Airway smooth muscle and airway hyperresponsiveness in asthma: mechanisms of airway smooth muscle dysfunction

Abstract

Airway smooth muscle plays a pivotal role in modulating bronchomotor tone. Modulation of contractile and relaxation signaling is critical to alleviate the airway hyperresponsiveness (AHR) associated with asthma. Emerging studies examining the phenotype of ASM in the context of asthma provide rich avenues to develop more effective therapeutics to attenuate the AHR associated with the disease.

A number of factors influence the development of asthma and increase the incidence of the disease. Airway smooth muscle (ASM) represents the pivotal cell modulating bronchomotor tone. A more thorough understanding of the phenotypic and genotypic changes to ASM in the context of asthma will help to elucidate mechanisms of the development of a hypercontractile phenotype that is characteristic of the disease and in identification of therapeutic targets to alleviate bronchoconstriction or augment bronchodilation with current therapies.

Asthma-derived ASM exhibits a hypercontractile phenotype

Altered mechanisms of ASM shortening in asthma

Asthma, an airway inflammatory disease, manifests as recurrent episodes of airflow obstruction. The inflammatory environment alters shortening of the ASM,^1–3 that in turn modulates bronchomotor tone. Modulation of intracellular kinases, enzymes, and ion channels underly three main modes of regulation of ASM contraction, and can be classified into four main categories: calcium-dependent (Figure 1), calcium-independent (Figure 1), calcium-sensitization (Figure 1), nerve-mediated mechanisms (Figure 1), and modulation of actin dynamics (Figure 2). Asthma, and the inflammatory milieu characteristic of the disease, can phenotypically alter how ASM shortens.

Figure 1.—

Calcium-dependent and –independent, and nerve-mediated, pathways which modulate ASM contraction and AHR. cADPR, cyclic-ADP-ribose; CaM, calmodulin; CD38, cluster of differentiation 38; CICR, Ca²⁺-induced Ca²⁺ release; DAG, sn-1,2-diacylglycerol; GEF, guanidine nucleotide exchange factors; IP₃, inositol-1,4,5-trisphosphate; IP₃R, inositol-1,4,5-trisphosphate receptor; MLC, myosin light chain; MLCK, myosin light-chain kinase; MYPT1, myosin phosphatase target subunit-1 of myosin light-chain phosphatase (MLCP); Orai1, store operated channel proteins; PIP₂, phosphatidylinositol (4,5 )-bis phosphate; RhoA-GDP, inactive form of RhoA; RhoA-GTP, active form of RhoA; ROCK, Rho-associated kinase; RyR, ryanodine receptor; SERCA, sarcoendoplasmic reticulum calcium ATPase; STIM1, stromal interaction molecule 1; BDNF, brain-derived neurotrophic factor; NT4, neurotrophin 4 receptor; TrkB, tropomyosin receptor kinase B/p75 neurotrophin receptor; TRPC, transient receptor potential cation channel 3/6; PLC beta, phospholipase C.

Figure 2.—

Actin remodeling pathways which modulate ASM contraction and AHR. C-Abl, c-abl tyrosine kinase; Abi1, c-Abl adaptor 1; N-WASP, neural wiskott-aldrich protein; Arp2/3, actin-related protein 2/3; GMFγ, glia maturation factor γ; Pfn-1, profilin-1; CAS/CrkII, Crk-associated substrate.

Calcium-dependent signaling in ASM

Binding of contractile agonists to G-protein couple receptors (GPCRs) within the cell membrane of ASM activates calcium (Ca²⁺)-dependent signaling pathways that elicit contractile responses in ASM.^4–9 Described below are some of the calcium-dependent pathways that are activated/altered in asthma-derived ASM.

A role for the inositol triphosphate receptor, ryanodine receptor, and sarco/endoplasmic reticulum ca²⁺-atpase

These contractile responses are elicited through producing Ca²⁺ oscillations due to functions of sarcoplasmic reticulum (SR) membrane Ca²⁺ channels such as the inositol triphosphate receptors (IP₃Rs),^9–12 ryanodine receptors (RyRs),^{5, 8, 11, 13} and sarco/endoplasmic reticulum Ca²⁺-atpase (SERCA).^{11, 14} These changes in intracellular Ca²⁺ concentrations [Ca²⁺]_i ultimately induce myosin light-chain kinase phosphorylation (pMLC) and contraction of ASM,⁷ and dysregulation of [Ca²⁺]_i can produce airway hyper-responsiveness that is characteristic of asthma.^{5, 6, 11, 14}

Agonist binding to GPCRs induces activation of phospholipase Cβ (PLCβ) via dissociation of the GPCR G_αq and G_γβ subunits and subsequent binding of the G_α-subunit to PLCβ (as reviewed in^{9, 11}). Activation of PLCβ induces hydrolysis of cytosolic phosphatidylinositol (4,5)-bis phosphate (PIP₂) into second messenger products inositol-1,4,5-trisphosphate (IP₃) and sn-1,2-diacylglycerol (DAG).^{9, 11, 12} IP₃Rs on the membrane of the SR bind IP₃ and release Ca²⁺ into the cytosol (as reviewed in 2). It has long been known that IP₃ induces [Ca²⁺]_i release from SR stores in ASM.^{10, 12} Interestingly, not all contractile agonists have been shown to behave similarly with respect to eliciting [Ca²⁺]_i release. It was demonstrated that asthma-derived ASM exhibited a rapid increase in [Ca²⁺]_i release then a slower decline in concentration in response to histamine, which was posited to be a result of IP₃-mediated [Ca²⁺]_i mobilization from intracellular stores.¹⁵ This study also showed that not all contractile agonists elicited augmented [Ca²⁺]_i release from intracellular stores, where bradykinin (Bk), but not thrombin or histamine, induced more [Ca²⁺]_i release from asthma-derived ASM. Tumor necrosis factor α (TNFα) has been shown to promote increased [Ca²⁺]_i in response to contractile agonists such as Bk, thrombin, and carbachol.⁴ This suggests that TNFα may elicit increased responses at the GPCR level, leading to increased [Ca²⁺]_i by promoting formation of IP₃ in ASM.^{4, 9} Additionally, it was noted that TNFα induced hyperresponsiveness of human small airways with carbachol stimulation.¹⁶

RyRs, located in the SR membrane of ASM, function similarly to IP₃Rs and serve to elicit release of [Ca²⁺]_i.^{5, 6, 8, 11, 13} Following agonist binding to contractile GPCRs, IP₃ increases [Ca²⁺]_i and evokes Ca²⁺-induced Ca²⁺ release (CICR), that in turn increases the probability of [Ca²⁺]_i release through RyR.⁸ Mathematical analysis of agonist-induced [Ca²⁺]_i oscillations suggest that the RyR is critical in inducing [Ca²⁺]_i store depletion, however, following induction of store emptying from the SR and induction of CICR through RyR, the likelihood of RyR channel opening decreases and IP₃R mediates prolonged [Ca²⁺]_i store emptying.^{8, 11}

This increase in [Ca²⁺]_i following contractile agonist stimulation is attenuated through activation of SERCA, which removes [Ca²⁺]_i from the cytosol and replenishes the SR [Ca²⁺]_i stores.^{11, 14} Expression of SERCA2 is reduced in ASM derived from patients with moderate asthma via immunoblot and immunohistochemistry, and the duration of agonist-induced [Ca²⁺]_i transients is amplified in ASM derived from asthma donors. These data suggest that that [Ca²⁺]_i dysfunction in ASM due to SERCA2 expression may contribute to hyperresponsiveness of the airways that is associated with asthma.¹⁴ Additionally, application of Bk, ryanodine, and thapsigargin, three contractile agonists that induce [Ca²⁺]_i store emptying in ASM, resulted in reduced SR [Ca²⁺]_i store refilling following Bk stimulation, suggesting impaired SERCA function in asthma-derived ASM.¹³

The pro-inflammatory cytokine interleukin (IL)-13 appears key in inducing hyperreactivity of ASM.^{13, 17, 18} In a mouse ASM, blockade of interleukin 13 (IL-13), completely abrogates AHR responses and administration of IL-13 was capable of inducing AHR over time.¹⁸ IL-13 increased [Ca²⁺]_i oscillations in ASM and augmented oscillations following treatment with cysteinyl leukotrienes, key metabolites of arachidonic acid.¹³ IL-13 was demonstrated to engender hyperresponsiveness to contractile agonists in both ASM and human small airways.^{16, 19–21} Additionally, inhibitors of RyR and IP₃R decreased [Ca²⁺]_i oscillations in ASM following agonist treatment ± IL-13 pre-treatment, suggesting that IL-13 augments signaling pathways involved in production of [Ca²⁺]_i oscillations through activation of the IP₃R and RyR.¹³ Interestingly, irrespective of its role in modulation of exatation-contraction (E-C) coupling in ASM, IL-13 can elicit greater eotaxin-1 release and that SERCA2 siRNA treatment of asthma-derived ASM further increased IL-13-induced eotaxin 1 release from the cells.¹⁴

CD38

In airway smooth muscle (ASM), CD38 functions as a transmembrane protein which has been implicated in pathways that mediate [Ca²⁺]_i responses which control contraction and relaxation of ASM and modulate AHR.^22–26 Specifically, CD38 influences [Ca²⁺]_i release from the SR via the RyR channel pathway through a secondary messenger, cyclic-ADP-ribose (cADPR).^{26, 27} cADPR is regulated by the ADP-ribosyl cyclase and cADPR hydrolase enzymatic activity within the membrane-bound CD38 and that cADPR expression promotes release of [Ca²⁺]_i stores from the SR following agonist treatment.^{22, 26, 27} CD38 has been shown to be critical in maintaining [Ca²⁺]_i stores in ASM cells in response to store-emptying in human ASM (HASM).²⁴

Inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukins (IL) such as IL-13 can contribute to asthma²⁶ through eliciting changes in [Ca²⁺]_i, eliciting AHR. TNFα can modulate the function of HASM cells derived from both asthma and non-asthma donors.²⁸ Studies of CD38 in store-operated [Ca²⁺]_i have revealed that HASM cells treated with TNFα increased expression of CD38 mRNA^{21, 24, 26} and cADPR enzymatic activity²⁴ and increased store-operated calcium entry (SOCE) in response to SR store emptying,^{24, 26} and ASM derived from asthma donors exhibits higher levels of CD38 expression with TNFα stimulation. Overexpression of CD38 also increased expression of CD38 mRNA, as well as increased cADPR activity following treatment with TNF-α.²⁴ Additionally, increased expression of CD38, as well as increased cADPR activity in HASM treated with IL-13 has been observed, suggesting a role for CD38 in AHR observed in asthma through modulation of [Ca²⁺]_i in ASM.²⁵

Calcium/Calmodulin and myosin light chain kinase

Smooth muscle contraction is dependent on the interactions between free [Ca²⁺]_i and CaM, which associate and cause a conformational change which allows calcium/calmodulin (CaM) to bind and activate myosin light chain kinase (MLCK).^{7, 29–31} Decreases in [Ca²⁺]_I dissociate between [Ca²⁺]_i-CaM complexes that induce inactivation of MLCK.³⁰ MLCK regulates contraction within airway smooth muscle cells through phosphorylation of 20-kD regulatory myosin light chains (MLC₂₀), and subsequent actin activation through actomyosin adenosine triphosphate (ATPase), allowing for actin-myosin filaments to elicit ASM contraction.^{9, 32–34} The interaction between actin and myosin filaments is dependent on increases in [Ca²⁺]_i caused by contractile agonist activation of GPCRs.^{9, 35} Increased [Ca²⁺]_i activates Ca²⁺/calmodulin-dependent kinase II (CaMKII) which phosphorylates MLCK, allowing for further phosphorylation of MLC₂₀ and subsequent contraction of ASM.^{7, 9}

MLCK protein expression and activity is increased in human ASM derived from sensitized airways^{32, 36} and in asthma-derived ASM. Additionally, presence of transforming growth factor β1 (TGF-β1), a cytokine commonly found in the lungs of asthma subjects, increased basal and contractile agonist-induced phosphorylation of MLC₂₀ in HASM³¹ and both basal and contractile agonist-stimulated narrowing of human small airways, suggesting that the effect of TGF-β1 may contribute to the hyperresponsiveness observed in asthma-derived ASM.

STIM/Orai1

Refilling of SR Ca²⁺ stores (Ca²⁺_SR) is critical to maintaining homeostasis within airway smooth muscle cells.^37–39 Stromal interaction molecules (STIM) proteins, located within the SR, have been implicated in controlling [Ca²⁺]_i following store emptying.^{38, 40} The N-terminus of STIM acts as a luminal Ca²⁺ sensor within the SR through a low affinity Ca²⁺ EF-hand domain which senses Ca²⁺ store emptying.³⁹ Following store depletion, STIM1 proteins rapidly associate and translocate to junctions between the ER and plasma membrane where STIM1 interacts with Orai1 channel proteins.^{38, 39, 41} Orai channels that span the plasma membrane of the cells act as store operated channel (SOC) proteins which transduce calcium-release activated [Ca²⁺]_i currents to mediate selective Ca²⁺ ion entry across the plasma membrane following translocation and coupling with STIM.³⁹ This process is known as SOCE and is critical in maintaining [Ca²⁺]_i homeostasis in the cytosol of airway smooth muscle (ASM) cells.^{37, 38, 41}

Proinflammatory cytokines IL-13 and TNFα have been shown to increase SOCE in ASM following agonist stimulation through increased STIM1 aggregation, increasing contractility and SOCE.⁴¹ Additionally, application of TGF-β1 (24 hours) increased basal [Ca²⁺]_i compared to control ASM cells, as well as a larger SOCE Ca²⁺ influx following store-depletion.^{41, 42} Interestingly, HASM cells pretreated with IL-13 do not display increased mRNA expression for neither STIM1 nor Orai1 despite displaying increased SOCE.³⁷ This suggests that increased expression of STIM1 and Orai1 may not factor in increased SOCE in HASM cells.

Calcium-independent sensitization pathways

Along with pathways that elicit calcium release, there are pathways by which ASM contracts that are deemed calcium-sensitization pathways. Described below are some of these pathways that are altered in asthma-derived ASM.

Rho/ROCK/MYPT1

Exposure to pro-inflammatory cytokines, such as TNFα and TGF-β, has been shown to increase AHR in ASM activation of pathways that include RhoA^43–45 and Rho-associated kinase (ROCK)⁴⁴ by activating a pathway known as Ca²⁺ sensitization.^{44, 46} As discussed previously, exposure to contractile agonists increases [Ca²⁺]_i through Ca²⁺-dependent pathways that induce SR Ca ²⁺ store release through channels such as IP₃R and RyR. These Ca²⁺-dependent pathways ultimately induce contractility and AHR in ASM through actin-myosin filament cross bridging following activation of MLCK. Phosphorylation of MLC₂₀ is also regulated in a somewhat Ca²⁺-independent manner by myosin light-chain phosphatase (MLCP), that dephosphorylates MLC₂₀, stimulating ASM relaxation.³³ Under normal conditions, contractility and relaxation of ASM is balancing of expression of MLCK and MLCP.^{33, 35, 46}

Activation of p115-RhoGEF by G_α12/13 and the exchange of inactive RhoA-GDP for RhoA-GTP activates ROCK.^{46, 47} Ca²⁺-sensitization occurs through ROCK-dependent phosphorylation of the myosin-binding subunit of MLCP, myosin phosphatase target subunit-1 (MYPT1), leading to inactivation of MLCP.^{33, 43, 44} Within ASM, inactivated MLCP leads to increased MLC₂₀ phosphorylation by MLCK and enhanced ASM contraction.^{33, 35, 44, 46} Asthma-derived ASM was found to have greater pMYPT1 than non-asthma ASM.¹⁹ Pretreatment with TNF-α in human bronchi has also shown to increase contraction following treatment with contractile agonist histamine.⁴⁸

A study from Ojiaku et al. showed that treatment of human precision cut lung slices (hPCLS) with TGF-β1 has augments both basal and agonist-induced constriction through increased phosphorylation of MLC₂₀.³¹ Additionally, this study found that HASM treated with TGF-β1 exhibited increased MYPT1 phosphorylation through increased ROCK activation.³¹ Other studies have also shown that pretreatment with TGF-β1 increased Rho-associated GEF expression and activity of RhoA, as well as increased expression of MYPT1 and phosphorylation of MLC₂₀ in response to bradykinin-induced contraction.⁴⁵

Exposure of HASM to TNFα has been shown to increase levels of RhoA-GTP and phosphorylated MYPT1, both of which was completely ablated in HASM pre-treated with a ROCK inhibitor, indicating the effects of TNFα to induce Ca²⁺ sensitization and AHR through the RhoA/ROCK pathway.⁴⁸

Actin remodeling-dependent pathways of contraction

As discussed previously, both Ca²⁺-dependent and Ca²⁺-sensitization pathways contribute to the phosphorylation state of MLC₂₀ and subsequent actin-myosin interactions which control ASM contraction and relaxation. Additionally, in response to a contractile stimulus, actin filaments polymerize in ASM independent of myosin phosphorylation.^{49, 50} As a result, both actin polymerization and MLC₂₀ phosphorylation are necessary for ASM contractions⁵⁰ (2). In response to a contractile agonist, soluble actin (G-actin) is converted into insoluble actin (F-actin).⁴⁹

In ASM, actin polymerization occurs following the production of a contractile stimulus⁵⁰ which produces phosphorylation and activation of c-Abl tyrosine kinase (c-Abl) leading to a signal cascade.^{50, 51} C-Abl expression has been found to be increased in ASM derived from severe asthma subjects, indicating a role in regulation of actin remodeling in asthma-derived ASM and AHR development.⁵¹ Activation of c-Abl leads the phosphorylation and activation of c-Abl adaptor 1 (Abi1) which associates with neural Wiskott-Aldrich syndrome protein (N-WASP).⁵⁰ N-WASP, under non-contractile conditions, is found in a conformationally inactive state.⁵⁰ Contractile forces increase Abi1 activity, leading Abi1 and N-WASP association, rendering N-WASP active and capable of association with actin-related protein 2/3 (Arp2/3) complex which drives actin polymerization.⁵⁰ Additionally, Crk-associated substrate (CAS) acts as an adaptor protein which helps regulate actin polymerization in ASM.⁴⁹ Experimentally, Wang et al. demonstrated that acetylcholine (Ach) treatment of ASM and human bronchial rings increased N-WASP/Abi1 association, as well as the formation of a multiprotein complex involving c-Abl, CAS, and Abi1.⁵⁰ Knockdown of c-Abl or CAS resulted in decreased Abi1/N-WASP association, indicating that both CAS and c-Abl are critical in inducing actin polymerization and could play a role in development of AHR.⁵⁰

Profolin-1 (Pfn-1), an actin regulatory protein, and Cortactin have also been shown to induce and regulate, respectively, actin polymerization and modulate contraction in ASM.⁵² Following exposure to ACh, HASM increase associated Pfn-1 and Cortactin as compared to unstimulated HASM, suggesting a time-dependent association of these proteins following induction of contraction.⁵² In a mouse model, association of Pfn-1 and Cortactin was correlated with increased force generation as well as increased F-actin/G-actin rations in HASM.⁵² Moreover, disruption of Pfn-1/Cortactin association decreased F-actin/G-actin ratios in HASM, and decreased contraction in human bronchial rings independent of myosin phosphorylation. Interestingly, c-Abl knockdown, but not knockdown of Abi1, reduced phosphorylation of Cortactin and Pfn-1/Cortactin association, indicating that c-Abl may regulate Pfn-1/Cortactin induced actin polymerization in a separate capacity from Abi1 induced actin polymerization.⁵²

Opposingly, an actin depolymerizing factor (ADF)/cofilin family protein, glia maturation factor-γ (GMF-γ), has been shown to bind Arp2/3 under resting conditions.^{49, 52–54} This binding of an ADF to Arp2/3 may block binding of N-WASP and induction of actin polymerization.⁵⁵ However, c-Abl activation following contraction induced by ACh phosphondates GMF-γ at Tyr-104 residue that disassociates from Arp2/3 complexes.^{49, 54, 55} This effect increases levels of F-actin and decreases actin depolymerization within HASM, allowing for increased actin polymerization and contraction independent of myosin phosphorylation.⁵⁵

Nerve-meditated mechanisms of contraction

Contraction of HASM can be modulated through nerve-mediated responses that are generated through ASM innervation.^{56, 57} Parasympathetic nerves, arising from brain stem and vagal origins, release ACh through muscarinic receptors (mainly M₃) which induce cholinergic contractile responses from ASM tissue.^{56, 57} Electric field stimulation (EFS) has been used to demonstrate nerve-mediated bronchoconstriction in human precision-cut lung slices (hPCLS)^{56, 58} and human bronchi tissue⁵⁷ through parasympathetic cholinergic pathways. Notably, non-cholinergic responses to EFS have also been demonstrated in hPCLS, though less effectively.⁵⁸ Release of 5-hydroxytryptamine (5HT) from postganglionic cholinergic nerves has also been shown to induce bronchoconstriction in human airway tissue through release of ACh.⁵⁹

Non-cholinergic contraction within ASM can be attributed to nerve-derived factors such as neurotrophins (NT) and neurotrophin receptors (NTR),^{56, 60, 61} which are produced through ASM innervation with postganglionic non-adrenergic non-cholinergic (NANC) nerves.^{56, 60, 62} In bronchial asthmatic patient serum, enhanced production of NT nerve growth factor (NGF) was found.^{63, 64} Neurotrophins such as brain derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4) and their receptor, Tropomyosin receptor kinase B (TrkB), have also been associated with asthmatic airways.^{56, 60, 64}

The ability for nerve-mediated signaling to modulate Ca²⁺-dependent and Ca²⁺-sensitization pathways within ASM such as PLC/IP₃ and RhoA, respectively, has been reviewed.^{65, 66} TrkB can upregulate Ca²⁺-dependent signaling pathways including PLC/IP3 within ASM,⁶⁶ that could promote development of AHR in asthma. Additionally, the p75NTR receptor, a low affinity BDNF receptor, has been observed to modulate RhoA signaling within ASM.⁶⁶ PLC has been implicated in regulation of BDNF release in ASM⁶⁵ showing that activation of PLC is necessary for release and regulation of BDFN within ASM, leading to further activation of Ca²⁺-dependent pathways⁶⁵ and the potential for development of contractility and AHR.

Experimentally, application of NTs including BDNF and neurotrophin 4 (NT4) enhances Ca²⁺ response in HASM following exposure to ACh, histamine, and brandykinin in a dose-dependent manner.^{61, 62} Exposure to the proinflamatory cytokine TNF-α, which is often upregulated in asthmatic tissue, further modulated Ca²⁺ response to BDNF following exposure to ACh or histamine.⁶¹ Interestingly, ASM has been shown to secrete BDNF through transient receptor potential channels (TRPC), which assist in regulating [Ca²⁺]_i, with higher BDNF secretion in asthmatic ASM, as well as with increased BDNF secretion following prolonged treatment with TNF-α.⁶⁵

Additionally, exposure of ASM to BDNF significantly increased the force response generated after exposure to histamine or ACh.⁶² Following Ca²⁺ store depletion, enhanced SOCE was also seen in ASM acutely exposed to 1 nM or 10 nM BDNF, as well as ASM exposed to 1 nm BDNF or NT4, indicating that NTs can induce Ca²⁺ entry.⁶² Increased [Ca²⁺]_i release from the SR or Ca²⁺ influx following exposure to BDNF, as well as increased expression of TrkB in human asthmatic bronchial samples,⁶⁰ suggests a potential for increased contractility in HASM from NANC nerve-mediated pathways. Additionally, though not well studied in humans, inhibitory NANC pathways may mediate bronchodilation in ASM to attenuate bronchoconstriction and AHR.⁵⁶

Taken together, these three mechanisms by which ASM contract have been shown to be altered in some way in asthma-derived ASM. These findings suggest that AHR induced in asthma is likely due to a combination of these mechanisms rather than amplification of one process alone.

Asthma-derived ASM displays attenuated responses to agonist-induced relaxation

With alterations in contractile signaling, there exist alterations in relaxation signaling that can be precipitated by the inflammatory environment present in asthma. Figure 3 illustrates signaling downstream of the β₂ adrenergic receptor (β₂AR), with the following section addressing phenotypic and genotypic changes to ASM that alter β₂AR function and downstream signaling.

Figure 3.—

β₂ adrenergic receptor (β₂AR)-mediated inhibition of contractile signaling, signaling pathways modulating relaxation, and mechanisms of β₂AR downregulation. RGS2/4/5, regulator of g protein signaling 2/4/5; βarr, β arrestin 1/2; PDE3/4, phosphodiesterase 3/4; GRK2/3, G protein receptor kinase 2/3; Epac, exchange factor directly activated by cAMP; PKA, protein kinase A; ADP, adenosine diphosphate; cAMP, cyclic adenosine monophosphate; AC, adenylyl cyclase.

Altered mechanisms of ASM relaxation in asthma

Downregulation of contractile pathways

Relaxation of ASM can modulate contractile pathways through activation of kinases and ion channels to reverse the effects of depolarization of the membrane following contractile GPCR activation. The most commonly targeted pathway to induce bronchodilation, that is also the target of current bronchodilator therapies used to treat asthma, is the β₂-adrenergic receptor (β₂AR). The β₂AR belongs to a GPCR family that has been found to G_αs-coupled in order to promote dilation in ASM cells. Activation of G_αs by the β₂AR then activates adenylate cyclase, which converts ATP to cAMP. Generation of cAMP induces activation of protein kinase A (PKA).⁴⁴ Activation of these mechanisms can inhibit increases in [Ca²⁺]_i elicited by contractile agonist receptor activation or reduce calcium sensitivity of the cell,⁶⁷ through impairment of myosin light chain phosphorylation. Phosphorylation of HSP20 has been identified to inhibit contraction of ASM.^{68, 69} Additionally, generation of cGMP following nitric oxide stimulation of protein kinase G (PKG) can also induce ASM relaxation. It has been shown that cAMP, through PKG, is able to directly inhibit SOCE.⁷⁰ Epac, a guanine nucleotide exchange factor, is an effector molecule downstream of cAMP. A cAMP analog that is targets activation of Epac was demonstrated to decrease contraction of ASM, MLC phosphorylation, and RhoA activities that are activated upon acetylcholine stimulation.⁷¹ This shows that cAMP, through Epac, is able to modulate both calcium-dependent and -independent sensitization pathways to induce relaxation of ASM. Activation of a PKC-dependent kinase, 17-kD myosin phosphatase inhibitor protein (CPI-17), has been shown to increase calcium sensitivity in ASM, illustrating another effect on calcium-dependent signaling that induces contraction, and subsequent knockdown of CPI-17 inhibited contraction of ASM.^{72, 73} Additionally, PKA has been shown to regulate membrane-proximal events including G_αq and PLC activation through phosphorylation to inhibit contractile agonist-induced [Ca²⁺]_i mobilization and hydrolysis of phosphoinositides.⁶⁷ To reverse the effects of [Ca²⁺]_i flux induced by contractile agonists, it has been shown that cAMP, agonists of the β₂AR, and PKA can all regulate gating of large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in ASM.⁷⁴ Additionally, components of the calcium-dependent signaling like SERCA2 have been shown to be downregulated in asthma-derived ASM¹⁴ and with chronic stimulation with β₂ agonists.⁶⁷ Relaxation of ASM is dependent on myosin light-chain phosphatase, which dephosphorylates MLC₂₀ when [Ca²⁺]_i is low (1, 6). Thus, dysregulation of [Ca²⁺]_i within the cytosol of asthmatic patients can lead to a buildup of [Ca²⁺]_i and increased contraction of ASM through activation of MLCK. A large component of this dysregulation of Ca²⁺ homeostasis may be SERCA that, when lowly expressed in asthma-derived ASM, can lead to buildup of [Ca²⁺]_i (8) and contraction of ASM through MLCK activity. Additionally, SERCA expression has been shown to be downregulated by ASM exposure to IL-13 and TNFα.²⁶

Upregulation of regulators of G protein signaling (RGS) proteins by β₂ agonists has also been shown to attenuate agonist-induced contraction of ASM. It was demonstrated that both β₂ agonist and glucocorticoid treatment of ASM increased RGS2 expression, that reduced [Ca²⁺]_i flux elicited by histamine, methacholine, and leukotrienes.⁷⁵ RGS2 is known to target G_αq-dependent signaling, a G protein that is known to be associated with multiple contractile GP-CRs. Additionally, RGS4 expression was found to be elevated in asthma-derived ASM and that overexpression of RGS4 was shown to attenuate muscarinic receptor-induced constriction.⁷⁶ Prolonged exposure to β₂ agonists has been shown to attenuate expression of RGS5 and siRNA-mediated knockdown of RGS5 reduced myosin light chain phosphorylation and [Ca²⁺]_i flux.⁷⁷ Levels of RGS5 are elevated in asthma-derived ASM.¹⁵ Reversal of actin polymerization is also affected by activation of β₂AR signaling. It was demonstrated that activation of cAMP-PKA signaling inhibits modulation of the actin cytoskeleton. PKA induced HSP20 phosphorylation, thereby decreasing cofilin phosphorylation and disrupting actin polymerization resulting in ASM relaxation.^{69, 78}

Regulation of β₂AR signaling

To prevent aberrant/chronic signaling through the β₂AR, there are mechanisms by which down-regulation of function/receptor number occurs. Desensitization of the β₂AR has also been identified both clinically and experimentally. This downregulation of the receptor and functions has been problematic with respect to the widespread utilization of β₂ agonists in the treatment of asthma,^{19, 20, 79–84} as prolonged use of these therapeutics results in downregulation of the receptor and attenuation of agonist-induced bronchodilation. Polymorphisms of the β₂AR have been shown to downregulate the function of the receptor via targeting the receptor for degradation, by decreasing affinity of the receptor for β₂ agonists, or through uncoupling of the receptor to adenylyl cyclase (as reviewed in⁸⁵), with some evidence that the specific polymorphisms Arg16Gly and Gln27Glu having been associated with asthma severity and response to β₂ agonist therapy.⁸⁶ Natural antagonism of cAMP production occurs via a family of enzymes called phosphodiesterases (PDEs). The PDE3 inhibitor SKF94120 significantly inhibits contraction of human bronchi.⁸⁷ Interestingly, multiple groups have found that PDE4D enzymes, including PDE4D5, are increased in expression in ASM derived from asthma subjects, as well as in TGF-β1-stimulated non-asthma ASM.^88–92 When β₂AR is stimulated, activation of the Gαβγ complex induces signaling downstream of the G_αs and Gβγ subunits. The Gβγ subunits can recruit βarrestins to the β₂AR through activation of G protein receptor kinases (GRKs), which then lead to internalization of the receptor. It has been demonstrated that ASM express GRKs 2 and 3, as well as βarrestin 1 and 2, but predominantly βarrestin 1.^93–95 Interestingly, inverse agonism of the β₂AR was found to be important for the therapeutic actions of β blockers. A study showed that the β blocker nadalol increased PC₂₀ and decreased FEV1 values over the study period, helping to partially reverse the AHR associated with asthma.⁹⁶ In a letter to the editor, it was suggested that a biased ligand that inhibits β₂ downregulation, leading to increased AHR, would be useful to antagonize the increased bronchoconstriction associated with asthma.⁹⁷ The idea of biased agonism or antagonism has been postulated to “dial out” the unwanted effects of β₂ agonists to provide more effective therapies for asthma.⁹⁸ Additionally, β₂ agonist stimulation of ASM has been shown to downregulate β₂AR expression through up-regulation of let-7f miRNA.⁹⁹ Asthma-derived ASM stimulation with a β₂ agonist showed that this mechanism of downregulation of the β₂AR is relevant in a disease model, suggesting that modulation of let-7f miRNA may be a valid target for therapeutic intervention in the case of β₂ agonist-insensitivity.¹⁰⁰

Cytokine-mediated alterations in β₂AR signaling may also contribute to the hyporesponsiveness to bronchodilators. IL-13 treatment has been shown to decrease β2 agonist-induced cAMP production as well as attenuate β₂ agonist-induced relaxation of human small airways^{16, 19, 20, 101} and cAMP production in ASM.^{21, 102} It was also demonstrated that following ASM stimulation with TGF-β1, β₂ agonist-induced cAMP production and relaxation was attenuated via a PDE4-dependent mechanism.⁹¹ IL1-β, a factor associated with viral infections that can exacerbate underlying asthma, was shown to attenuate β₂ agonist-induced cAMP production in ASM.²¹

Interestingly, activation of protein kinase G (PKG) can be achieved through stimulation of the β₂AR, but it also occurs through activation of an enzyme similar to adenylate cyclase called guanylate cyclase (GC). Investigators have noted that direct activation of GC with nitric oxide (NO) donors including NOC18, sodium nitroprusside, BAY 41-2272, and BAY 60-2770 induced dilation of human small airways.^{103, 104} Even in the face of β₂AR desensitization, the GC activators BAY 41 and BAY 60 were able to induce dilation of human small airways. Overnight treatment with the NO donor NOC18 augmented GC-dependent bronchodilation.¹⁰⁴

With agonism of the β₂AR being a main component of current asthma therapy, a greater understanding of mechanisms by which receptor stimulation dampens contractile signaling and how the inflammatory environment that is characteristic of asthma contributes to dysfunction of the receptor is key to future development of novel therapeutics to enhance signaling through the β₂AR and associated pathways.