Abstract

Aim:To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).Methods:Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.Results:Thrombin (0.3-3.0 U/mL) caused a concentration-and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH 2 (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH 2 (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH 2 (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3-30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3-3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1-10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.Conclusion:Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.

Original languageEnglish
Pages (from-to)49-56
Number of pages8
JournalActa Pharmacologica Sinica
Volume33
Issue number1
DOIs
Publication statusPublished - Jan 2012

Fingerprint

PAR-1 Receptor
Connective Tissue Growth Factor
JNK Mitogen-Activated Protein Kinases
Transcription Factor AP-1
Vascular Smooth Muscle
Thrombin
Smooth Muscle Myocytes
antineoplaston A10
Luciferases
Curcumin
Reporter Genes
Transfection
Western Blotting
Phosphorylation
Cell Line

Keywords

  • activator protein-1 (AP-1)
  • c-Jun N-terminal kinase (JNK)
  • connective tissue growth factor
  • mitogen-activated protein kinase (MAPK)
  • protease-activated receptor
  • thrombin
  • vascular smooth muscle cell

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)

Cite this

Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway. / Ko, Wen Chin; Chen, Bing Chang; Hsu, Ming Jen; Tsai, Chia Ti; Hong, Chuang Ye; Lin, Chien Huang.

In: Acta Pharmacologica Sinica, Vol. 33, No. 1, 01.2012, p. 49-56.

Research output: Contribution to journalArticle

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title = "Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway",
abstract = "Aim:To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).Methods:Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.Results:Thrombin (0.3-3.0 U/mL) caused a concentration-and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH 2 (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH 2 (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH 2 (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3-30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3-3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1-10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.Conclusion:Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.",
keywords = "activator protein-1 (AP-1), c-Jun N-terminal kinase (JNK), connective tissue growth factor, mitogen-activated protein kinase (MAPK), protease-activated receptor, thrombin, vascular smooth muscle cell",
author = "Ko, {Wen Chin} and Chen, {Bing Chang} and Hsu, {Ming Jen} and Tsai, {Chia Ti} and Hong, {Chuang Ye} and Lin, {Chien Huang}",
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T1 - Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway

AU - Ko, Wen Chin

AU - Chen, Bing Chang

AU - Hsu, Ming Jen

AU - Tsai, Chia Ti

AU - Hong, Chuang Ye

AU - Lin, Chien Huang

PY - 2012/1

Y1 - 2012/1

N2 - Aim:To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).Methods:Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.Results:Thrombin (0.3-3.0 U/mL) caused a concentration-and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH 2 (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH 2 (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH 2 (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3-30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3-3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1-10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.Conclusion:Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.

AB - Aim:To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).Methods:Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.Results:Thrombin (0.3-3.0 U/mL) caused a concentration-and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH 2 (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH 2 (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH 2 (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3-30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3-3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1-10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.Conclusion:Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.

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KW - c-Jun N-terminal kinase (JNK)

KW - connective tissue growth factor

KW - mitogen-activated protein kinase (MAPK)

KW - protease-activated receptor

KW - thrombin

KW - vascular smooth muscle cell

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