Varadharajan, S.

Abstract Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Background: Multiple asthma phenotypes are driven by pathobiological heterogeneity. Almost 25% of asthma patients do not respond well to current treatments like inhaled corticosteroids, urging researchers and clinicians to propose novel treatment targets and strategies. Orphan type C G-protein coupled receptor GPRC5B is one such novel target abundantly expressed in various tissues, but there are no known ligands for GPRC5B. Previously, we demonstrated that orphan type C G-protein coupled receptor GPRC5B is increasingly expressed in human asthmatic airways and plays a crucial role in regulating intracellular calcium ([Ca2+]i) in human airway smooth muscle (ASM) cells. However, the role of GPRC5B activation on airway contraction is not identified yet. Our objective for this study is to design and synthesize a novel ligand for GPRC5B and determine the effect of GPRC5B activation on [Ca2+]i responses in ASM cells and airway contractility in ex vivo murine airways. Methods: ZincPharmer was used for pharmacophore search. After energy minimization, the top hit compound was synthesized using a standard amide coupling reaction, purified by flash chromatography, and characterized by NMR spectroscopy. Human ASM cells loaded with Fura-2 AM were imaged for [Ca2+]i responses. Silencer select siRNA was used for transient silencing of GPRC5B in ASM cells. Myosin light chain (MLC) phosphorylation was measured using immunoblot analysis. Precision-cut lung slices (PCLS) (~200 μm thickness) of wild-type C57BL6 mice were used for contractile studies. Results: Based on molecular docking data, the top compound N-(5-chloro-2-hydroxybenzyl)-2-((tetrahydro-2H-pyran-4-yl)oxy) acetamide (L1) was synthesized. Concentration-dependent [Ca2+]i responses were observed on human ASM cells stimulated with L1 (1 nM - 1 μM) that were comparable to the known contractile agonist histamine (10 μM). L1-induced [Ca2+]i response was significantly reduced in GPRC5B knockdown ASM cells. GPRC5B knockdown in ASM cells was verified by Western analysis. Further, L1 exposure increased the MLC phosphorylation (Thr18/Ser19) in human ASM cells compared to vehicle. Acute exposure of L1 (1 μM) increased airway contractility of PCLS (~17%-25%) compared to vehicle exposure. Conclusion: These findings suggest that GPRC5B activation modulates intracellular calcium and airway contractility in human ASM cells and murine PCLS. Thus, GPRC5B could potentially be considered for a novel therapeutic regimen to tackle asthma. This work was partly supported by NIH grants R01-HL146705 (Venkatachalem), R01-HL088029 (Prakash), and R01-HL142061 (Pabelick, Prakash). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.