John M. Lim's research while affiliated with Thomas Jefferson University and other places

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors (GPCRs) causes airway hyperresponsiveness (AHR) in asthma. Activation of Gq-coupled GPCRs leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKα and ζ isoform knockout mice are protected from the development of allergen-induced AHR. Here we aimed at determining the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζwhile pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Further, we employed human precision-cut lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pre-treatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and myosin light chain phosphatase in ASM cells. Further, DGK inhibition decreased Gq agonist-induced calcium elevation, generation of IP3, and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β‐agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β‐agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c‐Abl). The cooperative effect of β‐agonists (isoproterenol) and c‐Abl inhibitors (GNF‐5, or imatinib) on contractile agonist (methacholine, or histamine) ‐induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c‐Abl inhibitors and β‐agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c‐Abl inhibitors and β‐agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β‐agonists is near optimal and is not increased by c‐Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β‐agonists and c‐Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.