Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.
Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.
Benyon
R C
RC
University Medicine, Southampton General Hospital, Southampton, United Kingdom. rcb@soton.ac.uk
Hovell
C J
CJ
Da Gaça
M
M
Jones
E H
EH
Iredale
J P
JP
Arthur
M J
MJ
D000818:Animals; D001672:Biocompatible Materials; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen; D004338:Drug Combinations; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D018093:Gelatinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D007797:Laminin; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011509:Proteoglycans; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism
D001672:Biocompatible Materials; D004338:Drug Combinations; D004792:Enzyme Precursors; D007527:Isoenzymes; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase