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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3185-3190.)
© 1997 American Heart Association, Inc.

Articles

cAMP Involvement in the Expression of MMP-2 and MT-MMP1 Metalloproteinases in Human Endothelial Cells

Francesca Peracchia; Antonio Tamburro; Cesaria Prontera; Barbara Mariani; ; Domenico Rotilio

From the Istituto di Ricerche Farmacologiche Mario Negri, "G. Paone" Environmental Health Center, Department of Vascular Medicine and Pharmacology, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy.

Correspondence to Dr Francesca Peracchia, "G. Paone" Environmental Health Center, Consorzio Mario Negri Sud, Via Nazionale, 66030 S. Maria Imbaro, Italy. E-mail peracchia{at}cmns.mnegri.it

Abstract Matrix metalloproteinases (MMPs) are a multigene family of enzymes secreted by a variety of cells, including human umbilical vein endothelial cells (HUVECs). Because metalloproteinases are potentially destructive agents, their production is tightly controlled at several levels. Rather little is known about the presence and regulation of MMPs in endothelial cells. In this study, we investigated the expression and regulation of MMP-2 and membrane type-matrix metalloproteinase (MT-MMP1), a membrane metalloproteinase strictly related to MMP-2 activation. Zymographic analysis of conditioned medium (CM) of HUVECs showed the presence of gelatinolytic activity mainly at 72 and 64 and 62 kD. The 64- and 62-kD bands, respectively, represent the intermediate and the completely active forms of MMP-2. When HUVECs were treated with forskolin (FK) (100 and 25 µmol/l), there was a decrease in the appearance of the 64 to 62 kDa doublet, suggesting an inhibition of the fully activated form of MMP-2. FK raises intracellular cAMP in HUVECs. The same data were obtained using dibutyryl-cAMP. Northern analysis revealed that the expression of MMP-2 increased slightly after treatment with FK, in contrast with gelatin zymography results. Taking into consideration the mechanism of activation of MMP-2, we tested the hypothesis that this compound could modulate MT-MMP1. As expected, FK was able to decrease MT-MMP1 expression. These data correlate with experiments using membranes of FK-treated HUVECs and incubated with control CM. Zymography revealed that when CM was incubated with membranes prepared from FK-treated HUVECs, there was a decrease in the appearance of the 64-kDa band, suggesting that the expression of MT-MMP1 was negatively modified. These results correlate with the MT-MMP1 protein level, negatively modified after FK treatment.

Key Words: metalloproteinases • endothelial cells • cAMP

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