ABSTRACT
BACKGROUND AND PURPOSE: PI3K-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 APPROACH: Gα12 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 RESULTS: Knockdown 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 IMPLICATIONS: Gα12 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.
Subject(s)
GTP-Binding Protein alpha Subunits, G12-G13/metabolism , Muscle, Smooth/metabolism , Phosphatidylinositol 3-Kinase/metabolism , rhoA GTP-Binding Protein/metabolism , Airway Obstruction/physiopathology , Asthma/physiopathology , Carbachol/pharmacology , Cells, Cultured , GTP-Binding Protein alpha Subunits, G12-G13/genetics , Gene Knockdown Techniques , Humans , Muscle Contraction/physiology , Myosin Light Chains/metabolism , Phosphorylation , Receptor, Muscarinic M3/genetics , Receptor, Muscarinic M3/metabolism , Signal Transduction/physiologyABSTRACT
Asthma manifests as airway hyperresponsiveness and inflammation, including coughing, wheezing, and shortness of breath. Immune cells and airway structural cells orchestrate asthma pathophysiology, leading to mucus secretion, airway narrowing, and obstruction. Phosphoinositide 3-kinase, a lipid kinase, plays a crucial role in many of the cellular and molecular mechanisms driving asthma pathophysiology and represents an attractive therapeutic target. Here, we summarize the diverse roles of phosphoinositide 3-kinase in the pathogenesis of asthma and discuss novel therapeutic approaches to treatment.
