GPR40 receptor activation promotes tight junction assembly in airway epithelial cells via AMPK-dependent mechanisms

ABSTRACT

Tight junctions play key roles in the regulation of airway epithelial barrier function and promotion of tight junction integrity is beneficial to lung health. G-protein coupled receptor (GPR) 40 has been identified as a receptor of polyunsaturated fatty acids. This study aimed to investigate the function of GPR40 in regulating tight junction assembly in human airway epithelial cells (Calu-3 cells) using GW9508, a GPR40 agonist. Immunoblotting and immunofluorescence analyses showed that Calu-3 cells expressed both types of polyunsaturated fatty acid receptors including GPR40 and GPR120. Intracellular Ca²⁺ measurements confirmed that GW9508 stimulated GPR40, but not GPR120. In Ca²⁺ switch assays, GW9508 promoted the recovery of transepithelial electrical resistance and re-localization of zonula occludens (ZO)-1 to intercellular areas. These effects were suppressed by inhibitors of GPR40 and phospholipase C (PLC). Interestingly, GW9508 enhanced tight junction assembly in an AMP-activated protein kinase (AMPK)-dependent manner. The effect of GW9508 on inducing tight junction assembly was also confirmed in 16HBE14o- cells. Our results indicate that GPR40 stimulation by GW9508 leads to AMPK activation via calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Collectively, this study reveals an unprecedented role of GPR40 in facilitating airway epithelial tight junction assembly via PLC-CaMKKβ-AMPK pathways. GPR40 represents a novel regulator of airway epithelial integrity and its stimulation may be beneficial in the treatment of airway diseases.

KEYWORDS:

• AMPK
• GPR40
• airway epithelial cells
• polyunsaturated fatty acids
• tight junctions

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by the Thailand Research Fund through Mahidol University (grant BRG5980008) and Dean Research Novice Award from the Faculty of Medicine Ramathibodi Hospital. Financial support from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0113/2557) to A.M. and C.M. is acknowledged.