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Function of ion transport proteins in pancreatic stellate cells (PSCs)

Das Pankreaskarzinom ist durch ein fibrotisches und hypoxisches Tumormikromilieu gekennzeichnet. Daran sind vor allen pankreatische Sternzellen beteiligt. Sternzellen und Krebszellen aktivieren sich gegenseitig parakrin. Das führt auch zur Stimulation der Migration von Sternzellen, was Voraussetzung für die effiziente Kommunikation mit den Krebszellen ist. Transient receptor potential (TRP) Kanäle wirken dabei als Sensoren und Modifikatoren des (Tumor)Mikromilieus. Sie sind gleichzeitig wesentliche Komponenten der Transduktion und Effektormechanismen, die den zellulären Antworten auf das Mikromilieu wie z.b Hypoxie zugrunde liegen. Diese Studie zeigt zum ersten Mal, dass die Ca2+-permeablen TRPC1- und TRPC6-Kanäle für die Reaktion der Sternzellen auf Hypoxie erforderlich sind.

  Pancreatic cancer is characterized by the presence of a highly fibrotic and hypoxic tumour microenvironment, primarily formed by pancreatic stellate cells (PSCs). This activates both PSCs and cancer cells to secrete various stimulants attracting more PSCs to the tumour area. Thus, the ability of PSCs to migrate is recognized as a consequence of their activated state and as a requirement for efficient communication with cancer cells. Evidence supports a role of the transient receptor potential (TRP) channels as sensors and modifiers of the (tumour) microenvironment, together with being essential components of the transduction and effector mechanisms underlying cellular responses to microenvironmental cues such as hypoxia. This study shows for the first time that the Ca2+-permeable TRPC1- and TRPC6-channels are important for the response of PSCs to hypoxia through a sensor, modifier and/or transduction/effector mechanism thereby affecting their migratory behavior.

Titel: Function of ion transport proteins in pancreatic stellate cells (PSCs)
Verfasser: Nielsen, Nikolaj GND
Gutachter: Schwab, Albrecht
Organisation: FB 13: Biologie
FB 05: Medizinische Fakultät
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 25.06.2015
Datum der letzten Änderung: 27.07.2015
Schlagwörter: Pankreatische Sternzellen; TRP Kanäle; Migration; Tumormilieu; Mikromilieu- sensor/Modifikator/Effektor
PSC; TRP channels; migration; tumour microenvironment; microenvironmental sensors/modifiers/effectors
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-29229486062
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-29229486062
Onlinezugriff:
Inhalt:
DECLARATION AND CERTIFICATION OF ORIGINALITY ................................................................................... i
PREFACE ........................................................................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................................................................. iii
CURRICULUM VITAE ................................................................................................................................................... iv
CONTRIBUTIONS BY THE AUTHOR ....................................................................................................................... vi
OVERALL AIM OF THE IONTRAC PROJECT .......................................................................................................... ix
SUMMARY ........................................................................................................................................................................ x
GERMAN RESÚME......................................................................................................................................................... xi
TABLE OF CONTENTS ................................................................................................................................................... I
ABBREVIATIONS ........................................................................................................................................................... V
INTRODUCTION
1.1 Anatomy and Function of the Pancreas .........................................................................1
1.2 Pancreatic Ductal Adenocarcinoma ...............................................................................3
1.2.1 Clinical Manifestation and Risk Factors.................................................................................. 3
1.2.2 Characterisation and Molecular Pathogenesis of PDAC ........................................................ 4
1.3 Pancreatic Stellate Cells – Key Players in PDAC Progression and Metastasis .............6
1.3.1 The History and Characterization of Pancreatic Stellate Cells ............................................... 7
1.3.2 Pancreatic Stellate Cell Activation.......................................................................................... 8
1.3.3 Pancreatic Stellate Cells in the (PDAC) Microenvironment .................................................10
1.3.3.1 Hypoxia – A Major Feature in PDAC and PSC Activation ............................................................. 11
1.3.3.2 PSCs Mutual Signalling with PDAC Cells ...................................................................................... 12
1.3.4 Autocrine Stimulation in PSCs ..............................................................................................14
1.4 Cell Migration ................................................................................................................ 15
1.4.1 Basic Principles of Cell Migration .........................................................................................15
1.4.2 Calcium-dependent Signalling in Cell Migration ..................................................................17
1.5 TRP Channels in Migration and as Microenvironmental Sensors, Modifiers and
Effectors ............................................................................................................................... 19
1.5.1 TRP Channel Family ..............................................................................................................19
1.5.1.1 TRP Channel Function .................................................................................................................. 19
1.5.1.2 TRP Channels in Cell Migration and Interaction Partners ............................................................ 20
1.5.2 TRP Channels as Sensors and Effectors of the Tumour Microenvironment ........................23
1.5.2.1 TRP Channels as Mechanosensors and Modulators of the TME .................................................. 23
1.5.2.2 TRP Channels in Hypoxia and Oxidative Stress ............................................................................ 24
PROJECT AIM
2.1 Hypothesis, Objective and Strategy .............................................................................. 29
MATERIALS AND METHODS
3.1 Materials ........................................................................................................................ 32
3.1.1 Media ...................................................................................................................................32
3.1.2 Chemicals and Kits ................................................................................................................32
3.1.3 Instruments ..........................................................................................................................34
3.1.4 Oligonucleotides...................................................................................................................35
3.2 Mouse Strains ................................................................................................................ 36
3.2.1 Pancreatic Stellate Cells .......................................................................................................36
3.3 Cell Biological Methods ................................................................................................. 37
3.3.1 Reactivating Frozen RLT-PSCs ..............................................................................................37
3.3.2 Isolation of Mice Pancreatic Stellate Cells ...........................................................................37
3.3.2.1 Identification of mPSCs ................................................................................................................ 38
3.3.3 Cell culture, Passaging and Cell Count .................................................................................39
3.4 Molecular Biological Methods ...................................................................................... 39
3.4.1 Protein Biochemical Methods ..............................................................................................39
3.4.1.1 Protein Isolation and Estimation .................................................................................................. 39
3.4.1.2 Protein Detection and Analysis .................................................................................................... 40
3.4.2 RNA Extraction and Purification from PSCs ..........................................................................43
3.4.3 Reverse Transcriptase PCR ...................................................................................................43
3.4.4 Polymerase Chain Reaction (PCR) ........................................................................................44
3.4.5 DNA Electrophoresis ............................................................................................................45
3.4.6 Quantitative Real-time PCR ..................................................................................................46
3.5 In Vitro Migration Assays .............................................................................................. 48
3.5.1 Hypoxia treatment ...............................................................................................................48
3.5.2 Two-Dimensional Migration .................................................................................................49
3.5.3 Three-Dimensional Migration ..............................................................................................50
3.5.4 Chemotaxis Assay .................................................................................................................52
3.5.5 Autocrine Stimulation Assay ................................................................................................53
3.5.6 Analysis of Migration Data ...................................................................................................53
3.6 Oxygen Consumption and Extracellular Acidification Measurements ...................... 55
3.6.1 Estimation of Oxygen consumption in 3D chamber.............................................................56
3.7 Manganese Quenching .................................................................................................. 58
3.8 Calcium Entry................................................................................................................. 60
3.9 Calpain Activity .............................................................................................................. 60
3.10 Statistical Analysis ...................................................................................................... 62
RESULTS
4.1 mPSC Identification ....................................................................................................... 63
4.2 Quantitative Assessment of TRPC Channel Expression in mPSCs .............................. 64
4.3 mPSC Migration in Three Dimensional Space ............................................................. 65
4.3.1 Oxidative Cell Stress in Three Dimensional mPSC Migration ...............................................66
4.4 Hypoxia and mPSC Migration ....................................................................................... 68
4.4.1 Chemically Induced Hypoxia Stimulates mPSC Migration ...................................................68
4.4.1.1 DMOG Induces mPSC Migration in a Dose-Responsive Manner .................................................. 68
4.4.1.2 Chronic Treatment with DMOG Induces an Anaerobic mPSC Phenotype .................................... 69
4.4.2 mPSCs Morphology Upon Hypoxia Stimulation ...................................................................71
4.4.3 TRPC1-/- and TRPC6-/- mPSCs Display Diminished Motility Under Chemically Induced
Hypoxia ..........................................................................................................................................72
4.4.4 Attenuated Autocrine Stimulation of TRPC1-/- and TRPC6-/- mPSCs Under Chemically
Induced Hypoxia ............................................................................................................................74
4.4.5 TRPC1-/- and TRPC6-/- mPSCs Display Diminished Motility Under Hypoxia Stimulation .......75
4.5 TRPC Channel Expression in mPSCs under Hypoxia ................................................... 78
4.6 Assessment of Autocrine Stimulation of mPSCs under Hypoxia ................................ 80
4.7 Calcium Influx in Hypoxia Stimulated mPSCs .............................................................. 82
4.7.1 Calpain – a Downstream Effector in PSC Migration .............................................................83
4.8 PDGF Stimulation of mPSC Migration ........................................................................... 84
4.8.1 PDGF Stimulates mPSC Migration in Three Dimensional Space ..........................................84
4.8.2 PDGF Chemotaxis .................................................................................................................86
DISCUSSION
5.1 The “Ideal” Migration Setup .......................................................................................... 89
5.2 Hypoxia in PSC Activation ............................................................................................. 90
5.3 TRPC1 and TRPC6 Channels in the Hypoxia-Mediated Activation of mPSCs ............. 91
5.3.1 TRPC1 Channel: Part of an Indirect Sensor Mechanism to Hypoxia in mPSCs .....................91
5.3.1.1 The TRPC1 Channel is not Implicated in the Secretion of Autocrine Stimulants .......................... 94
5.3.2 TRPC6 Channel: A Central Component in the Response of mPSCs to Hypoxia....................94
5.3.2.1 The TRPC6 Channel in a Modifier Mechanism ............................................................................. 95
5.3.2.2 TRPC6 Channel: Important for Hypoxia-mediated Calcium Entry ................................................ 97
5.4 Future Objectives .......................................................................................................... 99
CONCLUDING REMARKS AND PERSPECTIVES
6.1 Conclusions .................................................................................................................. 102
6.2 Perspectives ................................................................................................................. 104
REFERENCES
7. Reference List ................................................................................................................ 107