Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases

C M Belham, R J Tate, P H Scott, A D Pemberton, H R Miller, R M Wadsworth, G W Gould, R Plevin

Research output: Contribution to journalArticle

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Abstract

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.
LanguageEnglish
Pages939-46
Number of pages8
JournalBiochemical journal
Volume320 ( Pt 3)
Publication statusPublished - 1996

Fingerprint

PAR-2 Receptor
seryl-leucyl-isoleucyl-glycyl-arginyl-leucine
Mitogen-Activated Protein Kinases
Trypsin
Chemical activation
Smooth Muscle Myocytes
Muscle
Cells
Fibroblasts
thrombin receptor peptide SFLLRNP
thrombin receptor peptide (42-55)
MAP Kinase Kinase 2
MAP Kinase Kinase 1
Peptides
Trypsin Inhibitors
JNK Mitogen-Activated Protein Kinases
p38 Mitogen-Activated Protein Kinases
Reverse Transcriptase Polymerase Chain Reaction
Vascular Smooth Muscle
Soybeans

Keywords

  • aorta
  • enzyme activation
  • fibroblasts
  • forskolin
  • lung
  • kinase
  • protein kinases
  • oligopeptides
  • protein-serine-threonine kinases
  • protein-tyrosine kinases
  • Proto-Oncogene Proteins c-sis
  • rna messenger
  • rats
  • receptors
  • trypsin
  • trypsin inhibitors

Cite this

Belham, C M ; Tate, R J ; Scott, P H ; Pemberton, A D ; Miller, H R ; Wadsworth, R M ; Gould, G W ; Plevin, R. / Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. In: Biochemical journal. 1996 ; Vol. 320 ( Pt 3). pp. 939-46.
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abstract = "We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1{\%} desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1{\%} desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.",
keywords = "aorta, enzyme activation, fibroblasts, forskolin, lung, kinase, protein kinases, oligopeptides, protein-serine-threonine kinases, protein-tyrosine kinases, Proto-Oncogene Proteins c-sis, rna messenger, rats, receptors, trypsin, trypsin inhibitors",
author = "Belham, {C M} and Tate, {R J} and Scott, {P H} and Pemberton, {A D} and Miller, {H R} and Wadsworth, {R M} and Gould, {G W} and R Plevin",
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Belham, CM, Tate, RJ, Scott, PH, Pemberton, AD, Miller, HR, Wadsworth, RM, Gould, GW & Plevin, R 1996, 'Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases' Biochemical journal, vol. 320 ( Pt 3), pp. 939-46.

Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. / Belham, C M; Tate, R J; Scott, P H; Pemberton, A D; Miller, H R; Wadsworth, R M; Gould, G W; Plevin, R.

In: Biochemical journal, Vol. 320 ( Pt 3), 1996, p. 939-46.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases

AU - Belham, C M

AU - Tate, R J

AU - Scott, P H

AU - Pemberton, A D

AU - Miller, H R

AU - Wadsworth, R M

AU - Gould, G W

AU - Plevin, R

PY - 1996

Y1 - 1996

N2 - We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.

AB - We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.

KW - aorta

KW - enzyme activation

KW - fibroblasts

KW - forskolin

KW - lung

KW - kinase

KW - protein kinases

KW - oligopeptides

KW - protein-serine-threonine kinases

KW - protein-tyrosine kinases

KW - Proto-Oncogene Proteins c-sis

KW - rna messenger

KW - rats

KW - receptors

KW - trypsin

KW - trypsin inhibitors

UR - http://www.biochemj.org/bj/320/0939/3200939.pdf

M3 - Article

VL - 320 ( Pt 3)

SP - 939

EP - 946

JO - Biochemical Journal

T2 - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -