title: Xanthine oxidase activates pro-matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells through non-free radical mechanisms.
abstract: Reactive oxygen species (ROS) have been implicated in the regulation of matrix metalloproteinases (MMPs). The xanthine/xanthine oxidase (X/XO) reaction has been widely used as a source of exogenous ROS in studying MMPs, but commercial XO has also been known to be contaminated by proteolytic activity, and MMPs are protease sensitive substrate. We have investigated the activation of proMMP-2 by X/XO in cultured vascular smooth muscle cells (SMCs). SMCs were incubated with X/XO (unpurified or purified) or XO alone for 24h. X/XO activated proMMP-2 in a dose-dependent manner. A similar profile was observed using XO. Purified XO produced lower amounts of active MMP-2 compared to unpurified XO. EPR study showed that X/XO, not XO itself, produced superoxide anion, which was completely scavenged by SOD. However, X/XO-induced proMMP-2 activation could not be inhibited by combination of SOD and catalase. Incubation with XO either in cell-free conditioned media or in cells resulted in similar amounts of active MMP-2, suggesting that membrane-type-MMPs were not involved in proMMP-2 activation. This was further confirmed by the lack of inhibitory effect of hydroxamate MMP inhibitor, BB1101. Aprotinin blocked unpurified XO-induced proMMP-2 activation in a dose-dependent manner, demonstrating the proteolytic activity contained in XO is essential. We conclude that proteolytic activity contained in XO, rather the ROS derived from X/XO, is responsible for proMMP-2 activation in cultured SMCs. The results also suggest that caution needs to be taken when interpreting the reported results on activation of MMPs where X/XO had been used as an "authentic" source of superoxide anion.
authors.affiliation: College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
authors.forename: Wenlan
authors.initials: W
authors.lastname: Liu
authors.forename: Gary A
authors.initials: GA
authors.lastname: Rosenberg
authors.forename: Honglian
authors.initials: H
authors.lastname: Shi
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Furuichi
authors.forename: Graham S
authors.initials: GS
authors.lastname: Timmins
authors.forename: Lee A
authors.initials: LA
authors.lastname: Cunningham
authors.forename: Ke Jian
authors.initials: KJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D007611:Aprotinin / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000378:metabolism / Q000494:pharmacology; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D016895:Culture Media, Serum-Free; D004792:Enzyme Precursors; D005609:Free Radicals; D011480:Protease Inhibitors; D019820:Xanthine; D007611:Aprotinin; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D020778:Matrix Metalloproteinase 2