Pabelick, C.M.

Abstract Rationale: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and cellular chemical sensor. AhR maintains cellular homeostatsis in respons to endogenous and exogenous organic molecules. Airway smooth muscle (ASM) cells are major structural cells in airway which is involved in characteristic features of asthma such as increased airway wall thickness, airway remodeling and airway hyperresponsiveness. Previously, we found notable expression of AhR in human ASM and its increased expression during inflammation and asthma. However, the mechanisms of enhanced AhR expression in ASM is unknown. Therefore, we examined the role of pro-inflammatory signaling pathways in enhanced AhR expression. Methods: Primary human non-asthmatic and asthmatic ASM cells isolated from surgical lung resections (Mayo IRB approved) were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. At ∼80% confluency, cells were serum deprived and treated with TNFα (20 ng/mL), IL13 (50 ng/mL) with or without PI3K/Akt inhibitor (Wortmannin: 50 nM), p38 inhibitor (SB203580: 600 nM), NFkB inhibitor (SN50 20 μM), and AP1 inhibitor (SR11302 10 μM). Subsequently, expression of AhR was determined using RNA sequencing, confocal microscopy, real-time qPCR, and Western blotting. AhR expression in isolated ASM fron lung tissue section was determined using laser capture microdissection (LCM). Moreover, role of AP1 in inducing AhR expression was confirmed using c-JUN (AP1) siRNA transfection in ASM cells. Additionally, binding of AP1 to the AhR promoter region was demonstrated by chromatin-immunoprecipitation (ChIP-qPCR) technique. Results: AhR expression was significantly high in asthmatic ASM cells and tissue than non-asthmatics. Exposure to pro-inflammatory cytokine TNFα increased AhR mRNA and protein expression, whereas IL-13 failed to do so. Furthermore, inhibition of p38 significantly reduced AhR expression whereas PI3K inhibition had no effect, indicating a role for p38 in AhR increased expression. AP1 inhibition significantly downregulated AhR, while NFkB inhibition showed no effect. Similarly, siRNA mediated silencing of c-JUN (AP1) also downregulated AhR, indicating AP1 role in AhR upregulation. Furthermore, transcription factor binding study suggested AhR promoter region has binding site for AP1 and NFkB. Interestingly, ChIP-qPCR study confirmed binding of AP1 at AhR promote region in response to TNFα. Conclusions: Collectively, the current study show that pro-inflammatory cytokine TNFα increases AhR expression through p38-AP1 pathway. The data shows that pro-inflammatory cytokines can regulate AhR expression in ASM, suggesting AhR in ASM has a role in asthma.