Production of matrix metalloproteinases in human cultured mast cells: involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway.
BACKGROUND
Matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components. To confirm the role of mast cells as a source of MMPs, we investigated the production of MMP and its pathway in human cultured mast cells (HCMC). We also investigated the production of tissue inhibitors of metalloproteinase (TIMPs).


METHODS
HCMC was stimulated with phorbor 12-miristate 13-acetate (PMA) and/or calcium ionophore A23187 (A23187), and the resulting MMP production was evaluated by gelatin zymography and western blotting. Expression of MMP and TIMP mRNA was also examined. Granulocyte macrophage-colony stimulating factor (GM-CSF) was measured by ELISA and activation of extracellular signal-regulated kinase (ERK) was evaluated by western blotting.


RESULTS
We detected the de novo synthesis of MMP-9 in HCMC after stimulation with PMA and found that the synthesis was mediated through protein kinase C-mitogen activated protein kinase kinase (MEK)-ERK pathway. The MMP-9 production induced by PMA was suppressed by simultaneous treatment with A23187, whereas GM-CSF production was potentiated. We also detected the expression of mRNA for membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 after stimulation with PMA. Glucocorticoids and flavonoids inhibited MMP-9 production, and TIMPs and MMP inhibitors inhibited the gelatinolytic activity of mast cell-derived MMP-9. Furthermore, phenylmethylsulfonyl fluoride, a protease inhibitor, inhibited the conversion from proMMP-9 to active MMP-9.


CONCLUSIONS
These results suggest that the human mast cell is a leading member of MMP production, and the production, activation and activity are controllable by pharmacological agents.
Kimata
Masahiro
M
Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan.
Ishizaki
Masayuki
M
Tanaka
Hiroyuki
H
Nagai
Hiroichi
H
Inagaki
Naoki
N
D018929:Cell Culture Techniques; D002478:Cells, Cultured / Q000201:enzymology / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000494:pharmacology; D008407:Mast Cells / Q000201:enzymology / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010704:Phorbols / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*
D061965:Matrix Metalloproteinase Inhibitors; D010704:Phorbols; D011480:Protease Inhibitors; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D007073:Immunoglobulin E; D010664:Phenylmethylsulfonyl Fluoride; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020782:Matrix Metalloproteinases