Regulation of Membrane‐Type Matrix Metalloproteinase‐1 Expression by Growth Factors and Phorbol 12‐Myristate 13‐Acetate

Abstract

Overexpression of membrane-type matrix metalloproteinase (MT-MMP-1) results in the activation of both endogenous and exogenous 72-kDa gelatinase. To understand the effects of MT-MMP-1 on 72-kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT-1080 fibrosarcoma cells. Both cell types expressed the MT-MMP-1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2-3-fold. Concanavalin A treatment increased MT-MMP-1 mRNA levels in fibroblasts about 4-fold. Induction of MT-MMP-1 by phorbol 12-myristate 13-acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT-MMP-1 mRNA stability using actinomycin D indicated that the halflife was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT-1080 cells had significant 72-kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT-MMP-1 was present mainly in a 60-kDa form. PMA and concanavalin A caused 2-4-fold increases in its protein levels, while in HT-1080 cells PMA, concanavalin A, or overexpression of MT-MMP-1 did not significantly enhance the level of the 60-kDa protein. Instead, an immunoreactive, proteolytically processed 43-kDa form was observed, and its appearance correlated to 72-kDa gelatinase processing activity. Thus 72-kDa gelatinase activation, while enhanced by MT-MMP-1 expression, needs additional co-operating factors.Keywords: matrix metalloproteinase; membrane-type matrix metalloproteinase-l ; 72-kDa gelatinase ; matrix degradation ; cell invasion.Matrix metalloproteinases (MMPs) comprise currently a group of at least 14 distinct enzymes capable of degrading most if not all components of the extracellular matrix (Woessner, 1991;Birkedal-Hansen et al., 1993;Sato et al., 1994; Will and Hinzmann, 1995;Takino et al., 1995;Puente et al., 1996). Physiological processes employing some of the MMPs include growth and differentiation, tissue morphogenesis and remodelling and trophoblast invasion during embryo implantation. Several pathophysiological events depend also on metalloproteinases such as wound healing, periodontitis, rheumatoid arthritis and invasion and metastasis of cancer cells. Even in malignant tumors the invasive process is tightly regulated and involves controlled secretion and activation of proteinases and their inhibitors in order to create an optimal proteolytic balance to allow both adhesion to the extracellular matrix and migration Correspondence to J. Keski-Oja, Dept. of Virology, University of Fax: +358 0 434 6491. Abbreviations. bFGF, basic fibroblast groth factor; ConA, concanavalin A ; EGF, epidermal growth factor; IL-1P, interleukin-1P; MT-MMP-1, membrane-type matrix metalloproteinase-1 ; PMA, phorbol 12-myristate 13-acetate; TGF-P, transforming growth factor-P; TTMP-2, tissue inhibitor of metalloproteinases type 2; ...

Type IV Collagen Induces Matrix Metalloproteinase 2 Activation in HT1080 Fibrosarcoma Cells

“…6A and 6B, lanes 2-4). The occurrence of this latter form has been associated with the ability of the cells to activate MMP-2 and to degrade gelatin [13,29,59]. Consistent with these data, our results also demonstrated an obvious association between the TPA-and ConA-induced pro-MMP-2 activation and MT1-MMP processing into a 43-kDa form.…”

“…As previously reported by others [6,13,15,[56][57][58], we showed that TPA-or ConA-treated HT1080 cells increased MT1-MMP mRNA expression and substantially upregulated MT1-MMP protein synthesis. Furthermore, both treatments induced the formation of a 43-kDa MT1-MMP species (Figs.…”

“…6B, lane 3). This latter MT1-MMP species was recently identified as a cell-surface-associated inactive form which results from an MMP-dependent processing of the 60-kDa form and whose occurrence was related to pro-MMP-2 activation [13,52]. In contrast to TPA, ConA did not increase the level of the 63-kDa pro-MT1-MMP but it dramatically increased both 60-and 43-kDa species (Figs.…”

“…MMP-2 activation is mediated by a membrane-associated mechanism that can be induced in vitro by tumor necrosis factor-α, concanavalin A (ConA), cytochalasin D, and phorbol esters such as tetradecanoylphorbol 13-ace-tate (TPA) [13][14][15][16][17][18][19][20][21]. Sato and co-workers have identified MT1-MMP (membrane type 1 MMP or MMP-14): a MMP possessing a hydrophobic C-terminal transmembrane domain, which is considered as a physiological MMP-2 activator [22][23][24][25].…”

Stimulation of Matrix Metalloproteinase-9 Expression in Human Fibrosarcoma Cells by Synthetic Matrix Metalloproteinase Inhibitors

“…2A). TPA also induced the production of a 45-kDa protein with a weak gelatinolytic activity corresponding to MMP-1 [46], and increased the amount of 62 and 59-kDa active MMP-2 forms, accounting for the well-documented stimulatory effect of TPA on the MT1-MMP-dependent maturation process of pro-MMP-2 [25,46,47]. Similarly to TPA, treatment of the cells with GI129471 dose dependently stimulated the secretion of pro-MMP-9 with a maximal 6.6-fold stimulation after 48 h and 1 µM GI129471 (Fig.…”