Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis

Jan Görtzen, Robert Schierwagen, Jeanette Bierwolf, Sabine Klein, Frank E Uschner, Peter F van der Ven, Dieter O Fürst, Christian P Strassburg, Wim Laleman, Jörg-Matthias Pollok, Jonel Trebicka

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INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.

METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.

RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.

CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

OriginalsprogEngelsk
Artikelnummer359
TidsskriftFrontiers in Physiology
Vol/bind6
Antal sider9
ISSN1664-042X
DOI
StatusUdgivet - 2015

Fingeraftryk

Sarcoma
Hepatic Stellate Cells
Liver
Liver Cirrhosis
Hepatocytes
Albumins
Phospho-Specific Antibodies
rho-Associated Kinases
Messenger RNA
Acrylamide
Extracellular Matrix Proteins
Liver Transplantation
Transcriptional Activation
Ligation
Cultured Cells
Up-Regulation
Polymerase Chain Reaction

Citer dette

Görtzen, J., Schierwagen, R., Bierwolf, J., Klein, S., Uschner, F. E., van der Ven, P. F., ... Trebicka, J. (2015). Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis. Frontiers in Physiology, 6, [359]. https://doi.org/10.3389/fphys.2015.00359
Görtzen, Jan ; Schierwagen, Robert ; Bierwolf, Jeanette ; Klein, Sabine ; Uschner, Frank E ; van der Ven, Peter F ; Fürst, Dieter O ; Strassburg, Christian P ; Laleman, Wim ; Pollok, Jörg-Matthias ; Trebicka, Jonel. / Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis. I: Frontiers in Physiology. 2015 ; Bind 6.
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title = "Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis",
abstract = "INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.",
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author = "Jan G{\"o}rtzen and Robert Schierwagen and Jeanette Bierwolf and Sabine Klein and Uschner, {Frank E} and {van der Ven}, {Peter F} and F{\"u}rst, {Dieter O} and Strassburg, {Christian P} and Wim Laleman and J{\"o}rg-Matthias Pollok and Jonel Trebicka",
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Görtzen, J, Schierwagen, R, Bierwolf, J, Klein, S, Uschner, FE, van der Ven, PF, Fürst, DO, Strassburg, CP, Laleman, W, Pollok, J-M & Trebicka, J 2015, 'Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis', Frontiers in Physiology, bind 6, 359. https://doi.org/10.3389/fphys.2015.00359

Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis. / Görtzen, Jan; Schierwagen, Robert; Bierwolf, Jeanette; Klein, Sabine; Uschner, Frank E; van der Ven, Peter F; Fürst, Dieter O; Strassburg, Christian P; Laleman, Wim; Pollok, Jörg-Matthias; Trebicka, Jonel.

I: Frontiers in Physiology, Bind 6, 359, 2015.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis

AU - Görtzen, Jan

AU - Schierwagen, Robert

AU - Bierwolf, Jeanette

AU - Klein, Sabine

AU - Uschner, Frank E

AU - van der Ven, Peter F

AU - Fürst, Dieter O

AU - Strassburg, Christian P

AU - Laleman, Wim

AU - Pollok, Jörg-Matthias

AU - Trebicka, Jonel

PY - 2015

Y1 - 2015

N2 - INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

AB - INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

KW - Journal Article

U2 - 10.3389/fphys.2015.00359

DO - 10.3389/fphys.2015.00359

M3 - Journal article

VL - 6

JO - Frontiers in Physiology

JF - Frontiers in Physiology

SN - 1664-042X

M1 - 359

ER -

Görtzen J, Schierwagen R, Bierwolf J, Klein S, Uschner FE, van der Ven PF et al. Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis. Frontiers in Physiology. 2015;6. 359. https://doi.org/10.3389/fphys.2015.00359