SECRETION OF 72 KDA TYPE-IV COLLAGENASE GELATINASE BY CULTURED HUMAN LIPOCYTES - ANALYSIS OF GENE-EXPRESSION, PROTEIN-SYNTHESIS AND PROTEINASE ACTIVITY

被引:112
作者
ARTHUR, MJP
STANLEY, A
IREDALE, JP
RAFFERTY, JA
HEMBRY, RM
FRIEDMAN, SL
机构
[1] STRANGEWAYS RES LAB,CAMBRIDGE CB1 4RN,ENGLAND
[2] UNIV CALIF SAN FRANCISCO,LIVER CTR LAB,SAN FRANCISCO,CA 94110
基金
英国惠康基金;
关键词
D O I
10.1042/bj2870701
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [S-35]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [C-14]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.
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页码:701 / 707
页数:7
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