Deshpande, Deepak A.

Penn, Raymond B.

Jefferson Digital Commons

Thomas Jefferson University Jefferson Digital Commons Center for Translational Medicine Faculty Papers Center for Translational Medicine 12-1-2023 Reactive Oxygen Species Behaving Badly: Oxidized Phosphatidylcholines Corrupt Ca2+ Signaling in Airway Smooth Muscle Deepak A. Deshpande Raymond B. Penn Follow this and additional works at: https://jdc.jefferson.edu/transmedfp  Part of the Translational Medical Research Commons Let us know how access to this document benefits you This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Center for Translational Medicine Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu. EDITORIALSReactive Oxygen Species Behaving Badly: OxidizedPhosphatidylcholines Corrupt Ca21 Signaling in AirwaySmooth MuscleIncreasing evidence points to oxidative stress affecting multiple celltypes as an important driver of obstructive lung diseases. Reactiveoxygen species and reactive nitrogen species damage lipid and proteincomponents of extracellular fluids, cell membranes, organelles, andthe cell cytosol. Oxidized phosphatidylcholines (OxPCs) are amongthe bioactive agents generated by oxidation of the unsaturated fattyacyl chain, leading to hundreds of potential fragmented and cyclizedvariants and breakdown products such as malondialdehyde (1).Pascoe and colleagues previously reported that OxPCs disruptmitochondrial metabolic activity in human airway epithelial cells topromote reactive oxygen species generation, reduce cell viability, andimpair epithelial barrier function and barrier recovery after a wound(2). More recently, the same group identified specific OxPCs thatassociate with airway hyperresponsiveness in BAL samples fromasthmatic cohorts, thus establishing OxPCs to be effectors ofoxidative stress in asthma (3). Moreover, treatment of human airwaysmooth muscle (ASM) cells with a subset of OxPCs (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholines[OxPAPCs]) induced the expression of multiple inflammatorycytokines and oxylipins, suggesting a proinflammatory role of OxPCsin asthma. In addition, OxPAPCs caused airway narrowing in murineprecision-cut lung slices, consistent with the correlation of OxPAPCexpression and airway hyperresponsiveness in human subjects.In this issue of the Journal, Vaghasiya and colleagues (pp. 649–665)provide mechanistic insight into the procontractile effect of OxPCs onASM (4). Employing human ASM cultures, murine lung slices, andhuman lung tissue sections, they dissect the sources of intracellularcalciummobilization by OxPCs in ASM and causally link them toASM contraction (Figure 1). Specifically, they demonstrate that, inASM cells, OxPAPCs induce an immediate and sustained Ca2þ flux.The former is independent of extracellular Ca2þ and does not involveinositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), but does involveryanodine receptors (RyRs) and cyclic ADP ribose. The latter requiresextracellular Ca2þ as well as TRPA1 (transient receptor potentialankyrin 1), whose expression was verified in human ASM.From a functional perspective, this study describes the interesting(and somewhat surprising) discovery that inhibition of peak orsustained Ca2þ by RyR or TRPA1 inhibition, respectively, wassufficient to inhibit OxPC-induced ASM 20-kD regulatory myosinlight chain phosphorylation andmurine precision-cut lung slicecontraction. Further experiments revealed that peak and sustainedCa2þ induction were not interdependent, as approaches that inhibiteach fail to significantly affect the other. The unique observation thatinhibition of peak or sustained Ca2þ alone is sufficient to inhibitOxPC-stimulated contraction demonstrates that these phases ofmobilized Ca2þ are cooperative and are both required to induce ASMcontraction. Interestingly, features of Ca2þ induction stimulated byOxPCs are qualitatively distinct from those promoted by theGq-coupledM3muscarinic acetylcholine receptor. For example, IP3Rinhibition prevents Ca2þ flux stimulated by acetylcholine but not byOxPCs, whereas RyR inhibition affects peak Ca2þ by OxPCs but notby acetylcholine. TRPA1 activity induced by OxPAPCs is not a resultof the release of RyR-regulated intracellular Ca2þ pools.A wide variety of molecules that are spatially and temporallydistinct elicit Ca2þ signals in airway cells (5). For example, localizedCa2þ increases remain confined whereas other Ca2þ signals graduallydiffuse to form Ca2þ waves or oscillations. Each of these patternsinvolves distinct cellular Ca2þ sources and downstream effectors toeffect specific cellular functions. Previous studies in ASM cells havedemonstrated distinct patterns of Ca2þ increases (oscillations,transient, biphasic, sparks), subcellular localizations (mitochondria,cytosolic), and sources of Ca2þ (IP3R, RyR, VDCC, and SOCE), albeitusing ionotropic andmetabotropic ligands (6–9). Vaghasiya andcolleagues demonstrate that oxidized lipids, OxPCs, induce Ca2þsignals via distinct sources of Ca2þ in ASM cells. These findings furtherunderscore the complex nature by which the spatial and temporalfeatures of intracellular Ca2þ affect the ASM contractile state.Classical pharmacomechanical coupling by which procontractileG protein–coupled receptors (GPCRs) cause ASM contractioninvolves increases of Ca2þ and phosphorylation of myosin light chain.Most importantly, Gq-coupled GPCR-mediated Ca2þ increase isinitiated with the production of IP3 and the release of Ca2þ via IP3R onsarcoplasmic reticulum, with a subsequent activation of RyR channelsmediated via Ca2þ or cyclic ADP ribose (6, 10). Uniquely, OxPC-induced Ca2þ increases do not involve IP3Rs. Moreover, studies duringthe past decade note diverse patterns of Ca2þ increases that can becoupled or decoupled fromASM contraction and, in some instances(e.g., activation of bitter tastant–stimulated receptors), cause ASMrelaxation (11, 12). Although the details of which spatiotemporalfeatures of intracellular Ca2þ regulate effectors of ASM contractionremain poorly understood, advances in microscopic/analytical toolsand the development of genetically engineered calcium indicators (13)will likely enable mechanistic insight in the near future. Geneticapproaches involving deletion of Ca2þ sensing proteins and channels(TRP/STIM1) andmolecular description of posttranslationalmodifications of Ca2þ channels and transporters by OxPCs are critical.This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0.For commercial usage and reprints, please e-mail Diane Gern.Supported by grants HL137030, HL146645, and HL150560 (D.A.D.) and by National Heart, Lung, and Blood Institute grant HL58506, NationalInstitute of Allergy and Infectious Diseases grant AI161296, and grants HL136209, HL169522, HL145392, and HL114471 (R.B.P.).Originally Published in Press as DOI: 10.1165/rcmb.2023-0295ED on September 6, 2023Editorials 605 Given the dependence on reductionist cell-based models toexplore the mechanisms mediating ASM contraction by OxPCs,insight into the effect of OxPCs under integrative (and chronic)conditions will require future studies. OxPCs in vivowill undoubtedlyaffect multiple resident airway cells, as well as the interactions amongresident and infiltrating cells, in the context of allergic inflammation.Moreover, it is likely that OxPCs will influence airway remodelingeffected by allergic inflammation. Finally, whether OxPCs affectreceptor-mediated ASM contraction (e.g., M3 muscarinicacetylcholine receptor) or relaxation (e.g., b-2 adrenoceptor) isanother important question; it is likely that various GPCRs, andpossibly their downstream signaling partners, are directly modified byOxPCs, similar to modifications such as protonation andnitrosylation that occur during inflammation and are believed toinfluence airway function and disease (14–16).In summary, Vaghasiya and colleagues contribute to a growingbody of evidence indicating that molecules generated as the result ofoxidative stress in the lung play a role in asthma. Thus, OxPCs couldrepresent a component of persistent inflammation that preventsoptimized control in many patients. In this light, a focus on fullyextrapolating the pathobiological effects of OxPCs and otheroxidative stress mediators in the lung may yield some unexpectednew avenues for future therapeutic approaches. Oral antioxidantssuch as N-acetylcysteine have not proven to be effective in clinicaltrials, but the data generated here indicate that a more targeteddelivery of specific inhibitors of the receptors and pathways activatedby mediators such as OxPCs may hold future promise. One of themost telling observations of the study by Vaghasiya and colleagues isthe finding that TRPA1 is activated by OxPCs. Notably, priorpreclinical studies have identified TRPA1 as a target to suppressallergic inflammation and bronchospasm, and this has led to thedevelopment of a TRPA1 inhibitor that is already being tested inphase I human trials (17). Thus, the work by Vaghasiya andcolleagues has identified a mechanism that is consistent with thetherapeutic potential of TRPA1 inhibition for asthma.Author disclosures are available with the text of this article atwww.atsjournals.org.Deepak A. Deshpande, Ph.D.Raymond B. Penn, Ph.D.Center for Translational MedicineandDepartment of MedicineThomas Jefferson UniversityPhiladelphia, PennsylvaniaORCID IDs: 0000-0002-6187-5332 (D.A.D.); 0000-0001-8452-5880 (R.B.P.).Ca2+Indirect activationTRPA1OxPAPC PAPCROSAllergens, VirusesSmokeEnvironmental pollutantsCa2+Ca2+Ca2+Ca2+Ca2+Ca2+ Ca2+CD38cADPRCa2+Ca2+ Ca2+pMLC20p* p*p* p* p*ContractionMyosin thick filamentNADRyRSarcoplasmic reticulumFigure 1. Intracellular signaling mechanisms for oxidized phosphatidylcholine–induced airway smooth muscle (ASM) contraction. Inhaledbiological and chemical environmental factors can overwhelm intrinsic antioxidant pathways, resulting in accumulation of ROS, which oxidizebiomolecules. This includes phosphatidylcholine (e.g., 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [PAPC]), which is abundant incell membranes and extracellular fluid such as lung surfactant. Oxidation generates myriad oxidized PAPC (OxPAPC) variants, which inducebronchial narrowing. In human ASM cells, OxPAPC triggers concomitant, but mutually exclusive, activation of the ryanodine receptor (RyR) andtransient receptor potential ankyrin 1 (TRPA1) that mediates flux of Ca21 from sarcoplasmic reticulum and extracellular stores, respectively,resulting in an acute and sustained increase in intracellular Ca21. OxPAPC may activate TRPA1directly or indirectly, e.g., by altering cellmembrane properties. RyR activation is dependent on cADPR, a product derived from NAD by cyclase activity of the ectoenzyme CD38, whichcan be induced by OxPAPC. These phases of mobilized Ca21 work cooperatively, as both are required to induce ASM contraction. Inhibition ofeither pool is sufficient to prevent pMLC20, which is essential for activation of actomyosin cross-bridge cycling, after OxPAPC exposure, as wellas for airway contraction in murine thin-cut lung slices. cADPR=cyclic ADP ribose; NAD=nicotinamide adenine dinucleotide;pMLC20=phosphorylation of 20 kDa myosin light chain; ROS= reactive oxygen species.EDITORIALS606 American Journal of Respiratory Cell and Molecular Biology Volume 69 Number 6 | December 2023 References1. Pascoe CD, Vaghasiya J, Halayko AJ. Oxidation specific epitopes inasthma: new possibilities for treatment. Int J Biochem Cell Biol 2020;129:105864.2. Pascoe CD, Roy N, Turner-Brannen E, Schultz A, Vaghasiya J, RavandiA, et al. Oxidized phosphatidylcholines induce multiple functionaldefects in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol2021;321:L703–L717.3. Pascoe CD, Jha A, Ryu MH, Ragheb M, Vaghasiya J, Basu S, et al.Canadian Respiratory Research N. Allergen inhalation generates pro-inflammatory oxidised phosphatidylcholine associated with airwaydysfunction. Eur Respir J 2021;57:2000839.4. Vaghasiya J, Dalvand A, Sikarwar A, Mangat D, Ragheb M, Kowatsch K,et al. Oxidized phosphatidylcholines trigger TRPA1 and ryanodinereceptor–dependent airway smooth muscle contraction. Am J RespirCell Mol Biol 2023;69:649–665.5. Prakash YS. Airway smooth muscle in airway reactivity and remodeling:what have we learned? Am J Physiol Lung Cell Mol Physiol 2013;305:L912–L933.6. Bai Y, Edelmann M, Sanderson MJ. The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca(21)signaling of airway smooth muscle cells. Am J Physiol Lung Cell MolPhysiol 2009;297:L347–L361.7. Chen J, Sanderson MJ. Store-operated calcium entry is required forsustained contraction and Ca21 oscillations of airway smooth muscle. JPhysiol 2017;595:3203–3218.8. Delmotte P, Yang B, Thompson MA, Pabelick CM, Prakash YS, SieckGC. Inflammation alters regional mitochondrial Ca21 in human airwaysmooth muscle cells. Am J Physiol Cell Physiol 2012;303:C244–C256.9. Prakash YS, Pabelick CM, Kannan MS, Sieck GC. Spatial and temporalaspects of ACh-induced [Ca21]i oscillations in porcine tracheal smoothmuscle. Cell Calcium 2000;27:153–162.10. Prakash YS, Kannan MS, Walseth TF, Sieck GC. Role of cyclic ADP-ribose in the regulation of [Ca21]i in porcine tracheal smooth muscle.Am J Physiol 1998;274:C1653–C1660.11. Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM,An SS, et al. Bitter taste receptors on airway smooth musclebronchodilate by localized calcium signaling and reverse obstruction.Nat Med 2010;16:1299–1304.12. Huang J, Lam H, Koziol-White C, Limjunyawong N, Kim D, Kim N, et al.The odorant receptor OR2W3 on airway smooth muscle evokesbronchodilation via a cooperative chemosensory tradeoff betweenTMEM16A and CFTR. Proc Natl Acad Sci USA 2020;117:28485–28495.13. Ji G, Feldman ME, Deng KY, Greene KS, Wilson J, Lee JC, et al.Ca21-sensing transgenic mice: postsynaptic signaling in smoothmuscle. J Biol Chem 2004;279:21461–21468.14. Fonseca FV, Raffay TM, Xiao K, McLaughlin PJ, Qian Z, Grimmett ZW,et al. S-nitrosylation is required for b2 AR desensitization andexperimental asthma. Mol Cell 2022;82:3089–3102.e7.15. Isom DG, Sridharan V, Baker R, Clement ST, Smalley DM, DohlmanHG. Protons as second messenger regulators of G protein signaling.Mol Cell 2013;51:531–538.16. Nayak AP, Deshpande DA, Shah SD, Villalba DR, Yi R, Wang N, et al.OGR1-dependent regulation of the allergen-induced asthma phenotype.Am J Physiol Lung Cell Mol Physiol 2021;321:L1044–L1054.17. Balestrini A, Joseph V, Dourado M, Reese RM, Shields SD, Rouge L,et al. A TRPA1 inhibitor suppresses neurogenic inflammation and airwaycontraction for asthma treatment. J Exp Med 2021;218:e20201637.EDITORIALSEditorials 607 

Reactive Oxygen Species Behaving Badly: Oxidized Phosphatidylcholines Corrupt Ca2+ Signaling in Airway Smooth Muscle

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Reactive Oxygen Species Behaving Badly: Oxidized Phosphatidylcholines Corrupt Ca2+ Signaling in Airway Smooth Muscle

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