Sathish, Venkatachalem

Background and PurposeThe aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor that maintains cellular homeostasis. AhR in airway fibroblasts, epithelial and immune cells inhibit inflammatory responses. Nevertheless, its expression and role in airway smooth muscle (ASM), an airway structural cell indispensable in asthma pathophysiology, are obscure. This study uncovers AhR expression, underlying mechanisms, and activity in ASM during inflammation and asthma.Experimental ApproachCultured primary human nonasthmatic and asthmatic ASM cells were treated with TNFα/IL‐13, with/without pharmacological inhibitors targeting PI3K, p38MAPK, JNK, NFkB, and AP1. AhR expression was analysed using RNA‐sequencing, confocal microscopy, qPCR, and immunoblotting. AP1 specific role was confirmed using c‐JUN (AP1) siRNA and ChIP‐qPCR. AhR activity was determined by Nano luciferase in AhR agonist‐treated cells.Key ResultsWe found ubiquitous expression of AhR in ASM with up‐regulation in asthmatic ASM. TNFα increased AhR expression, whereas IL‐13 did not. Furthermore, p38 and JNK inhibition significantly reduced AhR expression with TNFα exposure, whereas PI3K inhibition had no effect. AP1 inhibition and c‐JUN knockdown significantly down‐regulated AhR expression, whereas NFkB inhibition showed no effect. TNFα promoted c‐JUN binding to AhR promoter and increased AhR mRNA expression. Additionally, AhR agonists significantly increased the xenobiotic response element (XRE) activity, CYP1B1, and AhR nuclear expression. TNFα exposure reduced XRE activity and AhR nuclear expression.Conclusion and ImplicationsOur findings suggest inflammation and asthma transcriptionally up‐regulates AhR through the p38/JNK‐AP1 pathway in ASM, identifying a potential therapeutic target for modulating AhR and its downstream effects in asthma.

Abstract Nicotine exposure in the context of smoking or vaping worsens airway function. Although nicotinic acetylcholine receptors (nAChRs) are commonly thought to exert effects through the peripheral nervous system, we previously showed that airway smooth muscle (ASM) expresses them, particularly α7 subtype nAChR (α7nAChR), with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using ASM cells from people without asthma versus people with mild to moderate asthma, we tested the hypothesis that nAChR-specific endoplasmic reticulum (ER) chaperones, resistance to inhibitors of cholinesterase 3 (RIC-3) and transmembrane protein 35A (TMEM35A) promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild to moderate asthma and exposure to proinflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, intracellular Ca2+ regulation, and ASM cell proliferation. Overall, our data highlight the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM toward airway contractility and remodeling.

This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

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.

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.

Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role for estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. We hypothesize that kisspeptin (Kp)/KISS1R signaling serves this role. Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We explored the role of Kp/KISS1R in regulating ASM proliferation. We report potentially novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10, mitigated PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The antiproliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G 0 /G 1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM proliferation and a potential therapeutic avenue to blunt remodeling in asthma.

AbstractAirway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain‐derived neurotrophic factor (BDNF). The glial cell‐derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+]i. Serum‐deprived ASM cells from non‐asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura‐2 loaded cells, acute GDNF or NRTN alone induced [Ca2+]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1‐Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1‐Fc chelator markedly decreased [Ca2+]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine‐induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

The incidence, severity, and mortality of ongoing coronavirus infectious disease 19 (COVID-19) is greater in men compared with women, but the underlying factors contributing to this sex difference are still being explored. In the current study, using primary isolated human airway smooth muscle (ASM) cells from normal males versus females as a model, we explored the effect of estrogen versus testosterone in modulating the expression of angiotensin converting enzyme 2 (ACE2), a cell entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using confocal imaging, we found that ACE2 is expressed in human ASM. Furthermore, Western analysis of ASM cell lysates showed significantly lower ACE2 expression in females compared with males at baseline. In addition, ASM cells exposed to estrogen and testosterone for 24 h showed that testosterone significantly upregulates ACE2 expression in both males and females, whereas estrogen downregulates ACE2, albeit not significant compared with vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)‐β, which upon activation down‐regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER‐α vs. ER‐β activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E 2 , an ER‐α agonist [propylpyrazoletriol (PPT)], and an ER‐β agonist [WAY‐200070 (WAY)] with TNF‐α or platelet‐derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT‐PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP‐1) and NF‐κB. TNF‐α or PDGF significantly ( P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF‐α‐ or PDGF‐induced ECM gene expression in ASM cells was significantly reduced with WAY ( P < 0.001). Moreover, WAY significantly down‐regulated the activation of NF‐κB( P < 0.001) and AP‐1 ( P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER‐β diminishes ECM deposition via suppressing the NF‐κB pathway activity and might serve as a novel target to blunt airway remodeling.—Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen‐receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF‐κB pathway. FASEB J. 33, 13935‐13950 (2019). www.fasebj.org