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A7266 - Role of KISS1/GPR54 Signaling in PDGF Induced Human Airway Smooth Muscle Remodeling
Author Block: R. Kalidhindi1, N. S. Ambhore1, M. Thompson2, C. M. Pabelick2, Y. Prakash2, S. Venkatachalem1; 1Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, United States, 2Departments of Anesthesiology and Perioperative Medicine, and Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States.
Rationale: Increased airway smooth muscle (ASM) mass and changes in extracellular matrix (ECM) contribute to airway remodeling in asthma. In addition to ECM proteins, ASM cells also produce a range of matrix metalloproteinases (MMP’s) and adhesion molecules that are further modulated by cytokines. Recent studies in cancer suggest that KISS1 receptor (KISS1R/ GPR54) is involved in metastasis suppressing function and influences NFκB and p38 MAPK signaling pathways. It was originally identified as a key protein regulating GnRH release in female puberty, suggesting a potential sex-specific role. GPR54 has been found to downregulate proliferation and migration in some cell types. There is currently no information on KISS1 receptors in asthma but its potential relevance lies in the fact that GPR54 is influenced by estrogen receptor signaling and there are sex differences in asthma. In the present study, we tested the hypothesis that KISS1R signaling plays a role in airway remodeling of asthma. Methods: Asthmatic and non-asthmatic primary human airway smooth muscle cells isolated from 3rd to 6th generation bronchi of surgical lung resections were cultured in DMEM-F12 (approved by Mayo Clinic IRB). Cells were serum deprived for 24h before initiating experiments. Immunofluorescence analysis (Olympus FV500 confocal microscope) was used to determine expression and subcellular localization of KISS1R/GPR54. ASM cells exposed to pro-inflammatory cytokines (TNFα, 20ng/mL or IL-13 50ng/mL) were subjected to Western analysis to quantify expression of KISS1R. Cell proliferation assay using MTT and Lionheart FX cell counting were performed in the presence of PDGF (2ng/mL) followed by 24h treatment with the endogenous KISS1R ligand metastin (1µM). Results: KISS1R/GPR54 levels were significantly decreased in asthmatic human ASM cells compared to non-asthmatics. The protein was localized to the plasma membrane. Furthermore, TNFα and IL-13 significantly upregulated GPR54 in both asthmatic and non-asthmatic ASM cells compared to vehicle. Interestingly, expression of GPR54 was greater in cells exposed to IL-13 compared to TNFα. In addition, PDGF-induced ASM cell proliferation was significantly decreased by metastin compared to PDGF alone. This was further confirmed by expression of proliferative markers such as PCNA and Cyclin-D. Conclusion: Expression of GPR54 is altered during asthma and inflammation in human ASM cells. Metastin, a GPR54 agonist reduces PDGF stimulated proliferation of human ASM cells. Thus, KISS1/GPR54 signaling may play an important role in airway remodeling during asthma.