A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14

Abstract

TAS2Rs (bitter taste receptors) are GPCRs (G protein–coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical G βγ -mediated stimulation of Ca 2+ release from intracellular stores by activation of PLCβ (phospholipase Cβ). Alone, this [Ca 2+ ] i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at G q -coupled receptors also increase [Ca 2+ ] i . We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCβ that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca 2+ ] i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca 2+ ] i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery

“…Drugs that inhibit pathways underlying contraction promote relaxation of pre-constricted airways [150,155,162,168,178,180,[203][204][205][206][207], and testing of selective molecules that either elicit or enhance bronchodilation have been used in PCLS [81,[208][209][210][211][212]. Other non-canonical pathways have also been targeted to reverse, or inhibit, airway contraction (specific studies listed in Table 4) [155,165,202,[213][214][215][216][217][218][219][220][221][222][223][224][225][226][227][228]. Compounds targeting orphan, or non-β 2 receptors, can serve as new classes of bronchodilators, or have been suggested to be useful as add-on therapy for existing therapeutics [164,165,[214][215][216][217][218][219]…”

“…Despite the limitations of mouse models in the study of airways diseases, rodent strains offer the opportunity to genetically modify the animal to assess the function of cell-specific gene deficiency on the development and progression of lung diseases. Two studies have utilized siRNA-mediated knockdown of specific transcripts in human PCLS [ 225 , 270 , 271 ], but the siRNA used in each study was not cell-type specific. Despite the limitations of working with animal model systems, these platforms can augment our understanding of disease pathogenesis when combined with observations using human cells and tissues.…”

“…To understand how histone modifications alter inflammation of the lung, the histone acetyltransferase inhibitor MG149 was found to attenuate LPS and Interferon γ (IFNγ)-stimulated proinflammatory gene expression [ 283 ]. Others have also optimized siRNA-mediated knockdown of multiple gene targets to decrease protein expression [ 225 , 270 , 271 ], with one of the studies also assessing the functional effects of the knockdown [ 225 ]. Receptor localization has also been studied in the epithelium of the airways by fluorescent microscopy following adenoviral transduction of hPCLS following desensitization of the β 2 AR [ 199 ].…”

Unlocking TAS2R14 activation through intricate multi-ligand binding networks

“…Notably, its activation in ASM induces bronchodilation, surpassing the effects triggered by β2-adrenergic receptor 13 . Consequently, TAS2R14 emerges as a potential therapeutic target for conditions like asthma 14 or chronic obstructive pulmonary disease (COPD) 15 . For taste perception, TAS2Rs typically couple to the G protein gustducin 16,17 , leading to increased intracellular calcium.…”

Demystifying Bitter Taste Receptor Relaxation of Airway Smooth Muscle

“…Woo and colleagues (pp. 417–429 ) provide evidence to suggest that agonist activation of TAS2R14 (the most abundant TAS2R in HASM) affects the polarity protein Par3, which inhibits the LIMK-cofilin pathway and induces F-actin severing and HASM relaxation ( 8 ). Smooth muscle contraction/relaxation is mainly regulated by two pathways: Myosin activation and actin cytoskeletal reorganization ( 9 – 13 ).…”