Posttranscriptional Stimulation of Endothelial Cell Matrix Metalloproteinases 2 and 1 by Endothelioma Cells Giulia Taraboletti, ,1 Laura Sonzogni, Veronica Vergani, Ghamartaj Hosseini,* Roberta Ceruti, , † Carmen Ghilardi, Antonio Bastone,‡ Elena Toschi,§ Patrizia Borsotti, Eugenio Scanziani,† Raffaella Giavazzi, Michael S. Pepper,* William G. Stetler-Stevenson,§ and Maria Rosa Bani Department of Oncology, Mario Negri Institute for Pharmacological Research, 24125 Bergamo and ‡20157 Milan, Italy; †Istituto di Anatomia Patologica Veterinaria e Patologia Aviare, Universita ` degli Studi, Milan, Italy; *Department of Morphology, University Medical Center, 1211 Geneva 4, Switzerland; and §Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothe- lioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24.24) and MMP-9 (92-kDa gelati- nase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggest- ing that stroma cells, particularly endothelial cells re- cruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endo- thelial cells, but not of the inhibitor TIMP-2. The in- creased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analy- sis), occurred through a posttranscriptional mecha- nism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endo- thelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis. © 2000 Academic Press Key Words: matrix metalloproteinases; endothelial cells; endothelioma; angiogenesis; hemangioma; pro- teases secretion. INTRODUCTION Matrix-degrading proteases play a critical role in the formation of the stromal compartment of tumors by contributing to the recruitment of inflammatory and connective cells and in particular of endothelial cells during the process of angiogenesis [1–3]. The degrada- tion of the extracellular matrix (ECM) contributes to cell recruitment through different mechanisms. It cre- ates a permissive environment for the migration of invading cells; furthermore, it results in the release of growth and chemotactic factors stored within the ma- trix and of proteolytically derived, biologically active fragments of matrix components. During angiogenesis, proteases are also involved in the final morphogenetic changes which lead to the formation of the new capil- lary vessels. Most of the degradative enzymes involved in tumor angiogenesis belong to two main families: the serine proteases, in particular the plasminogen activator (PA)/plasmin system, and the matrix metalloprotein- ases (MMPs) [1, 2, 4]. Matrix-degrading MMPs are a family of secreted or transmembrane zinc-dependent proteolytic enzymes, which degrade ECM at physiological pH [5]. They are involved in normal and pathological processes of tissue remodeling and cell invasion. Evidence indicates that MMPs are also involved in angiogenesis. Endothelial cells produce MMPs, and their production is increased by angiogenic factors, cytokines, and tumor-derived factors [4, 6 –9]. Natural and synthetic inhibitors of MMPs block angiogenesis [10 –13], and synthetic MMP inhibitors are currently being developed as antiangio- genic and antineoplastic drugs [14, 15]. 1 To whom reprint requests should be addressed. Fax: (39) 035 319331. E-mail: taraboletti@irfmn.mnegri.it. 384 0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 258, 384 –394 (2000) doi:10.1006/excr.2000.4936, available online at http://www.idealibrary.com on