在先前的研究中我們得知,膠原蛋白凝膠誘導正常上皮細胞凋亡是由於細胞內鈣離子恆定失調所引起,並且進一步發現膠原蛋白凝膠引發細胞內鈣離子上升的主要原因是藉由活化STIM1分子與Orai1結合所導致。此外，TRPC1在先前的文獻中已經被證實為一種機械力感受性的離子通道，TRPC1也被報導可藉由組合形成STIM-Orai1-TRPC 複合體可引發更大量的鈣池調控鈣離子流。然而TRPC1是否能和STIM以及Orai1結合並且參與在膠原蛋白凝膠所誘導的鈣池調控鈣離子流上升仍然未知。為了證實此假說，我們將wild-type或dominat-negative STIM1 轉染至LLC-PK1上皮細胞穩定表現: STIM1-ΔCAD 在先前報導指出可阻斷STIM1與Orai1結合; STIM1-ΔERM 則可阻斷STIM與Orai1以及TRPC1的結合。在免疫螢光影像的結果中，我們發現膠原蛋白凝膠可增加STIM1與Orai1或TRPC1 的colocalization，但是這個現象則會在穩定表現STIM1-ΔCAD或STIM1-ΔERM所抑制。我們接著進一步分析並發現LLC-PK1上皮細胞培養在膠原蛋白凝膠上則會提升鈣池調控鈣離子流，但是鈣離子流在穩定表現STIM1-ΔCAD或STIM1-ΔERM的細胞則會分別受到抑制70% 及完全抑制。此外我們進一步轉染STIM1或TRPC1 小片段RNA至LLC-PK1上皮細胞，實驗結果指出抑制TRPC1或STIM1蛋白表現量可阻斷30%及80%之鈣池調控鈣離子流。除了SOC 離子通道蛋白外，我們也發現當LLC-PK1上皮細胞培養在膠原蛋白凝膠上會造成細胞骨架中的肌動蛋白及微管蛋白結構破壞,先前的研究也指出細胞骨架的破壞會促進STIM1，Orai1和TRPC1的組裝並且提升鈣池調控鈣離子流。試證實假說，我們使用CytochalasinD或Nocodazole分別破壞肌動蛋白或微管蛋白，結果顯示無論破壞肌動蛋白或微管蛋白皆會造成細胞鈣池調控鈣離子流的增加。最後我們使用四種方法分析細胞凋亡的比率，在低分子DNA電泳的結果中我們發現當LLC-PK1上皮細胞培養再膠原蛋白凝膠上則會造成明顯的DNA裂解，然而這個現象在穩定表現STIM1-ΔCAD或STIM1-ΔERM的細胞中不受抑制,反之在西方墨點法的結果中顯示,穩定表現wild-type STIM1的細胞會抑制cleavage-caspse-3的蛋白表現量。在Hoechst 33258 的螢光染色結果中顯示，穩定表現STIM1-ΔCAD或STIM1-ΔERM皆只能輕微降低細胞造成DNA裂解的比率,然而在穩定表現wild-type STIM1的細胞中則能大量降低細胞DNA裂解的比率。我們還更進一步使用STIM1及TRPC1小片段RNA或SOC channel 抑制劑處理細胞並且分析細胞凋亡的比率，結果顯示無論是用小片段RNA抑制TRPC1或STIM1的蛋白表現量皆能分別降低30%及50%的細胞凋亡比率。

綜合以上數據我們得到結論:當LLC-PK1上皮細胞培養在膠原蛋白凝膠上時，STIM1，Orai1及TRPC1皆會參與並促進調節鈣池調控鈣離子流上升，在過程中細胞骨架的破壞可能參與其中的調控。

Previously we demonstrated that collagen gel-induced apoptosis mediated by Ca2+ homeostasis disturbance which was resulted from STIM1 coupled to Orai1, a essential pore subunit of store-operated Ca2+ channels in normal epithelial cells. On the other hand, TRPC1 have been proposed as mechanosensitive ion channels, assembly of STIM1-Orai1-TRPC1 complex have reported to elevate store-operated Ca2+ entry (SOCE). However, whether TRPC1 coordinates with STIM-Orai1 and contributes to collagen gel-induced elevation of SOCE is still unclear. To test this hypothesis, we established LLC-PK1 cells stably expressed two types of dominant negative STIM1 construct : STIM1-ΔCAD, which was reported a mutant prevented STIM-Orai1 interaction ; STIM1-ΔERM, which blocked Orai1 and TRPC1 coupled to STIM1. In immunofluoresence results, LLC-PK1 cells cultured on collagen gel enhanced the colocalization of STIM-Orai1 and STIM1-TRPC1, and this phenomenon was inhibited in cells overexpressed STIM-ΔCAD or STIM-ΔERM. Furthermore, SOCE was increased when cells cultured on collagen gel, but inhibited by STIM1-ΔCAD for 70% and totally blocked by STIM1-ΔERM. Besides, knockdown of TRPC1 or STIM1 also inhibited collagen gel-induced Ca2+ influx by 30% and 80% respectively. In addition to SOC channel molecules, the actin filament and microtubule were disorganized when LLC-PK1 cells seeded on collagen gel. Previous studies indicated that disruption of actin filament or microtubule enhanced SOCE resulted from STIM1-Orai1-TRPC1 interaction. To test this hypothesis, we applied cytochalasin D and nocodazole to disrupt actin filament and microtubule, the result showed that either cytochalasin D or nocodazole were up-regulated SOCE of LLC-PK1 cells. Finally, we analyzed apoptosis ratio in four methods. In low molecular DNA electrophoresis result, DNA ladder obviously in LLC-PK1 cells cultured on collagen gel, but this phenomenon were not prevented from STIM-ΔCAD or STIM-ΔERM overexpressed cells. In contrast, western blot results showed that cells overexpressed wild-type STIM1 prevented cleavage caspase-3 expression under collagen gel stimulation. In Hoechst 33258 staining and flow cytometry results, overexpression of STIM1-ΔCAD or STIM-ΔERM were slightly decreased apoptosis ratio and nucleus fragmentation, but dramatically decreased in wild-type STIM1 overexpressed cells. Moreover, we also applied SOC channel inhibitors or siRNA for STIM1 and TRPC1 to analyzed the apoptosis ratio by flow cytometry, results showed that collagen gel-induced Ca2+ influx was prevented by knockdown of STIM1 or TRPC1 as well as SOC channel inhibitors.

Taken together, we concluded that coordination of STIM1, Orai1 and TRPC1 contributed to collagen gel-induced elevation of store-operated Ca2+ entry and apoptosis in LLC-PK1 cells. The disorganization of cytoskeleton may involved in the regulation of this process.

Abdullaev, I.F. et al. Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation. Circulation Research 103, 1289-1299 (2008).

Albert, A.P., Saleh, S.N., Peppiatt-Wildman, C.M. & Large, W.A. Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells. The Journal of Physiology 583, 25-36 (2007).

Aldrich, R.A. et al. Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions. PLoS Biology 9, e1001025 (2011).

Ambudkar, I.S. Ca2+ signaling microdomains:platforms for the assembly and regulation of TRPC channels. Trends Pharmacol Sci 27, 25-32 (2006).

Arnadottir, J. & Chalfie, M. Eukaryotic mechanosensitive channels. Annu Rev Biophys 39, 111-137 (2010).

Baba, Y. et al. Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum. Proceedings of the National Academy of Sciences 103, 16704-16709 (2006).

Bandyopadhyay, B.C. Apical Localization of a Functional TRPC3/TRPC6-Ca2+-Signaling Complex in Polarized Epithelial Cells: ROLE IN APICAL Ca2+ INFLUX. Journal of Biological Chemistry 280, 12908-12916 (2004).

Bezzerides, V.J., Ramsey, I.S., Kotecha, S., Greka, A. & Clapham, D.E. Rapid vesicular translocation and insertion of TRP channels. Nature Cell Biology 6, 709-720 (2004).

Bird, G.S. et al. STIM1 is a calcium sensor specialized for digital signaling. Curr Biol 19, 1724-1729 (2009).

Bollimuntha, S., Cornatzer, E. & Singh, B.B. Plasma membrane localization and function of TRPC1 is dependent on its interaction with beta-tubulin in retinal epithelium cells. Vis Neurosci 22, 163-170 (2005).

Brandman, O., Liou, J., Park, W.S. & Meyer, T. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131, 1327-1339 (2007).

Brazer, S.C., Singh, B.B., Liu, X., Swaim, W. & Ambudkar, I.S. Caveolin-1 contributes to assembly of store-operated Ca2+ influx channels by regulating plasma membrane localization of TRPC1. J Biol Chem 278, 27208-27215 (2003).

Carafoli, E. Calcium signaling: A tale for all seasons. Proceedings of the National Academy of Sciences 99, 1115-1122 (2002).

Chen, C.S., Tan, J. & Tien, J. Mechanotransduction at cell-matrix and cell-cell contacts. Annu Rev Biomed Eng 6, 275-302 (2004).

Chen, Y.F. et al. Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Proceedings of the National Academy of Sciences 108, 15225-15230 (2011).

Cheng, K.T., Liu, X., Ong, H.L. & Ambudkar, I.S. Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. J Biol Chem 283, 12935-12940 (2008).

Cheng, K.T., Liu, X., Ong, H.L., Swaim, W. & Ambudkar, I.S. Local Ca(2)+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca(2)+ signals required for specific cell functions. PLoS Biol 9, e1001025 (2011).

Covington, E.D., Wu, M.M. & Lewis, R.S. Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1. Molecular Biology of the Cell 21, 1897-1907 (2010).

DeHaven, W.I. et al. TRPC channels function independently of STIM1 and Orai1. The Journal of Physiology 587, 2275-2298 (2009).

Deng, X., Wang, Y., Zhou, Y., Soboloff, J. & Gill, D.L. STIM and Orai: dynamic intermembrane coupling to control cellular calcium signals. J Biol Chem 284, 22501-22505 (2009).

Di Capite, J., Ng, S.W. & Parekh, A.B. Decoding of Cytoplasmic Ca2+ Oscillations through the Spatial Signature Drives Gene Expression. Current Biology 19, 853-858 (2009).

Feng, M. et al. Store-independent activation of Orai1 by SPCA2 in mammary tumors. Cell 143, 84-98 (2010).

Feske, S. et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179-185 (2006).

Flourakis, M. et al. Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells. Cell Death and Disease 1, e75 (2010).

Follonier, L., Schaub, S., Meister, J.J. & Hinz, B. Myofibroblast communication is controlled by intercellular mechanical coupling. Journal of Cell Science 121, 3305-3316 (2008).

Formigli, L. et al. Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation. J Cell Sci 122, 1322-1333 (2009).

Freichel, M. Functional role of TRPC proteins in native systems: implications from knockout and knock-down studies. The Journal of Physiology 567, 59-66 (2005).

Fukushima, M., Tomita, T., Janoshazi, A. & Putney, J.W. Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities. Journal of Cell Science 125, 4354-4361 (2012).

Galán, C., Dionisio, N., Smani, T., Salido, G.M. & Rosado, J.A. The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1. Biochemical Pharmacology 82, 400-410 (2011).

Gross, S.A. et al. Murine ORAI2 splice variants form functional Ca2+ release-activated Ca2+ (CRAC) channels. J Biol Chem 282, 19375-19384 (2007).

Gwack, Y. et al. Biochemical and functional characterization of Orai proteins. J Biol Chem 282, 16232-16243 (2007).

Hajkova, Z. et al. STIM1-Directed Reorganization of Microtubules in Activated Mast Cells. The Journal of Immunology 186, 913-923 (2010).

Huang, G.N. et al. STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels. Nature Cell Biology 8, 1003-1010 (2006).

Jardin, I., Lopez, J.J., Salido, G.M. & Rosado, J.A. Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1-forming Ca2+ Channels. Journal of Biological Chemistry 283, 25296-25304 (2008).

Ji, W. et al. Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proceedings of the National Academy of Sciences 105, 13668-13673 (2008).

Kar, P., Nelson, C. & Parekh, Anant B. CRAC Channels Drive Digital Activation and Provide Analog Control and Synergy to Ca2+-Dependent Gene Regulation. Current Biology 22, 242-247 (2012).

Kim, M.S. et al. Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1. Journal of Biological Chemistry 284, 9733-9741 (2009).

Kiselyov, K. et al. Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478-482 (1998).

Kohler, R. et al. Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler Thromb Vasc Biol 26, 1495-1502 (2006).

Krapivinsky, G., Krapivinsky, L., Stotz, S.C., Manasian, Y. & Clapham, D.E. POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters. Proceedings of the National Academy of Sciences 108, 19234-19239 (2011).

Lee, J., Ishihara, A., Oxford, G., Johnson, B. & Jacobson, K. Regulation of cell movement is mediated by stretch-activated calcium channels. Nature 400, 382-386 (1999).

Lewis, R.S. The molecular choreography of a store-operated calcium channel. Nature 446, 284-287 (2007).

Liao, Y. et al. Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proceedings of the National Academy of Sciences 105, 2895-2900 (2008).

Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol 15, 1235-1241 (2005).

Maroto, R. et al. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol 7, 179-185 (2005).

Maroto, R. et al. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nature Cell Biology 7, 179-185 (2005).

Mehta, D. et al. RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability. J Biol Chem 278, 33492-33500 (2003).

Muik, M. et al. A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels. J Biol Chem 284, 8421-8426 (2009).

Muik, M. et al. STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation. The EMBO Journal 30, 1678-1689 (2011).

Mullins, F.M., Park, C.Y., Dolmetsch, R.E. & Lewis, R.S. STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels. Proceedings of the National Academy of Sciences 106, 15495-15500 (2009).

Murata, T. et al. Genetic Evidence Supporting Caveolae Microdomain Regulation of Calcium Entry in Endothelial Cells. Journal of Biological Chemistry 282, 16631-16643 (2007).

Oláh, T. et al. Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells. Cell Calcium 49, 415-425 (2011).

Ong, H.L. et al. Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 282, 9105-9116 (2007).

Oritani, K. & Kincade, P.W. Identification of stromal cell products that interact with pre-B cells. J Cell Biol 134, 771-782 (1996).

Pani, B. et al. Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1. Proc Natl Acad Sci U S A 106, 20087-20092 (2009).

Pani, B. et al. Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE). Journal of Biological Chemistry 283, 17333-17340 (2008).

Pani, B. & Singh, B.B. Lipid rafts/caveolae as microdomains of calcium signaling. Cell Calcium 45, 625-633 (2009).

Parekh, A. Mitochondrial regulation of store-operated CRAC channels. Cell Calcium 44, 6-13 (2008).

Parekh, A.B. On the activation mechanism of store-operated calcium channels. Pflügers Archiv - European Journal of Physiology 453, 303-311 (2006).

Parekh, A.B. Functional consequences of activating store-operated CRAC channels. Cell Calcium 42, 111-121 (2007).

Parekh, A.B. Ca2+ microdomains near plasma membrane Ca2+ channels: impact on cell function. The Journal of Physiology 586, 3043-3054 (2008).

Parekh, A.B. Store-operated channels: mechanisms and function. The Journal of Physiology 586, 3033-3033 (2008).

Parekh, A.B. Local Ca2+influx through CRAC channels activates temporally and spatially distinct cellular responses. Acta Physiologica 195, 29-35 (2009).

Parekh, A.B. Decoding cytosolic Ca2+ oscillations. Trends in Biochemical Sciences 36, 78-87 (2011).

Park, C.Y. et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876-890 (2009).

Parvez, S. et al. STIM2 protein mediates distinct store-dependent and store-independent modes of CRAC channel activation. FASEB J 22, 752-761 (2008).

Paszek, M.J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241-254 (2005).

Penna, A. et al. The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456, 116-120 (2008).

Penner, R., Matthews, G. & Neher, E. Regulation of calcium influx by second messengers in rat mast cells. Nature 334, 499-504 (1988).

Pleines, I. et al. Rac1 is essential for phospholipase C-gamma2 activation in platelets. Pflugers Arch 457, 1173-1185 (2009).

Putney, J.W. & Bird, G.S. Cytoplasmic calcium oscillations and store-operated calcium influx. The Journal of Physiology 586, 3055-3059 (2008).

Rao, Jaladanki N. et al. Induced TRPC1 expression sensitizes intestinal epithelial cells to apoptosis by inhibiting NF-κB activation through Ca2+ influx. Biochemical Journal 397, 77 (2006).

Redondo, P.C., Jardin, I., Lopez, J.J., Salido, G.M. & Rosado, J.A. Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1783, 1163-1176 (2008).

Ridley, A.J. et al. Cell migration: integrating signals from front to back. Science 302, 1704-1709 (2003).

Roos, J. et al. STIM1, an essential and conserved component of store-operated Ca2+ channel function. J Cell Biol 169, 435-445 (2005).

Sharma, P. et al. Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release. Journal of Cell Science 123, 3061-3070 (2010).

Smyth, Jeremy T., Beg, Amber M., Wu, S., Putney, James W. & Rusan, Nasser M. Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle. Current Biology 22, 1487-1493 (2012).

Smyth, J.T., DeHaven, W.I., Bird, G.S. & Putney, J.W. Ca2+-store-dependent and -independent reversal of Stim1 localization and function. Journal of Cell Science 121, 762-772 (2008).

Stathopulos, P.B., Zheng, L., Li, G.-Y., Plevin, M.J. & Ikura, M. Structural and Mechanistic Insights into STIM1-Mediated Initiation of Store-Operated Calcium Entry. Cell 135, 110-122 (2008).

Vandebrouck, A. et al. Regulation of capacitative calcium entries by 1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of 1-syntrophin. The FASEB Journal 21, 608-617 (2007).

Varga-Szabo, D. et al. Store-operated Ca(2+) entry in platelets occurs independently of transient receptor potential (TRP) C1. Pflugers Arch 457, 377-387 (2008).

Venkatachalam, K. & Montell, C. TRP channels. Annu Rev Biochem 76, 387-417 (2007).

Vig, M. et al. CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr Biol 16, 2073-2079 (2006).

Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nature Reviews Molecular Cell Biology 7, 265-275 (2006).

Wang, Y., Lin, L. & Zheng, C. Downregulation of Orai1 expression in the airway alleviates murine allergic rhinitis. Exp Mol Med 44, 177-190 (2012).

Woodard, G.E., Salido, G.M. & Rosado, J.A. Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion. Am J Physiol Cell Physiol 294, C1323-1331 (2008).

Yu, F., Sun, L. & Machaca, K. Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis. Proc Natl Acad Sci U S A 106, 17401-17406 (2009).

Yuan, J.P. et al. SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat Cell Biol 11, 337-343 (2009).

Yuan, J.P., Zeng, W., Huang, G.N., Worley, P.F. & Muallem, S. STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels. Nat Cell Biol 9, 636-645 (2007).

Zeng, B., Chen, G.-L. & Xu, S.-Z. Store-independent pathways for cytosolic STIM1 clustering in the regulation of store-operated Ca2+ influx. Biochemical Pharmacology 84, 1024-1035 (2012).

Zeng, W. et al. STIM1 Gates TRPC Channels, but Not Orai1, by Electrostatic Interaction. Molecular Cell 32, 439-448 (2008).