Gα12facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner (original) (raw)

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 G 12 family proteins activate ROCK pathways in other cell types, their role in M 3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. EXPERIMENTAL APPROACH Gα 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 M 3 receptors or Gα 12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα 12 coimmunoprecipitated with M 3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SREluciferase 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 Gα 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 Gα 12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Loading...

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (50)

  1. Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015a). The Concise Guide to PHARMACOLOGY 2015/16: G protein-coupled receptors. Br J Pharmacol 172: 5744-5869.
  2. Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015b). The Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024-6109.
  3. Alexander SPH, Kelly E, Marrion N, Peters JA, Benson HE, Faccenda E et al. (2015c). The Concise Guide to PHARMACOLOGY 2015/16: Other proteins. Br J Pharmacol 172: 5734-5143.
  4. Amrani Y, Tliba O, Deshpande DA, Walseth TF, Kannan MS, Panettieri RA (2004). Bronchial hyperresponsiveness: insights into new signaling molecules. Curr Opin Pharmacol 4: 230-234.
  5. An SS, Fabry B, Trepat X, Wang N, Fredberg JJ (2006). Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness? Am J Respir Cell Mol Biol 35: 55-64.
  6. Balenga N a, Klichinsky M, Xie Z, Chan EC, Zhao M, Jude J et al. (2015). A fungal protease allergen provokes airway hyper- responsiveness in asthma. Nat Commun 6: 6763.
  7. Billington CK, Penn RB (2002). M3 muscarinic acetylcholine receptor regulation in the airway. Am J Respir Cell Mol Biol 26: 269-272.
  8. Billington CK, Penn RB (2003). Signaling and regulation of G protein- coupled receptors in airway smooth muscle. Respir Res 4: 2.
  9. Black JL, Panettieri RA, Banerjee A, Berger P (2012). Airway smooth muscle in asthma. Just a target for bronchodilation? Clin Chest Med 33: 543-558.
  10. Bradley SJ, Wiegman CH, Iglesias MM, Kong KC, Butcher AJ, Plouffe B et al. (2016). Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction. Proc Natl Acad Sci U S A 113: 4524-4529.
  11. Chiba Y, Misawa M (2001). Increased expression of G 12 and G 13 proteins in bronchial smooth muscle of airway hyperresponsive rats. Inflamm Res 50: 333-336.
  12. Chiba Y, Misawa M (2004). The role of RhoA-mediated Ca 2+ sensitization of bronchial smooth muscle contraction in airway hyperresponsiveness. J Smooth Muscle Res 40: 155-167.
  13. Chiba Y, Matsusue K, Misawa M (2010). RhoA, a possible target for treatment of airway hyperresponsiveness in bronchial asthma. J Pharmacol Sci 114: 239-247.
  14. Cooper PR, Lamb R, Day ND, Branigan PJ, Kajekar R, San Mateo L et al. (2009). TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation. Am J Physiol -Lung Cell Mol Physiol 297: L530-L537.
  15. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461-3471.
  16. Deshpande DA, Yan H, Kong K-C, Tiegs BC, Morgan SJ, Pera Tet al. (2014). Exploiting functional domains of GRK2/3 to alter the competitive balance of pro-and anticontractile signaling in airway smooth muscle. FASEB J 28: 956-965.
  17. Fabry B, Maksym GN, Butler JP, Glogauer M, Navajas D, Fredberg JJ (2001). Scaling the microrheology of living cells. Phys Rev Lett 87: 148102.
  18. Fisher JT, Vincent SG, Gomeza J, Yamada M, Wess J (2004). Loss of vagally mediated bradycardia and bronchoconstriction in mice lacking M 2 or M 3 muscarinic acetylcholine receptors. FASEB J 18: 711-713.
  19. Goncharova EA, Ammit AJ, Irani C, Carroll RG, Eszterhas AJ, Panettieri RA et al. (2002). PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L354-L363.
  20. Himes BE, Koziol-White C, Johnson M, Nikolos C, Jester W, Klanderman B et al. (2015). Vitamin D modulates expression of the airway smooth muscle transcriptome in fatal asthma. PLoS One 10: e0134057.
  21. Jude JA, Tirumurugaan KG, Kang BN, Panettieri RA, Walseth TF, Kannan MS (2012). Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases. Am J Respir Cell Mol Biol 47: 427-435.
  22. Kong KC, Gandhi U, Martin TJ, Anz CB, Yan H et al. (2008). Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization. Biochemistry 47: 9279-9288.
  23. Koziol-White CJ, Panettieri RA (2011). Airway smooth muscle and immunomodulation in acute exacerbations of airway disease. Immunol Rev 242: 178-185.
  24. Koziol-White CJ, Yoo EJ, Cao G, Zhang J, Papanikolaou E, Pushkarsky I et al. (2016). Inhibition of phosphoinositide 3-kinase (PI3K) promotes bronchodilation of human small airways in a Rho kinase- dependent manner. Br J Pharmacol 173: 2726-2738.
  25. Krymskaya VP, Orsini MJ, Eszterhas AJ, Brodbeck KC, Benovic JL, Panettieri RA et al. (2000). Mechanisms of proliferation synergy by receptor tyrosine kinase and G protein-coupled receptor activation in human airway smooth muscle. Am J Respir Cell Mol Biol 23: 546-554.
  26. Lee SJ, Lee WH, Ki SH, Kim YM, Lee SJ, Lee CH et al. (2009). Gα13 regulates methacholine-induced contraction of bronchial smooth muscle via phosphorylation of MLC20. Biochem Pharmacol 77: 1497-1505.
  27. Leopoldt D, Hanck T, Exner T, Maier U, Wetzker R, Nürnberg B (1998). Gbg stimulates phosphoinositide 3-kinase-g by direct interaction with two domains of the catalytic p110 subunit. JBiolChem 273: 7024-7029.
  28. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, Makhlouf GM (2003). Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3- mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylat. Biochem J 374: 145.
  29. Panettieri RA (2016). Neutrophilic and pauci-immune phenotypes in severe asthma. Immunol Allergy Clin North Am 36: 569-579.
  30. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI (1989a). A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol 256: C329-C335.
  31. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI (1989b). A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol 256: C329-C335.
  32. Penn RB, Benovic JL (2008). Regulation of heterotrimeric G protein signaling in airway smooth muscle. Proc Am Thorac Soc 5: 47-57.
  33. Pera T, Penn RB (2016). Bronchoprotection and bronchorelaxation in asthma: new targets, and new ways to target the old ones. Pharmacol Ther 164: 82-96.
  34. Pera T, Hegde A, Deshpande DA, Morgan SJ, Tiegs BC, Theriot BS et al. (2015). Specificity of arrestin subtypes in regulating airway smooth muscle G protein-coupled receptor signaling and function. FASEB J Off Publ Fed Am Soc Exp Biol 29: 4227-4235.
  35. Riobo NA, Manning DR (2005). Receptors coupled to heterotrimeric G proteins of the G 12 family. Trends Pharmacol Sci 26: 146-154.
  36. Roffel AF, Elzinga CRS, Amsterdam RGMV, Zeeuw RAD, Zaagsma J (1988). Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function. Eur J Pharmacol 153: 73-82.
  37. Roffel AF, Elzinga CR, Zaagsma J (1990). Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle. Pulm Pharmacol 3: 47-51.
  38. Roffel AF, Elzinga CRS, Zaagsma J (1993). Cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M2-like receptors. Eur J Pharmacol 250: 267-279.
  39. Rümenapp U, Asmus M, Schablowski H, Woznicki M, Han L, Jakobs KH et al. (2001). The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G 12 but not Gq-type G proteins. Regulators of G proteins as tools to dissect pertussis toxin- resistant G proteins in receptor-effector coupling. J Biol Chem 276: 2474-2479.
  40. Shaifta Y, Irechukwu N, Prieto-Lloret J, Mackay CE, Marchon KA, Ward JPT et al. (2015). Divergent modulation of Rho-kinase and Ca 2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle. Br J Pharmacol 172: 5265-5280.
  41. Shang X, Marchioni F, Sipes N, Evelyn CR, Jerabek-Willemsen M, Duhr S et al. (2012). Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases. Chem Biol 19: 699-710.
  42. Siehler S (2009). Regulation of RhoGEF proteins by G 12 / 13 -coupled receptors. Br J Pharmacol 158: 41-49.
  43. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SPH et al. (2016). The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucl Acids Res 44: D1054-D1068.
  44. Struckmann N, Schwering S, Wiegand S, Gschnell A, Yamada M, Kummer Wet al. (2003). Role of muscarinic receptor subtypes in the constriction of peripheral airways: studies on receptor-deficient mice. Mol Pharmacol 64: 1444-1451.
  45. Tseng P, Pushkarsky I, Di Carlo D (2014). Metallization and biopatterning on ultra-flexible substrates via dextran sacrificial layers. PLoS One 9 (8) e106091.
  46. Van Nieuwstadt RA, Henricks PA, Hajer R, van der Meer van Roomen WA, Breukink HJ, Nijkamp FP (1997). Characterization of muscarinic receptors in equine tracheal smooth muscle in vitro. Vet Q 19: 54-57.
  47. Wang Z (2016). Transactivation of epidermal growth factor receptor by G protein-coupled receptors: recent progress, challenges and future research. Int J Mol Sci 17: 95.
  48. Wells CD, Liu MY, Jackson M, Gutowski S, Sternweis PM, Rothstein JD et al. (2002). Mechanisms for reversible regulation between G 13 and Rho exchange factors. J Biol Chem 277: 1174-1181.
  49. Worzfeld T, Wettschureck N, Offermanns S (2008). G 12 /G 13 -mediated signalling in mammalian physiology and disease. Trends Pharmacol Sci 29: 582-589.
  50. Yang HW, Shin MG, Lee S, Kim JR, Park WS, Cho KH et al. (2012). Cooperative activation of PI3K by Ras and Rho family small GTPases. Mol Cell 47: 281-290.