蛋白酶激活受器二(PAR2)屬於G蛋白偶聯受器(GPCR)，常被胰蛋白酶(trypsin)等絲氨酸蛋白酶(serine proteases)所活化。目前已知此受器的活化能促進肺癌細胞增生、遷移及抑制細胞凋亡等生理現象。蛋白酶激活受器二的活化亦能透過抑制上皮鈣黏蛋白(E-cadherin)之間的黏附而降低肺上皮細胞屏障，使得肺上皮細胞組織間的通透性增加。由於上皮鈣黏蛋白表現量的改變為細胞是否有進行上皮細胞間質轉化(EMT)的依據，因此，此受器極可能參與肺癌細胞的上皮細胞間質轉化。雖然蛋白酶激活受器二已知能促進肺腺癌細胞的遷移，但其詳細機制尚未明確的被報導。在此研究，我探討蛋白酶激活受器二是否能調控肺腺癌細胞的遷移和上皮細胞間質轉化，並深入了解其分子機制。首先，蛋白酶激活受器二會高度表達在多種肺腺癌細胞株。當我利用兩株肺腺癌細胞株CL1-5和H1299進行實驗時，蛋白酶激活受器二的活化確實會誘導肺腺癌細胞的遷移及轉錄因子Slug所調控的上皮細胞間質轉化。當再利用CL1-5細胞進行分子機制的探討時，此受器調控肺腺癌細胞的遷移時會分別透過Src/ p38 MAPK和Src/Akt兩種訊號傳遞路線。而當中Src/ p38 MAPK訊號傳遞路線會由β-制動素1 (β-arrestin 1)調控，而非由G蛋白所調控。另外，在蛋白酶激活受器二調控肺腺癌細胞CL1-5的上皮細胞間質轉化時，亦發現ERK2會抑制肝醣合成酶激酶3β (GSK3β)的活性並進而讓轉錄因子Slug不被降解掉。於是，轉錄因子Slug就能進一步調控上皮細胞間質轉化相關的基因表現。最後，也發現當病患中的肺腺癌細胞的蛋白酶激活受器二表現量高時，其該病患的整體存活率及無腫瘤復發存活率都差於肺腺癌細胞的該受器表現量低的病患。總結來說，蛋白酶激活受器二會透過β-制動素1調控p38 MAPK相關訊號傳遞路線而促進肺腺癌細胞遷移。此外，此受器則會活化ERK2來促使轉錄因子Slug調控上皮細胞間質轉化相關的基因表現。根據我的研究成果推測，蛋白酶激活受器二可能可以當作標靶治療的目標基因來治療具有轉移能力的肺腺癌之病患並亦可當作生物標記來預測肺腺癌病患的預後。

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteases, such as trypsin. Activation of PAR2 has been shown to promote growth, prevent apoptosis, and induce migration in lung cancer. Because activation of PAR2 in airway epithelial cells reduces the airway epithelium barrier through the disruption of E-cadherin adhesion, PAR2 may be involved in epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Although PAR2 is known to regulate the migration of lung cancer cells, the detailed mechanism of this event is still unclear. Here, the underlying mechanisms of PAR2-mediated migration and EMT in lung adenocarcinoma cells were investigated. My results show that PAR2 is highly expressed in several lung adenocarcinoma cell lines. In two lung adenocarcinoma cell lines, CL1-5 and H1299 cells, activation of PAR2 promotes migration and Slug-mediated EMT. PAR2-induced migration of CL1-5 cells is mediated by the Src/p38 mitogen-activated protein kinase (p38 MAPK) and Src/Akt signaling pathways. β-arrestin 1, not G protein, is involved in this PAR2-mediated Src/p38 MAPK signaling pathway. PAR2-induced EMT in CL1-5 cells is dependent on the activation of extracellular-signal-regulated kinase 2 (ERK2). The activation of ERK2 further mediates Slug stabilization through suppressing the activity of glycogen synthase kinase 3β (GSK3β). In addition, a poor prognosis was observed in lung adenocarcinoma patients with a high expression of PAR2. Thus, in lung adenocarcinoma, PAR2 regulates its migration through β-arrestin 1-dependent activation of p38 MAPK and its EMT through ERK2-mediated stabilization of Slug. My findings also suggest that PAR2 might serve as a therapeutic target for metastatic lung adenocarcinoma and a potential biomarker for predicting the prognosis of lung adenocarcinoma.

Adams, M.N., Pagel, C.N., Mackie, E.J., and Hooper, J.D. (2012). Evaluation of antibodies directed against human protease-activated receptor-2. Naunyn-Schmiedeberg's archives of pharmacology. 385, 861-873.

Adams, M.N., Ramachandran, R., Yau, M.K., Suen, J.Y., Fairlie, D.P., Hollenberg, M.D., and Hooper, J.D. (2011). Structure, function and pathophysiology of protease activated receptors. Pharmacol Ther. 130, 248-282.

Ahn, S., Wei, H., Garrison, T.R., and Lefkowitz, R.J. (2004). Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2. The Journal of biological chemistry. 279, 7807-7811.

Al-Ani, B., Saifeddine, M., Kawabata, A., and Hollenberg, M.D. (1999). Proteinase activated receptor 2: Role of extracellular loop 2 for ligand-mediated activation. Br J Pharmacol. 128, 1105-1113.

Ando, S., Otani, H., Yagi, Y., Kawai, K., Araki, H., Fukuhara, S., and Inagaki, C. (2007). Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells. Respiratory research. 8, 31.

Asokananthan, N., Graham, P.T., Stewart, D.J., Bakker, A.J., Eidne, K.A., Thompson, P.J., and Stewart, G.A. (2002). House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. J Immunol. 169, 4572-4578.

Ay, C., Dunkler, D., Simanek, R., Thaler, J., Koder, S., Marosi, C., Zielinski, C., and Pabinger, I. (2011). Prediction of venous thromboembolism in patients with cancer by measuring thrombin generation: results from the Vienna Cancer and Thrombosis Study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 29, 2099-2103.

Bakin, A.V., Tomlinson, A.K., Bhowmick, N.A., Moses, H.L., and Arteaga, C.L. (2000). Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration. The Journal of biological chemistry. 275, 36803-36810.

Black, P.C., Mize, G.J., Karlin, P., Greenberg, D.L., Hawley, S.J., True, L.D., Vessella, R.L., and Takayama, T.K. (2007). Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer. The Prostate. 67, 743-756.

Bohm, S.K., Khitin, L.M., Grady, E.F., Aponte, G., Payan, D.G., and Bunnett, N.W. (1996). Mechanisms of desensitization and resensitization of proteinase-activated receptor-2. The Journal of biological chemistry. 271, 22003-22016.

Botham, A., Guo, X., Xiao, Y.P., Morice, A.H., Compton, S.J., and Sadofsky, L.R. (2011). Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis. The Biochemical journal. 438, 359-367.

Brown, J.K., Tyler, C.L., Jones, C.A., Ruoss, S.J., Hartmann, T., and Caughey, G.H. (1995). Tryptase, the dominant secretory granular protein in human mast cells, is a potent mitogen for cultured dog tracheal smooth muscle cells. American journal of respiratory cell and molecular biology. 13, 227-236.

Cairns, J.A., and Walls, A.F. (1996). Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J Immunol. 156, 275-283.

Camerer, E., Barker, A., Duong, D.N., Ganesan, R., Kataoka, H., Cornelissen, I., Darragh, M.R., Hussain, A., Zheng, Y.W., Srinivasan, Y., Brown, C., Xu, S.M., Regard, J.B., Lin, C.Y., Craik, C.S., Kirchhofer, D., and Coughlin, S.R. (2010). Local protease signaling contributes to neural tube closure in the mouse embryo. Dev Cell. 18, 25-38.

Camerer, E., Huang, W., and Coughlin, S.R. (2000). Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proceedings of the National Academy of Sciences of the United States of America. 97, 5255-5260.

Caruso, R., Pallone, F., Fina, D., Gioia, V., Peluso, I., Caprioli, F., Stolfi, C., Perfetti, A., Spagnoli, L.G., Palmieri, G., Macdonald, T.T., and Monteleone, G. (2006). Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation. The American journal of pathology. 169, 268-278.

Chen, J., Chen, J.K., and Harris, R.C. (2012). Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor-extracellular signal-regulated kinase signaling pathway. Mol Cell Biol. 32, 981-991.

Chen, W., and Bowden, G.T. (1999). Activation of p38 MAP kinase and ERK are required for ultraviolet-B induced c-fos gene expression in human keratinocytes. Oncogene. 18, 7469-7476.

Chou, T.Y., Chen, W.C., Lee, A.C., Hung, S.M., Shih, N.Y., and Chen, M.Y. (2009). Clusterin silencing in human lung adenocarcinoma cells induces a mesenchymal-to-epithelial transition through modulating the ERK/Slug pathway. Cell Signal. 21, 704-711.

Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R., and Wu, C.W. (1997). Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology. 17, 353-360.

Cisowski, J., O'Callaghan, K., Kuliopulos, A., Yang, J., Nguyen, N., Deng, Q., Yang, E., Fogel, M., Tressel, S., Foley, C., Agarwal, A., Hunt, S.W., 3rd, McMurry, T., Brinckerhoff, L., and Covic, L. (2011). Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer. The American journal of pathology. 179, 513-523.

Compton, S.J., Sandhu, S., Wijesuriya, S.J., and Hollenberg, M.D. (2002). Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling. The Biochemical journal. 368, 495-505.

Darmoul, D., Marie, J.C., Devaud, H., Gratio, V., and Laburthe, M. (2001). Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2. British journal of cancer. 85, 772-779.

DeFea, K.A., Zalevsky, J., Thoma, M.S., Dery, O., Mullins, R.D., and Bunnett, N.W. (2000). beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol. 148, 1267-1281.
Derynck, R., and Zhang, Y.E. (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 425, 577-584.

Ding, Q., Xia, W., Liu, J.C., Yang, J.Y., Lee, D.F., Xia, J., Bartholomeusz, G., Li, Y., Pan, Y., Li, Z., Bargou, R.C., Qin, J., Lai, C.C., Tsai, F.J., Tsai, C.H., and Hung, M.C. (2005). Erk associates with and primes GSK-3beta for its inactivation resulting in upregulation of beta-catenin. Mol Cell. 19, 159-170.

Elmariah, S.B., Reddy, V.B., and Lerner, E.A. (2014). Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist. PloS one. 9, e99702.

Ge, L., Ly, Y., Hollenberg, M., and DeFea, K. (2003). A beta-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. The Journal of biological chemistry. 278, 34418-34426.

Ge, L., Shenoy, S.K., Lefkowitz, R.J., and DeFea, K. (2004). Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2. The Journal of biological chemistry. 279, 55419-55424.

Gresner, P., Gromadzinska, J., and Wasowicz, W. (2009). Reference genes for gene expression studies on non-small cell lung cancer. Acta biochimica Polonica. 56, 307-316.

Grimsey, N., Soto, A.G., and Trejo, J. (2011). Regulation of protease-activated receptor signaling by post-translational modifications. IUBMB life. 63, 403-411.

Gupta, M.K., Mohan, M.L., and Naga Prasad, S.V. (2018). G Protein-Coupled Receptor Resensitization Paradigms. International review of cell and molecular biology. 339, 63-91.

Hansen, K.K., Sherman, P.M., Cellars, L., Andrade-Gordon, P., Pan, Z., Baruch, A., Wallace, J.L., Hollenberg, M.D., and Vergnolle, N. (2005). A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis. Proceedings of the National Academy of Sciences of the United States of America. 102, 8363-8368.

Hasdemir, B., Bunnett, N.W., and Cottrell, G.S. (2007). Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor. The Journal of biological chemistry. 282, 29646-29657.

Hasdemir, B., Murphy, J.E., Cottrell, G.S., and Bunnett, N.W. (2009). Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2. The Journal of biological chemistry. 284, 28453-28466.

Hein, L., Ishii, K., Coughlin, S.R., and Kobilka, B.K. (1994). Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. The Journal of biological chemistry. 269, 27719-27726.

Hong, J.H., Hong, J.Y., Park, B., Lee, S.I., Seo, J.T., Kim, K.E., Sohn, M.H., and Shin, D.M. (2008). Chitinase activates protease-activated receptor-2 in human airway epithelial cells. American journal of respiratory cell and molecular biology. 39, 530-535.

Hu, G.M., Mai, T.L., and Chen, C.M. (2017). Visualizing the GPCR Network: Classification and Evolution. Scientific reports. 7, 15495.

Huang, C.H., Yang, W.H., Chang, S.Y., Tai, S.K., Tzeng, C.H., Kao, J.Y., Wu, K.J., and Yang, M.H. (2009). Regulation of membrane-type 4 matrix metalloproteinase by SLUG contributes to hypoxia-mediated metastasis. Neoplasia. 11, 1371-1382.

Huang, S.H., Li, Y., Chen, H.G., Rong, J., and Ye, S. (2013). Activation of proteinase-activated receptor 2 prevents apoptosis of lung cancer cells. Cancer Invest. 31, 578-581.

Ishihara, H., Connolly, A.J., Zeng, D., Kahn, M.L., Zheng, Y.W., Timmons, C., Tram, T., and Coughlin, S.R. (1997). Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 386, 502-506.

Jacob, C., Cottrell, G.S., Gehringer, D., Schmidlin, F., Grady, E.F., and Bunnett, N.W. (2005a). c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2. The Journal of biological chemistry. 280, 16076-16087.

Jacob, C., Yang, P.C., Darmoul, D., Amadesi, S., Saito, T., Cottrell, G.S., Coelho, A.M., Singh, P., Grady, E.F., Perdue, M., and Bunnett, N.W. (2005b). Mast cell tryptase controls paracellular permeability of the intestine. Role of protease-activated receptor 2 and beta-arrestins. The Journal of biological chemistry. 280, 31936-31948.

Jahan, I., Fujimoto, J., Alam, S.M., Sato, E., Sakaguchi, H., and Tamaya, T. (2007). Role of protease activated receptor-2 in tumor advancement of ovarian cancers. Ann Oncol. 18, 1506-1512.

Jahan, I., Fujimoto, J., Alam, S.M., Sato, E., and Tamaya, T. (2008). Role of protease activated receptor-2 in lymph node metastasis of uterine cervical cancers. BMC Cancer. 8, 301.

Jin, E., Fujiwara, M., Pan, X., Ghazizadeh, M., Arai, S., Ohaki, Y., Kajiwara, K., Takemura, T., and Kawanami, O. (2003). Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas. Cancer. 97, 703-713.

Kasai, H., Allen, J.T., Mason, R.M., Kamimura, T., and Zhang, Z. (2005). TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respiratory research. 6, 56.

Kawabata, A., Saifeddine, M., Al-Ani, B., Leblond, L., and Hollenberg, M.D. (1999). Evaluation of proteinase-activated receptor-1 (PAR1) agonists and antagonists using a cultured cell receptor desensitization assay: activation of PAR2 by PAR1-targeted ligands. J Pharmacol Exp Ther. 288, 358-370.

Kim, J., Kong, J., Chang, H., Kim, H., and Kim, A. (2016). EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathway in breast cancer cells. Oncotarget. 7, 85021-85032.

Ko, J.C., Ciou, S.C., Cheng, C.M., Wang, L.H., Hong, J.H., Jheng, M.Y., Ling, S.T., and Lin, Y.W. (2008). Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells. Carcinogenesis. 29, 1448-1458.

Koshikawa, N., Hasegawa, S., Nagashima, Y., Mitsuhashi, K., Tsubota, Y., Miyata, S., Miyagi, Y., Yasumitsu, H., and Miyazaki, K. (1998). Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. The American journal of pathology. 153, 937-944.

Kozasa, T., Jiang, X., Hart, M.J., Sternweis, P.M., Singer, W.D., Gilman, A.G., Bollag, G., and Sternweis, P.C. (1998). p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science. 280, 2109-2111.

Kumar, S., Jiang, M.S., Adams, J.L., and Lee, J.C. (1999). Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase. Biochem Biophys Res Commun. 263, 825-831.

Kuo, F.T., Lu, T.L., and Fu, H.W. (2006). Opposing effects of beta-arrestin1 and beta-arrestin2 on activation and degradation of Src induced by protease-activated receptor 1. Cell Signal. 18, 1914-1923.

Lamouille, S., Xu, J., and Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 15, 178-196.

Li, M.D., and Yang, X. (2011). A Retrospective on Nuclear Receptor Regulation of Inflammation: Lessons from GR and PPARs. PPAR research. 2011, 742785.

Li, X., and Tai, H.H. (2014). Thromboxane A2 receptor-mediated release of matrix metalloproteinase-1 (MMP-1) induces expression of monocyte chemoattractant protein-1 (MCP-1) by activation of protease-activated receptor 2 (PAR2) in A549 human lung adenocarcinoma cells. Mol Carcinog. 53, 659-666.

Lin, S.C., Chou, Y.T., Jiang, S.S., Chang, J.L., Chung, C.H., Kao, Y.R., Chang, I.S., and Wu, C.W. (2016). Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell Plasticity. Cancer research. 76, 7036-7048.

Liu, C.W., Li, C.H., Peng, Y.J., Cheng, Y.W., Chen, H.W., Liao, P.L., Kang, J.J., and Yeng, M.H. (2014). Snail regulates Nanog status during the epithelial-mesenchymal transition via the Smad1/Akt/GSK3beta signaling pathway in non-small-cell lung cancer. Oncotarget. 5, 3880-3894.

Lourbakos, A., Chinni, C., Thompson, P., Potempa, J., Travis, J., Mackie, E.J., and Pike, R.N. (1998). Cleavage and activation of proteinase-activated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis. FEBS Lett. 435, 45-48.

Luo, R., Wang, X., Dong, Y., Wang, L., and Tian, C. (2014). Activation of protease-activated receptor 2 reduces glioblastoma cell apoptosis. J Biomed Sci. 21, 25.

Luo, W., Wang, Y., Hanck, T., Stricker, R., and Reiser, G. (2006). Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1. The Journal of biological chemistry. 281, 7927-7936.

Luo, W., Wang, Y., and Reiser, G. (2007). p24A, a type I transmembrane protein, controls ARF1-dependent resensitization of protease-activated receptor-2 by influence on receptor trafficking. The Journal of biological chemistry. 282, 30246-30255.

Ma, L., and Pei, G. (2007). Beta-arrestin signaling and regulation of transcription. J Cell Sci. 120, 213-218.

Macfarlane, S.R., Seatter, M.J., Kanke, T., Hunter, G.D., and Plevin, R. (2001). Proteinase-activated receptors. Pharmacol Rev. 53, 245-282.

Mansour, S.J., Candia, J.M., Gloor, K.K., and Ahn, N.G. (1996). Constitutively active mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 mediate similar transcriptional and morphological responses. Cell Growth Differ. 7, 243-250.

Massi, D., Naldini, A., Ardinghi, C., Carraro, F., Franchi, A., Paglierani, M., Tarantini, F., Ketabchi, S., Cirino, G., Hollenberg, M.D., Geppetti, P., and Santucci, M. (2005). Expression of protease-activated receptors 1 and 2 in melanocytic nevi and malignant melanoma. Human pathology. 36, 676-685.

Matej, R., Mandakova, P., Netikova, I., Pouckova, P., and Olejar, T. (2007). Proteinase-activated receptor-2 expression in breast cancer and the role of trypsin on growth and metabolism of breast cancer cell line MDA MB-231. Physiological research. 56, 475-484.

Mazharian, A., Ghevaert, C., Zhang, L., Massberg, S., and Watson, S.P. (2011). Dasatinib enhances megakaryocyte differentiation but inhibits platelet formation. Blood. 117, 5198-5206.

McCoy, K.L., Traynelis, S.F., and Hepler, J.R. (2010). PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology. Mol Pharmacol. 77, 1005-1015.

Medina, M., and Wandosell, F. (2011). Deconstructing GSK-3: The Fine Regulation of Its Activity. International journal of Alzheimer's disease. 2011, 479249.

Michel, N., Heuze-Vourc'h, N., Lavergne, E., Parent, C., Jourdan, M.L., Vallet, A., Iochmann, S., Musso, O., Reverdiau, P., and Courty, Y. (2014). Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor. Biol Chem. 395, 1015-1025.

Mihara, K., Ramachandran, R., Saifeddine, M., Hansen, K.K., Renaux, B., Polley, D., Gibson, S., Vanderboor, C., and Hollenberg, M.D. (2016). Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling. Mol Pharmacol. 89, 606-614.

Moilanen, M., Sorsa, T., Stenman, M., Nyberg, P., Lindy, O., Vesterinen, J., Paju, A., Konttinen, Y.T., Stenman, U.H., and Salo, T. (2003). Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen. Biochemistry. 42, 5414-5420.

Molino, M., Barnathan, E.S., Numerof, R., Clark, J., Dreyer, M., Cumashi, A., Hoxie, J.A., Schechter, N., Woolkalis, M., and Brass, L.F. (1997). Interactions of mast cell tryptase with thrombin receptors and PAR-2. The Journal of biological chemistry. 272, 4043-4049.

Moormann, C., Artuc, M., Pohl, E., Varga, G., Buddenkotte, J., Vergnolle, N., Brehler, R., Henz, B.M., Schneider, S.W., Luger, T.A., and Steinhoff, M. (2006). Functional characterization and expression analysis of the proteinase-activated receptor-2 in human cutaneous mast cells. The Journal of investigative dermatology. 126, 746-755.

Moraes, T.J., Martin, R., Plumb, J.D., Vachon, E., Cameron, C.M., Danesh, A., Kelvin, D.J., Ruf, W., and Downey, G.P. (2008). Role of PAR2 in murine pulmonary pseudomonal infection. American journal of physiology Lung cellular and molecular physiology. 294, L368-377.

Nyberg, P., Moilanen, M., Paju, A., Sarin, A., Stenman, U.H., Sorsa, T., and Salo, T. (2002). MMP-9 activation by tumor trypsin-2 enhances in vivo invasion of human tongue carcinoma cells. J Dent Res. 81, 831-835.

Nystedt, S., Emilsson, K., Larsson, A.K., Strombeck, B., and Sundelin, J. (1995). Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem. 232, 84-89.

Nystedt, S., Emilsson, K., Wahlestedt, C., and Sundelin, J. (1994). Molecular cloning of a potential proteinase activated receptor. Proceedings of the National Academy of Sciences of the United States of America. 91, 9208-9212.

Ohta, T., Shimizu, K., Yi, S., Takamura, H., Amaya, K., Kitagawa, H., Kayahara, M., Ninomiya, I., Fushida, S., Fujimura, T., Nishimura, G., and Miwa, K. (2003). Protease-activated receptor-2 expression and the role of trypsin in cell proliferation in human pancreatic cancers. International journal of oncology. 23, 61-66.

Oikonomopoulou, K., Hansen, K.K., Saifeddine, M., Vergnolle, N., Tea, I., Blaber, M., Blaber, S.I., Scarisbrick, I., Diamandis, E.P., and Hollenberg, M.D. (2006). Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs). Biol Chem. 387, 817-824.

Page, K., Strunk, V.S., and Hershenson, M.B. (2003). Cockroach proteases increase IL-8 expression in human bronchial epithelial cells via activation of protease-activated receptor (PAR)-2 and extracellular-signal-regulated kinase. J Allergy Clin Immunol. 112, 1112-1118.

Pallier, K., Cessot, A., Côté, J.F., Just, P.A., Cazes, A., Fabre, E., Danel, C., Riquet, M., Devouassoux-Shisheboran, M., Ansieau, S., Puisieux, A., Laurent-Puig, P., and Blons, H. (2012). TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma. PloS one. 7, e29954.

Park, M.S., Owen, B.A., Ballinger, B.A., Sarr, M.G., Schiller, H.J., Zietlow, S.P., Jenkins, D.H., Ereth, M.H., Owen, W.G., and Heit, J.A. (2012). Quantification of hypercoagulable state after blunt trauma: microparticle and thrombin generation are increased relative to injury severity, while standard markers are not. Surgery. 151, 831-836.

Qu, J., Li, M., An, J., Zhao, B., Zhong, W., Gu, Q., Cao, L., Yang, H., and Hu, C. (2015). MicroRNA-33b inhibits lung adenocarcinoma cell growth, invasion, and epithelial-mesenchymal transition by suppressing Wnt/beta-catenin/ZEB1 signaling. International journal of oncology. 47, 2141-2152.

Rajagopal, S., Rajagopal, K., and Lefkowitz, R.J. (2010). Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nature reviews Drug discovery. 9, 373-386.

Rakashanda, S., Rana, F., Rafiq, S., Masood, A., and Amin, S. (2012). Role of proteases in cancer: A review. Biotech Mol Biol Rev. 7, 12.

Ramachandran, R., Mihara, K., Chung, H., Renaux, B., Lau, C.S., Muruve, D.A., DeFea, K.A., Bouvier, M., and Hollenberg, M.D. (2011). Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2). The Journal of biological chemistry. 286, 24638-24648.

Ramsay, A.J., Dong, Y., Hunt, M.L., Linn, M., Samaratunga, H., Clements, J.A., and Hooper, J.D. (2008a). Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression. The Journal of biological chemistry. 283, 12293-12304.

Ramsay, A.J., Reid, J.C., Adams, M.N., Samaratunga, H., Dong, Y., Clements, J.A., and Hooper, J.D. (2008b). Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs). Biol Chem. 389, 653-668.

Redecha, P., Franzke, C.W., Ruf, W., Mackman, N., and Girardi, G. (2008). Neutrophil activation by the tissue factor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome. J Clin Invest. 118, 3453-3461.

Ricks, T.K., and Trejo, J. (2009). Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization. The Journal of biological chemistry. 284, 34444-34457.

Ruan, J.S., Zhou, H., Yang, L., Wang, L., Jiang, Z.S., Sun, H., and Wang, S.M. (2017). Ursolic Acid Attenuates TGF-beta1 Induced Epithelial-Mesenchymal Transition in NSCLC by Targeting Integrin alphaVbeta5/MMPs Signaling. Oncol Res. doi: 10.3727/096504017X15051723858706.

Ruoss, S.J., Hartmann, T., and Caughey, G.H. (1991). Mast cell tryptase is a mitogen for cultured fibroblasts. J Clin Invest. 88, 493-499.

Rydén, L., Grabau, D., Schaffner, F., Jönsson, P.E., Ruf, W., and Belting, M. (2010). Evidence for tissue factor phosphorylation and its correlation with protease activated receptor expression and the prognosis of primary breast cancer. Int J Cancer. 126, 2330-2340.

Schaffner, F., and Ruf, W. (2009). Tissue factor and PAR2 signaling in the tumor microenvironment. Arterioscler Thromb Vasc Biol. 29, 1999-2004.

Schioth, H.B., and Fredriksson, R. (2005). The GRAFS classification system of G-protein coupled receptors in comparative perspective. Gen Comp Endocrinol. 142, 94-101.

Schmidlin, F., Amadesi, S., Dabbagh, K., Lewis, D.E., Knott, P., Bunnett, N.W., Gater, P.R., Geppetti, P., Bertrand, C., and Stevens, M.E. (2002). Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. J Immunol. 169, 5315-5321.

Schmidlin, F., Amadesi, S., Vidil, R., Trevisani, M., Martinet, N., Caughey, G., Tognetto, M., Cavallesco, G., Mapp, C., Geppetti, P., and Bunnett, N.W. (2001). Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. Am J Respir Crit Care Med. 164, 1276-1281.

Schultheiss, M., Neumcke, B., and Richter, H.P. (1997). Endogenous trypsin receptors in Xenopus oocytes: linkage to internal calcium stores. Cell Mol Life Sci. 53, 842-849.

Shao, M., Cao, L., Shen, C., Satpathy, M., Chelladurai, B., Bigsby, R.M., Nakshatri, H., and Matei, D. (2009). Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tissue transglutaminase. Cancer research. 69, 9192-9201.

Shi, X., Gangadharan, B., Brass, L.F., Ruf, W., and Mueller, B.M. (2004). Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2, 395-402.

Smith, R., Jenkins, A., Lourbakos, A., Thompson, P., Ramakrishnan, V., Tomlinson, J., Deshpande, U., Johnson, D.A., Jones, R., Mackie, E.J., and Pike, R.N. (2000). Evidence for the activation of PAR-2 by the sperm protease, acrosin: expression of the receptor on oocytes. FEBS Lett. 484, 285-290.

Somwar, R., Koterski, S., Sweeney, G., Sciotti, R., Djuric, S., Berg, C., Trevillyan, J., Scherer, P.E., Rondinone, C.M., and Klip, A. (2002). A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation. The Journal of biological chemistry. 277, 50386-50395.

Song, J.S., Kang, C.M., Park, C.K., and Yoon, H.K. (2013). Thrombin induces epithelial-mesenchymal transition via PAR-1, PKC, and ERK1/2 pathways in A549 cells. Exp Lung Res. 39, 336-348.

Stalheim, L., Ding, Y., Gullapalli, A., Paing, M.M., Wolfe, B.L., Morris, D.R., and Trejo, J. (2005). Multiple independent functions of arrestins in the regulation of protease-activated receptor-2 signaling and trafficking. Mol Pharmacol. 67, 78-87.

Stambolic, V., and Woodgett, J.R. (1994). Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation. The Biochemical journal. 303 ( Pt 3), 701-704.

Su, S., Li, Y., Luo, Y., Sheng, Y., Su, Y., Padia, R.N., Pan, Z.K., Dong, Z., and Huang, S. (2009). Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration. Oncogene. 28, 3047-3057.

Su, X., Camerer, E., Hamilton, J.R., Coughlin, S.R., and Matthay, M.A. (2005). Protease-activated receptor-2 activation induces acute lung inflammation by neuropeptide-dependent mechanisms. J Immunol. 175, 2598-2605.

Suen, J.Y., Adams, M.N., Lim, J., Madala, P.K., Xu, W., Cotterell, A.J., He, Y., Yau, M.K., Hooper, J.D., and Fairlie, D.P. (2017). Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling. Pharmacol Res. 117, 328-342.

Suen, J.Y., Cotterell, A., Lohman, R.J., Lim, J., Han, A., Yau, M.K., Liu, L., Cooper, M.A., Vesey, D.A., and Fairlie, D.P. (2014). Pathway-selective antagonism of proteinase activated receptor 2. Br J Pharmacol. 171, 4112-4124.

Sugimoto, N., Takuwa, N., Okamoto, H., Sakurada, S., and Takuwa, Y. (2003). Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol. 23, 1534-1545.

Sun, G., Stacey, M.A., Schmidt, M., Mori, L., and Mattoli, S. (2001). Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells. J Immunol. 167, 1014-1021.

Sun, L., Li, P.B., Yao, Y.F., Xiu, A.Y., Peng, Z., Bai, Y.H., and Gao, Y.J. (2018). Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma. World journal of gastroenterology. 24, 1120-1133.

Sun, Z., Cao, B., and Wu, J. (2015). Protease-activated receptor 2 enhances renal cell carcinoma cell invasion and migration via PI3K/AKT signaling pathway. Exp Mol Pathol. 98, 382-389.

Suzuki, A., Palmer, G., Bonjour, J.P., and Caverzasio, J. (1999). Regulation of alkaline phosphatase activity by p38 MAP kinase in response to activation of Gi protein-coupled receptors by epinephrine in osteoblast-like cells. Endocrinology. 140, 3177-3182.

Svensson, K.J., Kucharzewska, P., Christianson, H.C., Sköld, S., Löfstedt, T., Johansson, M.C., Morgelin, M., Bengzon, J., Ruf, W., and Belting, M. (2011). Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. Proceedings of the National Academy of Sciences of the United States of America. 108, 13147-13152.

Takahashi-Yanaga, F., Shiraishi, F., Hirata, M., Miwa, Y., Morimoto, S., and Sasaguri, T. (2004). Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts. Biochem Biophys Res Commun. 316, 411-415.

Takeuchi, T., Harris, J.L., Huang, W., Yan, K.W., Coughlin, S.R., and Craik, C.S. (2000). Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. The Journal of biological chemistry. 275, 26333-26342.

Thornton, T.M., Pedraza-Alva, G., Deng, B., Wood, C.D., Aronshtam, A., Clements, J.L., Sabio, G., Davis, R.J., Matthews, D.E., Doble, B., and Rincon, M. (2008). Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science. 320, 667-670.

Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., and Jemal, A. (2015). Global cancer statistics, 2012. CA Cancer J Clin. 65, 87-108.

Tsai, C.C., Kuo, T.Y., Hong, Z.W., Yeh, Y.C., Shih, K.S., Du, S.Y., and Fu, H.W. (2015). Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells. Virulence. 6, 755-765.

Tsutsumi, R., Higashi, H., Higuchi, M., Okada, M., and Hatakeyama, M. (2003). Attenuation of Helicobacter pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase. The Journal of biological chemistry. 278, 3664-3670.

Ueda, H., Morishita, R., Yamauchi, J., Itoh, H., Kato, K., and Asano, T. (2001). Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading. The Journal of biological chemistry. 276, 6846-6852.

Uusitalo-Jarvinen, H., Kurokawa, T., Mueller, B.M., Andrade-Gordon, P., Friedlander, M., and Ruf, W. (2007). Role of protease activated receptor 1 and 2 signaling in hypoxia-induced angiogenesis. Arterioscler Thromb Vasc Biol. 27, 1456-1462.

Van der Velden, J., Barker, D., Barcham, G., Koumoundouros, E., and Snibson, K. (2012). Increased mast cell density and airway responses to allergic and non-allergic stimuli in a sheep model of chronic asthma. PloS one. 7, e37161.

Versteeg, H.H., Schaffner, F., Kerver, M., Ellies, L.G., Andrade-Gordon, P., Mueller, B.M., and Ruf, W. (2008). Protease-activated receptor (PAR) 2, but not PAR1, signaling promotes the development of mammary adenocarcinoma in polyoma middle T mice. Cancer research. 68, 7219-7227.

Vibhuti, A., Gupta, K., Subramanian, H., Guo, Q., and Ali, H. (2011). Distinct and shared roles of beta-arrestin-1 and beta-arrestin-2 on the regulation of C3a receptor signaling in human mast cells. PloS one. 6, e19585.

Wagner, E.F. (2010). Bone development and inflammatory disease is regulated by AP-1 (Fos/Jun). Ann Rheum Dis. 69 Suppl 1, i86-88.

Wang, M., Zhao, J., Zhang, L., Wei, F., Lian, Y., Wu, Y., Gong, Z., Zhang, S., Zhou, J., Cao, K., Li, X., Xiong, W., Li, G., Zeng, Z., and Guo, C. (2017). Role of tumor microenvironment in tumorigenesis. Journal of Cancer. 8, 761-773.

Wells, C.D., Liu, M.Y., Jackson, M., Gutowski, S., Sternweis, P.M., Rothstein, J.D., Kozasa, T., and Sternweis, P.C. (2002). Mechanisms for reversible regulation between G13 and Rho exchange factors. The Journal of biological chemistry. 277, 1174-1181.

Wilson, S., Greer, B., Hooper, J., Zijlstra, A., Walker, B., Quigley, J., and Hawthorne, S. (2005). The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells. The Biochemical journal. 388, 967-972.

Winter, M.C., Shasby, S.S., Ries, D.R., and Shasby, D.M. (2006). PAR2 activation interrupts E-cadherin adhesion and compromises the airway epithelial barrier: protective effect of beta-agonists. American journal of physiology Lung cellular and molecular physiology. 291, L628-635.

Witte, D., Zeeh, F., Gadeken, T., Gieseler, F., Rauch, B.H., Settmacher, U., Kaufmann, R., Lehnert, H., and Ungefroren, H. (2016). Proteinase-Activated Receptor 2 Is a Novel Regulator of TGF-beta Signaling in Pancreatic Cancer. J Clin Med. 5.

Wygrecka, M., Didiasova, M., Berscheid, S., Piskulak, K., Taborski, B., Zakrzewicz, D., Kwapiszewska, G., Preissner, K.T., and Markart, P. (2013). Protease-activated receptors (PAR)-1 and -3 drive epithelial-mesenchymal transition of alveolar epithelial cells - potential role in lung fibrosis. Thromb Haemost. 110, 295-307.

Xiang, Y., Masuko-Hongo, K., Sekine, T., Nakamura, H., Yudoh, K., Nishioka, K., and Kato, T. (2006). Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1beta, TNF-alpha and TGF-beta. Osteoarthritis and cartilage. 14, 1163-1173.

Xie, M., Zhang, L., He, C.S., Xu, F., Liu, J.L., Hu, Z.H., Zhao, L.P., and Tian, Y. (2012). Activation of Notch-1 enhances epithelial-mesenchymal transition in gefitinib-acquired resistant lung cancer cells. J Cell Biochem. 113, 1501-1513.

Xu, W.F., Andersen, H., Whitmore, T.E., Presnell, S.R., Yee, D.P., Ching, A., Gilbert, T., Davie, E.W., and Foster, D.C. (1998). Cloning and characterization of human protease-activated receptor 4. Proceedings of the National Academy of Sciences of the United States of America. 95, 6642-6646.

Xu, Z., Zhu, L., Yao, M., Zhong, G., Dong, Q., and Yu, A. (2015). PTEN plays an important role in thrombin-mediated lung cancer cell functions. Biomed Res Int. 2015, 459170.

Yang, C.C., Hsiao, L.D., Yang, C.M., and Lin, C.C. (2017). Thrombin Enhanced Matrix Metalloproteinase-9 Expression and Migration of SK-N-SH Cells via PAR-1, c-Src, PYK2, EGFR, Erk1/2 and AP-1. Mol Neurobiol. 54, 3476-3491.

Yang, L., Ma, Y., Han, W., Li, W., Cui, L., Zhao, X., Tian, Y., Zhou, Z., Wang, W., and Wang, H. (2015). Proteinase-activated receptor 2 promotes cancer cell migration through RNA methylation-mediated repression of miR-125b. The Journal of biological chemistry. 290, 26627-26637.

Yau, M.K., Liu, L., and Fairlie, D.P. (2013). Toward drugs for protease-activated receptor 2 (PAR2). J Med Chem. 56, 7477-7497.

Yue, D., Li, H., Che, J., Zhang, Y., Tseng, H.H., Jin, J.Q., Luh, T.M., Giroux-Leprieur, E., Mo, M., Zheng, Q., Shi, H., Zhang, H., Hao, X., Wang, C., Jablons, D.M., and He, B. (2014). Hedgehog/Gli promotes epithelial-mesenchymal transition in lung squamous cell carcinomas. J Exp Clin Cancer Res. 33, 34.

Zeeh, F., Witte, D., Gadeken, T., Rauch, B.H., Grage-Griebenow, E., Leinung, N., Fromm, S.J., Stolting, S., Mihara, K., Kaufmann, R., Settmacher, U., Lehnert, H., Hollenberg, M.D., and Ungefroren, H. (2016). Proteinase-activated receptor 2 promotes TGF-beta-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-beta type I receptor ALK5. Oncotarget. 7, 41095-41109.

Zhang, H.J., Wang, H.Y., Zhang, H.T., Su, J.M., Zhu, J., Wang, H.B., Zhou, W.Y., Zhang, H., Zhao, M.C., Zhang, L., and Chen, X.F. (2011). Transforming growth factor-beta1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition. Molecular and cellular biochemistry. 355, 309-314.

Zhang, X., Wang, F., Chen, X., Li, J., Xiang, B., Zhang, Y.Q., Li, B.M., and Ma, L. (2005). Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors. J Neurochem. 95, 169-178.

Zhao, P., Lieu, T., Barlow, N., Metcalf, M., Veldhuis, N.A., Jensen, D.D., Kocan, M., Sostegni, S., Haerteis, S., Baraznenok, V., Henderson, I., Lindstrom, E., Guerrero-Alba, R., Valdez-Morales, E.E., Liedtke, W., McIntyre, P., Vanner, S.J., Korbmacher, C., and Bunnett, N.W. (2014a). Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4. The Journal of biological chemistry. 289, 27215-27234.

Zhao, P., Metcalf, M., and Bunnett, N.W. (2014b). Biased signaling of protease-activated receptors. Frontiers in endocrinology. 5, 67.

Zhao, Y., Wang, H., Li, X., Cao, M., Lu, H., Meng, Q., Pang, H., Li, H., Nadolny, C., Dong, X., and Cai, L. (2014c). Ang II-AT1R increases cell migration through PI3K/AKT and NF-kappaB pathways in breast cancer. J Cell Physiol. 229, 1855-1862.

衛生福利部國民健康署 (2017). 癌症登記線上互動查詢系統 (衛生福利部國民健康署). https://cris.hpa.gov.tw/pagepub/Home.aspx?itemNo=cr.a.10.