Sathish, Venkatachalem

Abstract In asthma, pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) drive excessive mitochondrial fission in ASM cells through activating dynamin-related protein 1 (DRP1). Recently, we demonstrated that aryl hydrocarbon receptor (AhR) is expressed in human ASM and is upregulated during inflammation and asthma. This study explores the role of AhR in regulating mitochondrial fission in human ASM cells under pro-inflammatory and asthmatic conditions. Primary human nonasthmatic and asthmatic ASM cells were treated with 6-formylindolo[3,2-b]carbazole (FICZ: AhR agonist), with or without TNFα. Mitochondrial morphology was assessed using MitoTracker staining. DRP1 expression was evaluated in whole-cell and mitochondrial fractions. Loss- and gain-of-function studies (AhR inhibition, knockdown, and overexpression) were performed. AhR binding on the DRP1 promoter and promoter activity were assessed by ChIP-qPCR and luciferase reporter. The effects of AhR activation on oxygen consumption rate (OCR) were analyzed using seahorse XF-Pro. AhR activation significantly inhibited TNFα- and asthma-induced mitochondrial fission in ASM cells via inhibiting DRP1 in both inactive and active forms. In contrast, AhR inhibition or knockdown aggravated mitochondrial fission, while AhR overexpression failed to prevent TNFα-induced fission without ligand activation. Mechanistically, AhR bound the DRP1 promoter and suppressed its promoter activity, consistent with a genomic mode of action, while failing to alter ERK1/2 phosphorylation. Additionally, AhR activation also reduced TNFα- and asthma-induced increase in OCR. Collectively, this study shows that AhR activation prevents mitochondrial fission by inhibiting DRP1 in ASM during inflammation and highlights AhR as a promising therapeutic target for asthma and other airway diseases associated with mitochondrial dysfunction.

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.

Introduction: Throughout the lifetime of an asthmatic, the lung undergoes changes in the composition of its extracellular matrix (ECM) and becomes more fibrotic, a characteristic feature of airway remodeling. Due to this, lung functionality decreases as the lung becomes less elastic and airway resistance increases. The ECM proteins constantly network with their respective mechanosensory receptors and regulatory proteins on the cell membrane. These signaling interactions allow for continuous interactions and feedback between the ECM and airway smooth muscle cells (ASM). ASM cells are one of the major structural cells that regular airway remodeling and are the primary target for treating asthma. In this study, we investigated the specific alterations that occur in the ECM of ASM cells treated with TGF-β1. Methods: Human primary airway smooth muscle (ASM) cells were grown to confluency in DMEM/F-12 with 10% FBS and 1% AbAm in standard conditions. Cells were serum deprived for 48 hours prior to experiments. Cells were treated with TGF-β1 (2 ng/mL) in serum free media. Cells were harvested after 48 hours and 200 µg of protein lysate was prepared. Data-independent acquisition (DIA) mass spectrometry was performed at IDeA National Resource for Quantitative Proteomics. Fold changes for ECM class proteins were collected from DIA results. Pathway analysis was then done using Qiagen ingenuity pathway analysis. Furthermore, expression patterns of select proteins were confirmed via western blotting. Results: The effects of TGF-β1 on specific types of collagens, integrins, laminins, and additional ECM proteins were analyzed. Western blotting confirmed similar changes in expression as measured by DIA. TGF-β1 exposure upregulated LAMA4, LAMB2, and LAMC1 which is likely to increase the prevalence of Laminins 221 and 421. TGF-β1 augmented integrin signaling by upregulating ITGA7, ITGA11, ITGA1, ITGB1 and downregulating ITGA2 and ITGB8. TGF-β1 increased expression of TSP1, TSP2, TSP3, TNS-C, FB, Elastin and Versican while downregulating TNS-X. Several interactions between ECM proteins and their sensors and receptors were highlighted for their enrichment. Conclusion: The DIA analysis shows an upregulation of proteins involved in airway remodeling by TGF-β1 in ASM cells. These findings substantiate our previous results as well as provide insights into a more targeted approach in studying ECM-Cell interactions. NIH R01HL171245 (Venkatachalem), R01HL146705 (Venkatachalem), R01HL142061 (Pabelick, Prakash). and IDeA National Resource for Quantitative Proteomics (R24GM137786). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rationale: Asthma is a chronic inflammatory disease of conducting airways characterized by airway hyperresponsiveness and remodeling. Airway smooth muscle (ASM) is a major structural cell in the airway that contributes to airway hyperresponsiveness and remodeling. Mitochondrial dynamics is a fission/fusion process crucial for optimal function and signal transduction. Inflammation alters the mitochondrial dynamics in ASM, thereby disrupt mitochondrial function and contributes to asthma pathophysiology. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cellular homeostasis. Recently, we found upregulation of AhR in response to inflammation and asthma in ASM. Additionally, AhR activation inhibited inflammation-induced ASM proliferation and remodeling. However, the role of AhR in regulating mitochondrial dynamics in ASM during inflammation and asthma is obscure. Objective: The aim of this work is to unveil the role of AhR in regulating mitochondrial structure and function in human ASM during inflammation and asthma. Methods: Primary non-asthmatic and asthmatic human ASM cells isolated from surgical lung sections were cultured in DMEM-F12 medium supplemented with 10% FBS and 1% AbAm. Cells were serum deprived, treated with AhR agonist 6-Formylindolo[3,2-b]carbazole (FICZ: 10 nM) and antagonist CH223191 (10 μM), with/or without TNFα (20 ng/mL). Mitochondrial bioenergetics was determined by Seahorse XFp analyzer. Mitochondrial morphology was visualized using mitotracker dye. Expressions of fission and fusion mRNA and protein were determined by qPCR and immunoblotting. Additionally, AhR role in fusion and fission was confirmed in shRNA mediated AhR knockdown and AhR overexpressed ASM cells. Results: AhR activation by FICZ alleviated TNFα-induced disrupted mitochondrial bioenergetics in non-asthmatic ASM cells as well as in asthmatic ASM. Furthermore, TNFα-induced increased mitochondrial fission and reduced fusion was restored by AhR activation in both asthmatic and non-asthmatic ASM. AhR inhibition by CH223191 and knockdown promoted mitochondrial fission and blunted fusion in non-asthmatic ASM cells. Interestingly, AhR overexpression and activation by FICZ restored the mitochondrial dynamics altered by inflammation and asthma in ASM cells. Conclusion: Collectively, AhR activation promotes mitochondrial fusion and abolishes fission induced by inflammation and asthma in human ASM, thereby maintains mitochondrial structure and homeostasis. Therefore, AhR is a potential therapeutic target to mitigate altered mitochondrial dynamics in ASM during inflammation and asthma. Supported by NIH grants R01-HL171245 (Venkatachalem and Britt), R01-HL146705 (Venkatachalem), R01-HL155095 (Britt), R01-HL142061 (Pabelick, Prakash) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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.

Abstract Background: Kisspeptins (Kp) are a group of hypothalamic peptides that aid in regulating pubertal development and sex hormone signaling. In our recent studies, we observed the important role of Kp-10 (Kp peptide) and its receptor KISS1R signaling in regulating the airway smooth muscle (ASM) structure and function. In these studies, we observed significantly reduced expression of KISS1R during asthma pathophysiology suggesting a loss of protective mechanism and exogeneous Kp-10 may be protective in the context of asthma. Studies have shown that specific modifications to the amino acids of Kp peptides can result in increased binding efficiency and receptor activation. Accordingly, we developed three novel Kp peptides, named KpSATs (13A, 13B, and 13C), and tested the potential effects/efficiency on ASM cell remodeling. Methods: The Molecular docking of the KpSAT peptides (13A, 13B and 13C) with the receptor KISS1R were performed using HPEPDOCK. KpSATs were synthesized by GenScript (New Jersey, US) with purity of ≥97%. Primary human ASM cells isolated from surgical lung resections (Mayo Clinic IRB-approved) were cultured in DMEM-F12. Cells were treated with PDGF (2ng/mL) and/or TGF-β (2ng/mL) with/without treatments of KpSATs (13A, 13B, and 13C: 250nM-1µM) and Kp-10 (1µM). ASM cell proliferation was measured using MTT. Extracellular matrix (ECM) deposition (fibronectin and collagen-I/III) studies were performed using In-Cell Western (LiCor-odyssey). The mechanistic signaling pathway (phospho-PKC-δ) were measured using Western blot analysis. Results: In the novel KpSATs, we substituted three amino acids in the endogenous Kp-13 sequence, Y10W and P12T for KpSAT-13A; S6A and P12T for KpSAT-13B; and S6A and Y10W for KpSAT-13C. The peptides dock in the orthosteric ligand binding pocket attained with docking scores ranging from -250 to -280. The MTT assay indicated a reduction in PDGF-induced ASM cell proliferation upon exposure to KpSATs. Notably, we found that KpSAT-13A and KpSAT-13B had significant effects in reducing proliferation at lower concentrations of 500nM compared to 1µM of Kp-10. Furthermore, ECM deposition studies showed a significant reduction of TGF-β1-induced collagen-I/III and fibronectin in ASM cells exposed to KpSAT-13A and 13B. In addition, we observed significant inhibition of PDGF-induced phospho-PKC-δ by KpSAT-13A and 13B. Conclusion: Overall, these findings highlight that modification in amino acid sequence in endogenous Kp-13 sequence significantly increases the potency of synthesized novel KpSATs on ASM cells. In addition, KpSATs showed better inhibitory effects with a lower concentration compared to Kp-10 and could be considered as a promising therapeutic strategy to mitigate asthma.

Abstract Rationale: Within the asthmatic lung, inflammatory signaling alters the contractile capacity of airway smooth muscle (ASM). In chronic inflammation, ASM become hypercontractile and are a key contributor toward airway hyperresponsiveness (AHR). As a heterogenous disorder, several asthmatic endotypes have been defined. A severe ASM endotype is partly defined as being glucocorticoid insensitive. We have reported the role of Kisspeptin (Kp)/KISS1R signaling in the lung in the context of airway fibrosis. In this study, KISS1R and Kp-10 were investigated for a potential interactions with intracellular calcium [Ca2+]i signaling and ASM contractility in the in vitro setting of severe asthmatic endotypes. Methods: Primary ASM were obtained from non-pathological samples from the Mayo Clinic. ASM cells were treated with inflammatory cytokines (TNFα 20 ng/ml, or IL-13 50 ng/mL)± Kp-10 (1 μM). The steroid-insensitive asthma endotype was investigated using pro-inflammatory cytokines (TNFα 20 ng/ml and IFN-γ 25 ng/mL) ± a corticosteroid (Fluticasone Propionate 100 nM). Β2-Adrenergic Receptor (β2-AR) desensitized endotypes were developed by treating cells with a β2-AR agonist (Salbutamol 1 μM) for 24 hours prior to and along with cytokine treatments (TNFα 20 ng/ml and IFN-γ 25 ng/mL or IL-13 50 ng/mL) ± Kp-10. [Ca2+]i was measured via Fura-2AM (4 μM) labeling on an Olympus Fluoview 300 microscope. Myosin light chain (MLC) phosphorylation studies were used to quantify downstream mechanics of ASM contractility. Results: KISS1R activation in IL-13 treated cells reduced [Ca2+]i in response to histamine. In contrast, TNFα treated cells were responsive to Kp-10 for acute treatments, however this effect was lost after 24 hours of Kp-10 treatment. TNFα and IFN-γ treated cells showed a robust [Ca2+]i response to histamine, and both fluticasone (∼20% reduction) and Kp-10 (∼10% reduction) alone had a minimal effect in reducing [Ca2+]i. Fluticasone and Kp-10 co-exposure significantly reduced [Ca2+]i and significantly reduced the effect of TNFα/IFN-γ (∼45% reduction). In B2-AR desensitized cells, Kp-10 exposure maintained the effect of reducing [Ca2+]i, suggesting Kp-10/KISS1R signaling works through an alternate or independent pathway. Kp-10 treatments on cytokine exposed cells reduced p-MLC in response to histamine, suggesting a role in ASM contractility. Conclusion: The increase of [Ca2+]i in inflamed cells is associated with AHR; Kp-10/KISS1R signaling showed varying efficacy in reducing [Ca2+]i within inflammatory endotypes. These results also highlight that Kp-10 acts synergistically with the current therapeutics available for 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.