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

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

The Role and Mechanism of Transforming Growth Factor Beta 1 in Airway Hyperresponsiveness

Asthma, a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR) and airway remodeling, affects 300 million people worldwide. Conventional asthma management includes the use of glucocorticoids and β2-agonist bronchodilators to combat inflammation and reverse airway narrowing. In severe asthma patients, whose asthma is poorly controlled with conventional therapies, frequent asthma exacerbations can lead to sustained AHR that remains insensitive to bronchodilator therapy. Therefore, it is essential to determine the mechanisms contributing to AHR and irreversible airflow obstruction in asthma to reduce patient morbidity and mortality. Transforming growth factor beta 1 (TGF-β1), a growth factor elevated in the airway of patients with asthma, perpetuates airway inflammation and airway remodeling in airway structural cells. The role of TGF-β1 in mediating AHR, however, remains unclear. In this dissertation, we demonstrate that TGF-β1 primes agonist-induced contractile responses and attenuates agonist-induced relaxation pathways in human airway smooth muscle (HASM), contributing to AHR and irreversible airflow obstruction in asthma. The dynamics of AHR and single-cell excitation-contraction coupling were measured using supravital microscopy, magnetic twisting cytometry, and biochemical assays in human precision-cut lung slices (hPCLS) and isolated primary HASM cells. We demonstrate that overnight TGF-β1 treatment augmented basal and agonist-induced shortening in hPCLS and isolated HASM cells. Interestingly, TGF-β1 increased HASM cell shortening and myosin light chain phosphorylation in a Smad3-dependent manner, with little effect on intracellular calcium levels. Additionally, we find that overnight TGF-β1 treatment decreased β2-agonist-induced relaxation responses in HASM cells via induction of phosphodiesterase 4 (PDE4), an enzyme that negatively regulates β2-agonist signaling pathways. Pharmacological and siRNA inhibition of PDE4 and Smad3, respectively, restored β2-agonist sensitivity in TGF-β1-treated HASM cells. Together, our data suggest that increased levels of TGF-β1 in the airway contribute to airway narrowing, asthma exacerbations, and bronchodilator resistance in asthma. Furthermore, our results establish TGF-β1 as a novel therapeutic target to decrease airway exacerbations in patients with severe and treatment-resistant asthma

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

Background and purposePI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined.Experimental approachGα12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction.Key resultsKnockdown of M3 receptors or Gα12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα12 coimmunoprecipitated with M3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS.Conclusions and implicationsGα12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

Background and purposePI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined.Experimental approachGα12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction.Key resultsKnockdown of M3 receptors or Gα12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα12 coimmunoprecipitated with M3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS.Conclusions and implicationsGα12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma