Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2.
The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 microM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 microM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.
Darmoul
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Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichât, Paris, 75018, France.
Marie
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JC
Devaud
H
H
Gratio
V
V
Laburthe
M
M
D015152:Blotting, Northern; D018938:Caco-2 Cells; D002118:Calcium / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D019073:HT29 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014357:Trypsin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; C083115:activator protein-2 binding element; D014357:Trypsin; D002118:Calcium