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Br J Pharmacol. 2017 Dec;174(23):4383-4395. doi: 10.1111/bph.14040. Epub 2017 Nov 12.

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

Author information

1
Rutgers Institute for Translational Medicine and Science, Child Health Institute, Rutgers University, New Brunswick, NJ, USA.
2
Department of Medicine, Division of Pulmonary and Critical Care Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, PA, USA.
3
Department of Environmental Health and Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
4
Department of Bioengineering, University of California, Los Angeles, CA, USA.
5
Department of Molecular and Medicinal Pharmacology, University of California, Los Angeles, CA, USA.
6
California NanoSystems Institute, University of California, Los Angeles, CA, USA.
7
Department of Mechanical Engineering, University of California, Los Angeles, CA, USA.
8
National Institute of Mental Health, Bethesda, MD, USA.
9
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.

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:

12 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:

12 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.

PMID:
28921504
PMCID:
PMC5715591
DOI:
10.1111/bph.14040
[Indexed for MEDLINE]
Free PMC Article

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