FFAR1 may play a complex role in proliferation that is cell and tissue specific [
30].
Bronchodilators and corticosteroids are the critical components in mainstream asthma therapy. About 5–10% of asthma patients, with severe asthma, have suboptimal response to corticosteroids [
31]. Further, β
2AR receptor desensitization and tachyphylaxis leads to uncontrolled asthma symptoms [
32]. Therefore, there is an unmet need for novel bronchodilators to expand the current repertoire of treatments available for asthma. The FFAR1 agonist TAK875 attenuated MLC phosphorylation in β
2AR-desensitized HASM cells, indicating the potential of this compound to curb AHR in severe asthma.
ASM relaxation is mediated by a variety of cell signaling pathways activated by cAMP mobilizing agents. β
2AR, the major receptor responsible for ASM relaxation, activates adenylate cyclase activity and cAMP levels to mediate PKA-dependent inhibition of MLC kinase. Other mechanisms, such as inhibition of phospholipase activity, Ca
2+ mobilization and activation of large conductance K
+ channels are also implicated in ASM relaxation [
33]. Studies in pancreatic beta cells showed that, GW9508 activates ATP-sensing K
+ channels (K
ATP channels) to inhibit membrane depolarization and insulin secretion [
34]. In HASM cells, activation of K
ATP channels caused relaxation, suggesting that these channels are functionally similar to that of pancreatic beta cells [
35]. The regulatory roles of FFAR1 agonists on membrane potential remain to be elucidated, as are the K
ATP channels and other previously unrecognized Ca
2+-evoked HASM relaxation mechanisms.
Although this study originated from our interest in obesity, the FFAR1 agonist TAK875 has no differential effect on MLC phosphorylation in obese- and non-obese donor derived HASM cells. However, our in vitro findings may not predict whether FFAR1 function in HASM cells is altered in obesity in vivo. Plasma free fatty acids are elevated in obesity, therefore prolonged exposure to free fatty acids may desensitize and modulate FFAR1 functions in obese individuals [
36,
37]. Studies using ectopic expression systems demonstrated ligand-dependent and -independent FFAR1 receptor internalization, suggesting a desensitization mechanism similar to that of β
2AR tachyphylaxis [
38]. Collectively, how changes associated with an obesity phenotype, such as inflammation, proliferation and cell metabolism, are differentially modulated by FFAR1 or FFAR4 in obesity remains unknown [
39].
Our key findings identify a bronchoprotective role for these FFAR1 agonists. However, we acknowledge the following deficiencies in the study: (i) The expression levels of FFAR1 mRNA is low in HASM cells (Additional file
2: Figure S1). This low expression precluded siRNA-mediated silencing of FFAR1 as an additional approach to determine the necessity of this receptor in attenuated E–C coupling. It is plausible that the FFAR1 agonists elicit their bronchoprotective effect, not through the FFAR1 but through hitherto unidentified targets. (ii) We have not focused on the FFAR4 in this study [
40]. Based on Ca
2+ mobilization assays, GW9508 showed ~ 100-fold affinity to FFAR1 than FFAR4. The selectivity of GW9508 to FFAR1 was demonstrated in other cell types mostly in ectopic expression backgrounds [
41]. It is plausible that the relative selectivity of GW9508 towards FFAR1 is different in endogenous expression conditions seen in HASM cells. In light of the recent report on FFAR4 and ASM relaxation, this needs to be addressed in future studies [
18]. Although these limitations prevent us from confirming the necessity of FFAR1 to bronchoprotection, our findings suggest that TAK875 acts to reverse or prevent HASM shortening.
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