UCLA

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.