TRPC6 channels modulate the response of pancreatic stellate cells to hypoxia
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Pancreatic cancer is characterized by a massive fibrosis (desmoplasia), which is primarily caused by activated pancreatic stellate cells (PSCs). This leads to a hypoxic tumor microenvironment further reinforcing the activation of PSCs by stimulating their secretion of growth factors and chemokines. Since many of them elicit their effects via G-protein-coupled receptors (GPCRs), we tested whether TRPC6 channels, effector proteins of many G-protein-coupled receptor pathways, are required for the hypoxic activation of PSCs. Thus far, the function of ion channels in PSCs is virtually unexplored. qPCR revealed TRPC6 channels to be one of the most abundant TRPC channels in primary cultures of murine PSCs. TRPC6 channel function was assessed by comparing PSCs from TRPC6−/− mice and wildtype (wt) littermates. Cell migration, Ca2+ signaling, and cytokine secretion were analyzed as readout for PSC activation. Hypoxia was induced by incubating PSCs for 24 h in 1% O2 or chemically with dimethyloxalylglycine (DMOG). PSCs migrate faster in response to hypoxia. Due to reduced autocrine stimulation, TRPC6−/− PSCs fail to increase their rate of migration to the same level as wt PSCs under hypoxic conditions. This defect could not be overcome by the stimulation with platelet-derived growth factor. In line with these results, calcium influx is increased in wt but not TRPC6−/− PSCs under hypoxia. We conclude that TRPC6 channels of PSCs are major effector proteins in an autocrine stimulation pathway triggered by hypoxia.
KeywordsTRPC6 channel Pancreatic stellate cell Migration Tumor microenvironment Hypoxia
This work was supported by the Marie Curie Initial Training Network IonTraC (FP7-PEOPLE-2011-ITN grant agreement no. 289648) and Deutsche Forschungsgemeinschaft, Cells-in-Motion Cluster of Excellence (EXC 1003—CiM), University of Münster, Germany, and DFG SCHW 407/17-1.
Compliance with Ethical Standards
Conflicts of Interest
At the time of the project SC and IK were employees of Bayer Pharma AG. It did not impact the study design nor data interpretation. Other authors do not have a conflict of interest.
- 5.Chigurupati S, Venkataraman R, Barrera D, Naganathan A, Madan M, Paul L, Pattisapu JV, Kyriazis GA, Sugaya K, Bushnev S, Lathia JD, Rich JN, Chan SL (2010) Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness. Cancer Res 70:418–427CrossRefPubMedGoogle Scholar
- 6.Couvelard A, O'Toole D, Leek R, Turley H, Sauvanet A, Degott C, Ruszniewski P, Belghiti J, Harris AL, Gatter K, Pezzella F (2005) Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas. Histopathology 46:668–676CrossRefPubMedGoogle Scholar
- 7.Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, Obst M, Yildirim E, Salanova B, Kalwa H, Essin K, Pinkenburg O, Luft FC, Gudermann T, Birnbaumer L (2005) Increased vascular smooth muscle contractility in TRPC6−/− mice. Mol Cell Biol 25:6980–6989CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Eguchi D, Ikenaga N, Ohuchida K, Kozono S, Cui L, Fujiwara K, Fujino M, Ohtsuka T, Mizumoto K, Tanaka M (2013) Hypoxia enhances the interaction between pancreatic stellate cells and cancer cells via increased secretion of connective tissue growth factor. J Surg Res 181:225–233CrossRefPubMedGoogle Scholar
- 17.Ferdek PE, Jakubowska MA (2017) Biology of pancreatic stellate cells-more than just pancreatic cancer. Pflugers ArchGoogle Scholar
- 24.Jakubowska MA, Ferdek PE, Gerasimenko OV, Gerasimenko JV, Petersen OH (2016) Nitric oxide signals are interlinked with calcium signals in normal pancreatic stellate cells upon oxidative stress and inflammation. Open Biol 6Google Scholar
- 29.Liu X, Cheng KT, Bandyopadhyay BC, Pani B, Dietrich A, Paria BC, Swaim WD, Beech D, Yildrim E, Singh BB, Birnbaumer L, Ambudkar IS (2007) Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1−/− mice. Proc Natl Acad Sci U S A 104:17542–17547Google Scholar
- 30.Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ CT Method. Methods 25:402–408Google Scholar
- 36.Rebours V, Albuquerque M, Sauvanet A, Ruszniewski P, Levy P, Paradis V, Bedossa P, Couvelard A (2013) Hypoxia pathways and cellular stress activate pancreatic stellate cells: development of an organotypic culture model of thick slices of normal human pancreas. PLoS One 8:e76229CrossRefPubMedPubMedCentralGoogle Scholar
- 43.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
- 44.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 3:649CrossRefPubMedPubMedCentralGoogle Scholar
- 46.Zhou X, Ye Y, Sun Y, Li X, Wang W, Privratsky B, Tan S, Zhou Z, Huang C, Wei YQ, Birnbaumer L, Singh BB, Wu M (2015) Transient receptor potential channel 1 deficiency impairs host defense and proinflammatory responses to bacterial infection by regulating protein kinase C alpha signaling. Mol Cell Biol 35:2729–2739CrossRefPubMedPubMedCentralGoogle Scholar