Development of in situ zymography to localize active matrix metalloproteinase-7 (matrilysin-1).
Matrix metalloproteinase-7 (MMP-7) is upregulated during carcinogenesis and its expression correlates with metastasis of human endometrial and gastrointestinal carcinomas. In the present study, we have developed a new method to localize the activity of MMP-7 within tissues. Polyethylene terephthalate films were uniformly coated with crosslinked carboxymethylated transferrin (CCm-Tf) as a substrate and incubated with frozen tissue sections mounted on the films. CCm-Tf on the films was degraded selectively by MMP-7, but showed little or no susceptibility to MMP-1, -2, -3, -9, or -13; MT1-MMP; MT3-MMP; or ADAMTS4. Although some serine proteinases such as elastase also digested CCm-Tf, CCm-Tf films impregnated with serine proteinase inhibitors prevented the digestion. When frozen sections of human endometrial carcinoma and lung carcinoma tissues were incubated on CCm-Tf films or those treated with proteinase inhibitors, the activity was detected in the carcinoma cell nests, where MMP-7 was immunolocalized. The present in situ zymography using CCm-Tf may be a useful method to analyze the functions of MMP-7 in pathophysiological conditions.
Nemori
Ryoichi
R
Advanced Core Technology Laboratories, Fuji Photo Film Co., Ltd., Kanagawa, Japan.
Yamamoto
Masayoshi
M
Kataoka
Fumio
F
Hashimoto
Gakuji
G
Arakatsu
Hiroshi
H
Shiomi
Takayuki
T
Okada
Yasunori
Y
D003432:Cross-Linking Reagents; D016889:Endometrial Neoplasms / Q000201:enzymology; D005260:Female; D005629:Frozen Sections; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D011093:Polyethylene Terephthalates; D015203:Reproducibility of Results; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D013379:Substrate Specificity; D014168:Transferrin / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D011093:Polyethylene Terephthalates; D015842:Serine Proteinase Inhibitors; D014168:Transferrin; D012697:Serine Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7