Inhibition of proteases involved in embryo implantation by cholesterol sulfate.
BACKGROUND
Matrix metalloproteinases (MMPs) and the plasminogen activator (PA)/plasmin system are two major groups of proteases involved in the matrix degradation required for embryo implantation. We previously showed that the content of cholesterol sulfate (CS) in rabbit endometrium increases characteristically during the implantation period. Furthermore, CS has been reported to inhibit serine proteases. In this study, we investigated whether CS can regulate the activity of proteases in cultured human endometrial stromal cells.


RESULTS
CS (1-30 microM) and plasminogen (precursor of plasmin) were added to the culture media of human endometrial stromal cells and incubated for 24 h. Culture media were collected for analysis of plasmin and MMP-2, -3 and -9 enzyme activities using fluorescence assays. Plasmin and MMP-3 activities were significantly reduced by CS in a dose-dependent manner (P < 0.001). Western blot analysis of the culture media revealed that CS inhibited the conversion by plasmin of MMP-3 from the precursor form to the active form. Fluorescence assay using a common substrate of MMP-2 and MMP-9 showed that enzymatic activity remains at approximately 50%, even at 30 microM CS. Gelatin zymography demonstrated that CS inhibited the activation of MMP-9 but not MMP-2 from the precursor, suggesting that the activation of MMP-2 may be independent of plasmin.


CONCLUSIONS
CS inhibits not only plasmin activity but also MMP activities indirectly by inhibiting the plasmin-mediated process. These findings suggest that CS may be an important regulator of proteolysis during trophoblast invasion.
Koizumi
Minako
M
Obstetrics and Gynaecology, The University of Tokyo, Tokyo, Japan.
Momoeda
Mikio
M
Hiroi
Hisahiko
H
Nakazawa
Fumiko
F
Nakae
Hanako
H
Ohno
Tomoko
T
Yano
Tetsu
T
Taketani
Yuji
Y
D002478:Cells, Cultured; D002788:Cholesterol Esters / Q000494:pharmacology*; D010064:Embryo Implantation / Q000187:drug effects / Q000502:physiology*; D004717:Endometrium / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010958:Plasminogen / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D002788:Cholesterol Esters; D015842:Serine Proteinase Inhibitors; D010958:Plasminogen; D005341:Fibrinolysin; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; C007045:cholesteryl sulfate