TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia
Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca2+ ([Ca2+]i) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1−/−) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca2+]i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1−/− controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1−/− mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca2+]i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.
KeywordsTRPA1 mRNA expression Human lung tissues Murine lung tissues Hyperoxia Alveolar epithelial hyperplasia
We thank Bettina Braun, Benjamin Lukas Heinz, Christine Hollauer, Daniel Hofmann, and Heinz Janser for excellent technical expertise as well as Drs. Thomas Büch and Eva Schäfer for kindly providing the TRPA1 expressing HEK293 cell line. We are grateful for Dr. Jiong Zhang’s expertise in preparing dorsal root ganglia (DRG) and thank Dr. Claudia Staab-Weijnitz for her help in identifying lung epithelial cells.
M.K. and A.D. designed the study, R.L. and D.S. designed and performed the proteomic analysis, A.Ö.Y. designed and supervised the quantification of lung tissue sections, M.K. performed all other experiments, M.K. and A.D. analyzed the data, M.K., A.D., and T.G. wrote the manuscript, all authors edited and approved the manuscript.
Compliance with ethical standards
All animal experiments were approved by the local authorities (Regierung Oberbayern) and are in compliance with ethical standards.
Conflict of interest
The authors declare that they have no conflict of interest.
- 4.Buch T, Schafer E, Steinritz D, Dietrich A, Gudermann T (2013) Chemosensory TRP channels in the respiratory tract: role in toxic lung injury and potential as "sweet spots" for targeted therapies. Rev Physiol Biochem Pharmacol. https://doi.org/10.1007/112_2012_10
- 5.Buch TR, Schafer EA, Demmel MT, Boekhoff I, Thiermann H, Gudermann T, Steinritz D, Schmidt A (2013) Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants, in pulmonary epithelial cells. Chem Biol Interact 206:462–471. https://doi.org/10.1016/j.cbi.2013.08.012 CrossRefPubMedGoogle Scholar
- 6.Chubanov V, Ferioli S, Wisnowsky A, Simmons DG, Leitzinger C, Einer C, Jonas W, Shymkiv Y, Bartsch H, Braun A, Akdogan B, Mittermeier L, Sytik L, Torben F, Jurinovic V, van der Vorst EP, Weber C, Yildirim OA, Sotlar K, Schurmann A, Zierler S, Zischka H, Ryazanov AG, Gudermann T (2016) Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival. eLife 5:e20914. https://doi.org/10.7554/eLife.20914 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T, James JJ, Maysuria M, Mitton JD, Oliveri P, Osborn JL, Peng T, Ratcliffe AL, Webster PJ, Davidson EH, Hood L, Dimitrov K (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317–325. https://doi.org/10.1038/nbt1385 CrossRefPubMedGoogle Scholar
- 16.Hofmann K, Fiedler S, Vierkotten S, Weber J, Klee S, Jia J, Zwickenpflug W, Flockerzi V, Storch U, Yildirim AO, Gudermann T, Konigshoff M, Dietrich A (2017) Classical transient receptor potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis. Biochim Biophys Acta 1863:560–568. https://doi.org/10.1016/j.bbadis.2016.12.002 CrossRefPubMedGoogle Scholar
- 19.John-Schuster G, Hager K, Conlon TM, Irmler M, Beckers J, Eickelberg O, Yildirim AO (2014) Cigarette smoke-induced iBALT mediates macrophage activation in a B cell-dependent manner in COPD. Am J Physiol Lung Cell Mol Physiol 307:L692–L706. https://doi.org/10.1152/ajplung.00092.2014 CrossRefPubMedGoogle Scholar
- 21.Kalwa H, Storch U, Demleitner J, Fiedler S, Mayer T, Kannler M, Fahlbusch M, Barth H, Smrcka A, Hildebrandt F, Gudermann T, Dietrich A (2015) Phospholipase C epsilon (PLCepsilon) induced TRPC6 activation: a common but redundant mechanism in primary podocytes. J Cell Physiol 230:1389–1399. https://doi.org/10.1002/jcp.24883 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Kondrikov D, Caldwell RB, Dong Z, Su Y (2011) Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis. Free Radic Biol Med 50:1689–1698. https://doi.org/10.1016/j.freeradbiomed.2011.03.020 CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Mukhopadhyay I, Gomes P, Aranake S, Shetty M, Karnik P, Damle M, Kuruganti S, Thorat S, Khairatkar-Joshi N (2011) Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells. J Recept Signal Transduct Res 31:350–358. https://doi.org/10.3109/10799893.2011.602413 CrossRefPubMedGoogle Scholar
- 27.Mutze K, Vierkotten S, Milosevic J, Eickelberg O, Konigshoff M (2015) Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/beta-catenin-driven trans-differentiation of murine alveolar epithelial cells. Dis Model Mech 8:877–890. https://doi.org/10.1242/dmm.019117 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Nassini R, Pedretti P, Moretto N, Fusi C, Carnini C, Facchinetti F, Viscomi AR, Pisano AR, Stokesberry S, Brunmark C, Svitacheva N, McGarvey L, Patacchini R, Damholt AB, Geppetti P, Materazzi S (2012) Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation. PLoS One 7:e42454. https://doi.org/10.1371/journal.pone.0042454 CrossRefPubMedPubMedCentralGoogle Scholar
- 31.Schaefer EA, Stohr S, Meister M, Aigner A, Gudermann T, Buech TR (2013) Stimulation of the chemosensory TRPA1 cation channel by volatile toxic substances promotes cell survival of small cell lung cancer cells. Biochem Pharmacol 85:426–438. https://doi.org/10.1016/j.bcp.2012.11.019 CrossRefPubMedGoogle Scholar
- 33.Stenger B, Zehfuss F, Muckter H, Schmidt A, Balszuweit F, Schafer E, Buch T, Gudermann T, Thiermann H, Steinritz D (2015) Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents. Arch Toxicol 89:1631–1643. https://doi.org/10.1007/s00204-014-1414-4 CrossRefPubMedGoogle Scholar
- 34.Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A (2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112:819–829CrossRefPubMedGoogle Scholar
- 35.Takahashi N, Kuwaki T, Kiyonaka S, Numata T, Kozai D, Mizuno Y, Yamamoto S, Naito S, Knevels E, Carmeliet P, Oga T, Kaneko S, Suga S, Nokami T, Yoshida J, Mori Y (2011) TRPA1 underlies a sensing mechanism for O2. Nat Chem Biol 7:701–711. https://doi.org/10.1038/nchembio.640 CrossRefPubMedGoogle Scholar
- 38.Vyas-Read S, Wang W, Kato S, Colvocoresses-Dodds J, Fifadara NH, Gauthier TW, Helms MN, Carlton DP, Brown LA (2014) Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition. Am J Physiol Lung Cell Mol Physiol 306:L326–L340. https://doi.org/10.1152/ajplung.00074.2013 CrossRefPubMedGoogle Scholar
- 40.Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U, Mederos y Schnitzler M, Ghofrani HA, Schermuly RT, Pinkenburg O, Seeger W, Grimminger F, Gudermann T (2006) Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci U S A 103:19093–19098CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Weissmann N, Sydykov A, Kalwa H, Storch U, Fuchs B, Mederos y Schnitzler M, Brandes RP, Grimminger F, Meissner M, Freichel M, Offermanns S, Veit F, Pak O, Krause KH, Schermuly RT, Brewer AC, Schmidt HH, Seeger W, Shah AM, Gudermann T, Ghofrani HA, Dietrich A (2012) Activation of TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in mice. Nat Commun 3:649. https://doi.org/10.1038/ncomms1660 CrossRefPubMedPubMedCentralGoogle Scholar