TRPV4 activity modulates cell activities including receptor trafficking and transcriptional or translational regulations. We tested its CRISPR/Cas9 scissor efficacy in HepG2 (HEK293) cell noticed that it worked well in both cell lines to eliminate TRPV4 genome sequences. To confirm TRPV4 functions in the cell morphology maintenance and cell growth (beyond Ca 2+ channel), we compared its wound healing, cell surface area, survival property and soft agar growth ability after deletion of TRPV4 gene in the cells with its CRISPR/Cas9 system. With these experiments, we confirmed that TRPV4 is required not only to function as Ca 2+ channel but also to maintain its proper cell morphology as a corner stone protein on the cell adhesion junction.
KeywordsTRPV4Ca2+ Ion ChannelE-CadherinTubulinSoft Agar GrowthCRISPR/Cas9 Scissor System
Grace, M.S., Bonvini, S.J., Belvisi, M.G. and McIntyre, P. (2017) Modulation of the TRPV4 Ion Channel as a Therapeutic Target for Disease. Pharmacology & Therapeutics, 177, 9-22. https://doi.org/10.1016/j.pharmthera.2017.02.019
Kang, S.S., Shin, S.H., Auh, C.K. and Chun, J. (2012) Human Skeletal Dysplasia Caused by a Constitutive Activated Transient Receptor Potential Vanilloid 4 (TRPV4) Cation Channel Mutation. Experimental & Molecular Medicine, 44, 702-722. https://doi.org/10.3858/emm.2012.44.12.080
Nilius, B. and Owsianik, G. (2010) Channelopathies Converge on TRPV4. Nature Genetics, 42, 98-100. https://doi.org/10.1038/ng0210-98
Nilius, B. and Voets, T. (2013) The Puzzle of TRPV4 Channelopathies. EMBO Reports, 14, 152-163. https://doi.org/10.1038/embor.2012.219
Gold, M.S. and Gebhart, G.F. (2010) Nociceptor Sensitization in Pain Pathogenesis. Nature Medicine, 16, 1248-1257. https://doi.org/10.1038/nm.2235
Alessandri-Haber, N., Dina, O.A., Chen, X. and Levine, J.D. (2009) TRPC1 and TRPC6 Channels Cooperate with TRPV4 to Mediate Mechanical Hyperalgesia and Nociceptor Sensitization. Journal of Neuroscience, 29, 6217-6228. https://doi.org/10.1523/JNEUROSCI.0893-09.2009
Liedtke, W. and Friedman, J.M. (2003) Abnormal Osmotic Regulation in Trpv4-/- Mice. Proceedings of the National Academy of Sciences of the United States of America, 100, 13698-13703. https://doi.org/10.1073/pnas.1735416100
Liedtke, W., Tobin, D.M., Bargmann, C.I. and Friedman, J.M. (2003) Mammalian TRPV4 (VR-OAC) Directs Behavioral Responses to Osmotic and Mechanical Stimuli in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 2, 14531-14536. https://doi.org/10.1073/pnas.2235619100
Liedtke, W. (2005) TRPV4 Plays an Evolutionary Conserved Role in the Transduction of Osmotic and Mechanical Stimuli in Live Animals. The Journal of Physiology, 567, 53-58. https://doi.org/10.1113/jphysiol.2005.088963
Vriens, J., Watanabe, H., Janssens, A., Droogmans, G., Voets, T. and Nilius, B. (2004) Cell Swelling, Heat, and Chemical Agonists Use Distinct Pathways for the Activation of the Cation Channel TRPV4. Proceedings of the National Academy of Sciences of the United States of America, 101, 396-401. https://doi.org/10.1073/pnas.0303329101
Everaerts, W., Nilius, B. and Owsianik, G. (2010) The Vanilloid Transient Receptor Potential Channel TRPV4: From Structure to Disease. Progress in Biophysics and Molecular Biology, 103, 2-17. https://doi.org/10.1016/j.pbiomolbio.2009.10.002
Delany, N.S., Hurle, M., Facer, P., Alnadaf, T., Plumpton, C., Kinghorn, I., See, C.G., Costigan, M., Anand, P., Woolf, C.J., Crowther, D., Sanseau, P. and Tate, S.N. (2001) Identification and Characterization of a Novel Human Vanilloid Receptor-Like Protein, VRL-2. Physiological Genomics, 4, 165-174. https://doi.org/10.1152/physiolgenomics.2001.4.3.165
Watanabe, H., Vriens, J., Prenen, J., Droogmans, G., Voets, T. and Nilius, B. (2003) Anandamide and Arachidonic Acid Use Epoxyeicosatrienoic Acids to Activate TRPV4 Channels. Nature, 424, 434-438. https://doi.org/10.1038/nature01807
Suzuki, M., Hirao, A. and Mizuno, A. (2003) Microtubule-Associated [Corrected] Protein 7 Increases the Membrane Expression of Transient Receptor Potential Vanilloid 4 (TRPV4). The Journal of Biological Chemistry, 278, 51448-51453. https://doi.org/10.1074/jbc.M308212200
Strotmann, R., Schultz, G. and Plant, T.D. (2003) Ca2+-Dependent Potentiation of the Nonselective Cation Channel TRPV4 Is Mediated by a C-Terminal Calmodulin Binding Site. The Journal of Biological Chemistry, 278, 26541-26549. https://doi.org/10.1074/jbc.M302590200
Gagnon, K.B., England, R. and Delpire, E. (2006) Volume Sensitivity of Cation-Cl − Cotransporters Is Modulated by the Interaction of Two Kinases: Ste20-Related Proline-Alanine-Rich Kinase and WNK4. American Journal of Physiology-Cell Physiology, 290, C134-C142. https://doi.org/10.1152/ajpcell.00037.2005
Shin, S.H., Lee, E.J., Chun, J., Hyun, S. and Kang, S.S. (2015) Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1. The Open Biochemistry Journal, 9, 24-33. https://doi.org/10.2174/1874091X01509010024
Cuajungco, M.P., Grimm, C., Oshima, K., D’Hoedt, D., Nilius, B., Mensenkamp, A.R., Bindels, R.J., Plomann, M. and Heller, S. (2006) PACSINs Bind to the TRPV4 Cation Channel. PACSIN 3 Modulates the Subcellular Localization of TRPV4. The Journal of Biological Chemistry, 281, 18753-18762. https://doi.org/10.1074/jbc.M602452200
Palacio-Mancheno, P.E., Evashwick-Rogler, T.W., Laudier, D.M., Purmessur, D. and Iatridis, J.C. (2018) Hyperosmolarity Induces Notochordal Cell Differentiation with Aquaporin3 Upregulation and Reduced N-Cadherin Expression. Journal of Orthopaedic Research, 36, 788-798. https://doi.org/10.1002/jor.23715
Sokabe, T., Fukumi-Tominaga, T., Yonemura, S., Mizuno, A. and Tominaga, M. (2010) The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes. The Journal of Biological Chemistry, 285, 18749-18758. https://doi.org/10.1074/jbc.M110.103606
Becker, D., Bereiter-Hahn, J. and Jendrach, M. (2009) Functional Interaction of the Cation Channel Transient Receptor Potential Vanilloid 4 (TRPV4) and Actin in Volume Regulation. European Journal of Cell Biology, 88, 141-152. https://doi.org/10.1016/j.ejcb.2008.10.002
Fernandes, J., Lorenzo, I.M., Andrade, Y.N., Garcia-Elias, A., Serra, S.A., Fernandez-Fernandez, J.M. and Valverde, M.A. (2008) IP3 Sensitizes TRPV4 Channel to the Mechano- and Osmotransducing Messenger 5’-6’-Epoxyeicosatrienoic Acid. Journal of Cell Biology, 181, 143-155. https://doi.org/10.1083/jcb.200712058
Fusi, C., Materazzi, S., Minocci, D., Maio, V., Oranges, T., Massi, D. and Nassini, R. (2014) Transient Receptor Potential Vanilloid 4 (TRPV4) Is Downregulated in Keratinocytes in Human Non-Melanoma Skin Cancer. Journal of Investigative Dermatology, 134, 2408-2417. https://doi.org/10.1038/jid.2014.145
Doudna, J.A. and Charpentier, E. (2014) The New Frontier of Genome Engineering with CRISPR-Cas9. Science, 346, 1258096. https://doi.org/10.1126/science.1258096
Fiorio Pla, A., Ong, H.L., Cheng, K.T., Brossa, A., Bussolati, B., Lockwich, T., Paria, B., Munaron, L. and Ambudkar, I.S. (2012) TRPV4 Mediates Tumor-Derived Endothelial Cell Migration via Arachidonic Acid-Activated Actin Remodeling. Oncogene, 31, 200-212. https://doi.org/10.1038/onc.2011.231
Vriens, J., Janssens, A., Prenen, J., Nilius, B. and Wondergem, R. (2004) TRPV Channels and Modulation by Hepatocyte Growth Factor/Scatter Factor in Human Hepatoblastoma (HepG2) Cells. Cell Calcium, 36, 19-28. https://doi.org/10.1016/j.ceca.2003.11.006
Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A. and Zhang, F. (2013) Genome Engineering Using the CRISPR-Cas9 System. Nature Protocols, 8, 2281-2308. https://doi.org/10.1038/nprot.2013.143
Shin, S.H., Lee, E.J., Hyun, S., Chun, J., Kim, Y. and Kang, S.S. (2012) Phosphorylation on the Ser 824 Residue of TRPV4 Prefers to Bind with F-Actin than with Microtubules to Expand the Cell Surface Area. Cellular Signalling, 24, 641-651. https://doi.org/10.1016/j.cellsig.2011.11.002
Lee, W.H., Choong, L.Y., Mon, N.N., Lu, S., Lin, Q., Pang, B., Yan, B., Krishna, V.S., Singh, H., Tan, T.Z., Thiery, J.P., Lim, C.T., Tan, P.B., Johansson, M., Harteneck, C. and Lim, Y.P. (2016) TRPV4 Regulates Breast Cancer Cell Extravasation, Stiffness and Actin Cortex. Scientific Reports, 6, 27903. https://doi.org/10.1038/srep27903
Adapal, R.K., Thoppil, R.J., Ghosh, K., Cappelli, H.C., Dudley, A.C., Paruchuri, S., Keshamouni, V., Klagsbrun, M., Meszaros, J.G., Chilian, W.M., Ingber, D.E. and Thodeti, C.K. (2016) Activation of Mechanosensitive Ion Channel TRPV4 Normalizes Tumor Vasculature and Improves Cancer Therapy. Oncogene, 35, 314-322. https://doi.org/10.1038/onc.2015.83
Monteith, G.R., Davis, F.M. and Roberts-Thomson, S.J. (2012) Calcium Channels and Pumps in Cancer: Changes and Consequences. The Journal of Biological Chemistry, 287, 31666-31673. https://doi.org/10.1074/jbc.R112.343061
Sharma, S., Goswami, R., Zhang, D.X. and Rahaman, S.O. (2018) TRPV4 Regulates Matrix Stiffness and TGFβ1-Induced Epithelial-Mesenchymal Transition. Journal of Cellular and Molecular Medicine, 23, 761-774. https://doi.org/10.1111/jcmm.13972