Volume 383, Issue 1

Cell migration is a complex process requiring tight control of several mechanisms including dynamic reorganization of the actin cytoskeleton and adhesion to the extracellular matrix. The GPIanchored urokinase plasminogen activator receptor (uPAR) has an important role in the regulation of cell motility in many cell types. This is partly due to the localization of proteolytic activity on the cell surface by binding of the serine protease uPA. Results accumulated over the last decade suggest that uPAR is also involved in motility control through other mechanisms. These include induction of signal transduction events after ligation with uPA, binding to the extracellular matrix molecule vitronectin (VN), and association with integrins and other transmembrane partners. In this review these mechanisms will be discussed with a special emphasis on how the GPIlinked receptor transmits signals to the intracellular milieu and how uPAR participates in the regulation of actin cytoskeleton reorganization and cell adhesion during cell migration.

The serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1) is the primary physiological inhibitor of the tissuetype and the urokinasetype plasminogen activator (tPA and uPA, respectively) and as such an important regulator of proteolytic events taking place in the circulation and in the extracellular matrix. Moreover, a few nonproteolytic functions have been ascribed to PAI-1, mediated by its interaction with vitronectin or the interaction between the uPAPAI-1 complex bound to the uPA receptor and members of the low density lipoprotein receptor family. PAI-1 belongs to the serpin family, characterised by an unusual conformational flexibility, which governs its molecular interactions. In this review we describe the antiproteolytic and nonproteolytic functions of PAI-1 from both a biological and a biochemical point of view. We will relate the various biological roles of PAI-1 to its biochemistry in general and to the different conformations of PAI-1 in particular. We put emphasis on the intramolecular rearrangements of PAI-1 that are required for its antiproteolytic as well as its nonproteolytic functions.

Studies of extracellular matrix (ECM) biology in the nervous system have mainly focused on laminin, fibronectin and tenascinR, proteins that are present during nervous system development and normal function. However, during disease, fibrin, which physiologically is not present in the nervous tissue, is detected at nervous tissue lesions. This review summarizes evidence that correlates fibrin deposition with neuropathology and presents recent findings on cellular mechanisms and intracellular signaling pathways regulated by fibrin that might contribute to nervous system disease.

The urokinasemediated plasminogen activation (PA) system has been shown to play a key role in cell migration and tissue invasion by regulating both cellassociated proteolysis and cellcell and cellmatrix interactions. The expression and activity of the components of this complex system are strictly regulated. The control of the expression occurs both at transcriptional and posttranscriptional levels. This review is focused on the posttranscriptional regulation of gene expression of all components of the PA system.

Tumor progression occurs within a microecosystem, where cancer cells and myofibroblasts exchange proteinases and cytokines that promote growth directly through stimulation of proliferation and survival, as well as invasion through local proteolysis of the extracellular matrix and stimulation of motility. Myofibroblasts maintain the capacity of fibroblasts to induce differentiation. Fibroblasts are the main source of tumorassociated myofibroblasts. The transition to myofibroblasts also occurs in noncancerous situations. This transition is modulated by mechanical stress and cytokines, amongst which transforming growth factorβ. The crosstalk between cancer cells and myofibroblasts illustrates the microecosystem of tumor invasion. In order to consider myofibroblasts as a possibly new target for cancer therapy, further characterization of the molecular crosstalk between myofibroblasts and cancer cells is required.

Cell responses to exogenous stimuli often result in a rapid decrease of cell surface density of a wide range of diverse regulatory proteins, receptor and adhesion molecules in particular. This decrease may occur in a liganddependent fashion (downregulation), following endocytosis and degradation by lysosomal proteases, or by downmodulation, where molecules are targeted by endoproteases directly on cell surface. These proteases are recruited by transmodulating agents, different from ligand, which act via their own receptors and the related intracellularlygenerated signals. Endoproteolytic activity determines the release of large portions (shedding) of substrate proteins, called ectodomains, which are usually not ligandbound, and therefore represent biologicallyactive molecules. Ectodomain shedding is involved in a number of pathophysiological processes, such as inflammation, cell degeneration and apoptosis, and oncogenesis. Common features of the process, such as the involvement of protein kinase C and of transmembrane metalloproteases, have been identified. In this review, we summarize basic concepts on downmodulation and ectodomain shedding, and provide an update of the issue with respect to: (i) new entries to the list of molecules found involved in the process; (ii) current views about the upstream control of shedding, i.e. the pathways linking the signals triggered by the transmodulating agents to the activation of endoproteolytic activity on the cell surface.

The plasminogen activating system is important for extracellular proteolysis and plays a regulatory role in interactions with other tissue degrading systems. Studies on the plasminogen activating system in gingival crevicular fluid (GCF) as well as gingival tissue are reviewed. tPA, uPA, PAI-1 and PAI-2 have all been detected in GCF. Especially tPA and PAI-2 are found in high concentrations. In tissue studies fibrinolytic activity has been found in the gingival pocket epithelium in humans and in animal studies. tPA and PAI-2 have been detected there immunohistochemically. Local production of the PAs and PAIs has been verified with in situ hybridization. In inflammation, a more intense and widespread immunohistochemical staining of tPA and PAI-2 is seen. Higher concentrations of tPA and PAI-2 are found in GCF but the balance between them seems to be constant. A systemically disturbed balance of the plasminogen activating system in GCF has been observed during pregnancy, with a possible protective function of PAI-2. In studies of periodontitis, the production of PAI-2 seemed to be locally lowered at impaired sites. In a study of children, a higher inflammatory response to bacterial plaque was accompanied by a higher fibrinolytic ativity in GCF samples. Bacterial LPS has been found to change the ratio of tPA to PAI-2 in cultured gingival fibroblasts. Interactions between PAI-2 and a protease in the gingival epithelium has been verified through the immunohistochemical detection of relaxed PAI-2.

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogenlike kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysinebinding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysinebinding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysinebinding function of these kringles by a monoclonal antibody that recognises key components of the lysinebinding site. In contrast, the lysinebinding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissuetype plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A SerIle substitution at the activation cleavage site is present in apo(a). Reinstallation of the ArgVal peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissuetype plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).

Several reports have attributed to green tea chemopreventive and therapeutic properties. Epidemiological studies have linked the regular use of green tea to a reduced incidence of breast and colon carcinomas. Tea contains several antioxidants, including polyphenols of the catechin (green tea) and theaflavin (black tea) groups. Green tea derivatives have been shown to act in vitro and in vivo as antiinflammatory, antiviral and antitumor drugs. Despite the extensive body of data only few studies have investigated the molecular mechanisms underlying these effects. In this brief review we focus on the inhibitory activity of catechins derived from green tea toward proteases involved in tumor invasion.

We have previously reported that phosphorylation of human urokinase on Ser138/303 abolishes its catalyticindependent motogen and proadhesive abilities, whereas receptor binding is not affected. Here we show that substitution of the two relevant serines with glutamic acid residues impairs the ability of urokinase to mobilize a variety of human and mouse cell lines as well as human primary T lymphocytes. Accordingly, urokinase receptordependent signaling, leading to cytoskeletal rearrangements and paxillin redistribution, does not occur in MCF-7 breast carcinoma cells exposed to phosphorylationlike urokinase. Unlike the wildtype form, disubstituted urokinase is unable to induce the physical association of urokinase receptor with αvβ5 vitronectin receptor, which is required for MCF-7 urokinasedependent cell migration. Finally, the disubstituted variant fails to activate p55fgr, a member of the Src tyrosine kinase family, which mediates cell migration and adhesion of U937 myelomonocytic cells. In conclusion, the finding that specific amino acid substitutions strongly interfere with the ability of urokinase to stimulate cell migration, and the associated intracellular events uncover a novel way to regulate urokinase receptordependent signaling.

We have explored intracellular pathways involved in the urokinase type plasminogen activator (urokinase or uPA)stimulated migration of human airway smooth muscle cells (hAWSMC). Using a set of uPA mutants we found that protease activity, growth factorlike and kringle domains of uPA differentially contribute to activation of p42/p44erk1,2 and p38 MAPkinases. Consistent with our earlier data [Mukhina et al., J. Biol. Chem. 275 (2000), 16450 16458], the kringle domain of uPA was sufficient and required to stimulate cell motility. Here we report that uPA mutants containing the kringle domain specifically activate the p38 MAPkinase pathway and actomyosin by increasing phosphorylation of the critical Ser-19 on the myosin regulatory light chain and MAPkinase sites of the actinassociated regulatory protein caldesmon. While pharmacological inhibition of p38 MAPkinase activation did not affect myosin light chain phosphorylation, it blocked the increase in caldesmon phosphorylation and uPAstimulated migration of hAWSMC on a collagencoated surface. We conclude that activation of p38 MAPkinase and downstream phosphorylation of nonmuscle caldesmon is essential for urokinasestimulated smooth muscle cell migration.

The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factorβ (TGFβ) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cellassociated fibrinolytic system. We have observed that the urokinase plasminogen activator (uPA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly upregulated by TGFβ, mainly in dystrophic myoblasts. bFGF upregulated uPA in both normal and dystrophic myoblasts grown in primary culture, while a striking downregulation was observed with TGFβ. TGFβ was the only growth factor able to exceptionally upregulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factordependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cellassociated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of uPA and of its receptor. Noteworthy, TGFβ induced a dosedependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFβchallenged dystrophic myoblasts undergo an exceptional upregulation of the receptor and of PAI-1, we propose the possibility that the TGFβinduced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.

Human matrix metalloproteinase-2 (MMP-2) contains three intandem fibronectin type II (FII) repeats that bind gelatin. Here, we report the NMR solution structure of the first FII module of MMP-2 (col-1). The latter is described as a characteristic, globular FII fold containing two βsheets, a stretch of 3[1]helix, a turn of αhelix, and an exposed hydrophobic surface lined with aromatic residues. We show that col-1 binds (ProProGly)6, a mimic of gelatin, with a Ka of approx. 0.42 per mM, and that its binding site involves a number of aromatic residues as well as Arg34, as previously found for the second and third homologous repeats. Moreover, the affinity of the intandem col-1+2 construct (col-12) toward the longer ligand (ProProGly)12 is twice that for (ProProGly)6, as expected from mass action. A detailed structural comparison between FII and kringle domains indicates that four main conformational features are shared: two antiparallel βsheets, a central 3[1]helix, and the quasiperpendicular orientation of the two proximal CysCys bonds. Structure superposition by optimizing overlap of cystine bridge areas results in close juxtaposition of their main βsheets and 3[1]helices, and reveals that the gelatin binding site of FII modules falls at similar locations and exhibits almost identical topological features to those of the lysine binding site of kringle domains. Thus, despite the minor (<15%) consensus sequence relating FII modules to kringles, there is a strong folding and binding site structural homology between the two domains, enforced by key common conformational determinants.

The urokinase receptor is a multifunctional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1 48 (uPA1 48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1 48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin αchains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.

Matrix metalloproteases from the cell surface cleave an 80 kDa Ecadherin fragment (sECAD) that induces invasion of cancer cells into collagen type I and inhibits cellular aggregation. Conditioned media from MDCKts.srcCl2 cells at 40 C and 35 C, PCm.src5 and COLO-16 cells at 37 C contained spontaneously released sECAD; these 48 h old conditioned media were capable of inhibiting Ecadherin functions in a paracrine way. Here we show direct cleavage of the extracellular domain of Ecadherin by the serine protease plasmin. sECAD released by plasmin inhibits Ecadherin functions as evidenced by induction of invasion into collagen type I and inhibition of cellular aggregation. This functional inhibition by sECAD was reversed by aprotinin or by immunoadsorption on protein Sepharose 4 fast flow beads with antibodies against the extracellular part of Ecadherin. Our results demonstrate that plasmin produces extracellular Ecadherin fragments which regulate Ecadherin function in cells containing an intact Ecadherin/ catenin complex.

Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In nonconfluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by nonfibrillar FN deposits at lateral cell domains in regions of cellcell contacts. FRT cells secrete and expose on the plasma membrane the tissuetype plasminogen activator and, in serumfree cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are preincubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmindependent (tPA activated) and plasminindependent proteolytic activities are involved.

Plasminogen activators of the urokinase and tissuetype and fetal calf serum (uPA, tPA, FCS) exert their mitogenic effect on quiescent human dermal fibroblasts and modulate the mRNA expression of cellcycle related genes. The present study deals with the effects of PAs on the expression of fibronectin (FN), a heterodimeric extracellular matrix (ECM) protein that can be modulated in different ways by various mitogens. The kinetics of FN gene response was examined in quiescent fibroblasts upon PA stimulation (30 min 24 h). The results obtained evidenced that: (i) all mitogens tested (uPA, tPA and FCS) led to an increase of FN mRNA expression in early G1, as shown by the analysis of two sequences, III-9, common to all FN mRNAs, and EDA+, present only in the EDA+FN isoform; (ii) the kinetic profiles of FN mRNA stimulation were comparable for the three mitogens, although the effects on the FNECM assembly were distinct; (iii) tPA and FCS led to FN assembly in the ECM, which was absent or decreased in uPAtreated cultures. Immunobiochemical analysis of total FN and EDA + FN showed that FN induced by tPA was mainly dimeric (450 500 kDa), whereas FN induced by uPA was mainly monomeric (230 250 kDa). These differences are probably due to the differential enzymatic action of tPA and uPA on FN, which might be related to a differential role of the two PAs in several physiopathological conditions.

Platelet Activating Factor (PAF), an inflammatory bioactive lipid, has been shown to be involved in the regulation of the activity of matrix metalloproteinases (MMPs). In view of the role played by MMPs in tumor cell invasiveness, we investigated whether PAF influences MMP activity in a system of neuroblastoma clones, the AA5 and AE12 cells, isolated from the human LaN1 neuroblastoma cell line. These clones were characterized by an inverse relationship between invasiveness and differentiative capacity and by the expression of specific cell surface PAF receptors. We found that the levels of mRNAs specific for MMP-2 and for MT1-MMP, the MMP-2 activator, were reduced in both clones treated with 300 nM PAF. These changes are consistent with the reduced secretion and activation of MMP-2 found in the neuroblastoma clones exposed to PAF. These effects were accompanied by an inhibition of invasiveness through Matrigel and by a promotion of differentiation, as revealed by an increased percentage of cells with neurites. The finding that both neuroblastoma clones exposed to the metalloproteinase inhibitors, BB3103 and 1,10-phenanthroline, increased their differentiative capacity and reduced their invasiveness through Matrigel, represents a further indication that PAF modulates differentiation and invasiveness by affecting the activity of MMPs.

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sunexposed and nonexposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast like cells from specimens of sunexposed areas. Little or no reaction was found in biopsies of sunprotected skin. Fibroblast cultures from sunexposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sunprotected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sunprotected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastaselike activity expressed by fibroblasts can be attributed to at least two enzymes.

The urokinasetype plasminogen activation system, including the serine protease uPA (urokinasetype plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPAderived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SPuPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPApeptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 m in diameter to which HMWuPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SPuPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMWuPA, ATF) as well as highly effective, synthetic cyclic uPAderived peptides (cyclo21,29 uPA21 30, cyclo21,29uPA21 30, cyclo21,29[ DCys[21]2-Nal24Cys29]uPA21 30, and cyclo21,29uPA21 30. The results obtained with the noncellular SPuPA/uPAR system are highly comparable to those obtained with a cellular system involving FITCuPA and the promyeloid cell line U937 as the source of uPAR.

Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinasetype plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral bloodderived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPAinduced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture fivefold, whereas VSMC display an increased expression of cell surfaceassociated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

We have recently identified a gene (the WFIKKN gene) on human chromosome 16 (16p13.3) that encodes a secreted protein containing WAPtype, Follistatin/ Kazal type, Kunitztype and NTRtype proteaseinhibitory modules and an Immunoglobulin domain [Trexler et al., Proc. Natl. Acad. Sci. USA 98 (2001), 3705 3709]. In the present work we show that a gene on chromosome 17 encodes a WFIKKNrelated protein (WFIKKNRP) that has the same domain organization as the WFIKKN protein. The exonintron structure of the two genes is also similar as both genes have a single phase 0 intron that splits their WAP domains in equivalent positions. In view of the presence of several protease inhibitory modules in these proteins it seems likely that they serve to control the action of multiple types of proteases. The tissue expression pattern of the two proteins, however, is markedly different suggesting that they have distinct biological roles. Whereas the WFIKKN gene is expressed primarily in adult pancreas, liver and thymus but not in brain and ovary, significant expression of the WFIKKNRP gene is observed in ovary, testis and brain, but not in liver. Pronounced differences could also be seen in the case of fetal tissues: expression of the WFIKKN gene was highest in the lung, skeletal muscle and liver, whereas the WFIKKNRP gene was expressed primarily in brain, skeletal muscle, thymus and kidney.

Osteopontin, a noncollageneous bone matrix protein, is produced in several human tumors but its role in cancer progression has been only partially elucidated. In this study we investigated the potential role of osteopontin in the malignancy of prostate cancer cells. Chemotaxis and chemoinvasion analyses revealed a dosedependent increase in PC3 cell movement induced by osteopontin and a strict dependence of cell invasion on αvβ3 integrin function. The pattern of protease expression was modified by osteopontin and was characterized by an upregulation of plasminogen activators. Our findings suggest that osteopontin may confer selective malignant potential to prostate cancer cells through the enhancement of their invasive and proteolytic capability.

During testicular development and maturation, extracellular matrix (ECM) remodelling is a fundamental process which requires the presence of several proteases and protease inhibitors. Among the proteases, a pivotal role has been proposed for matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs). Here we report an analysis of MMP-2, MT1-MMP and TIMP-2 expression by rat Sertoli cells in culture using RTPCR and zymographic techniques. Stimulating Sertoli cells with folliclestimulating hormone (FSH) in vitro induced evident changes in the level of their mRNA in a timedependent manner. In the case of TIMP-2 and MT1-MMP, the respective transcripts were augmented up to threefold after 24 h of treatment, and MMP-2 transcripts increased by four times in the same period. MMP-2 activity determined by gelatin zymography showed an increase in enzyme secretion after FSH stimulation. The results of this study suggest that: (i) at the surface of Sertoli cells proMMP-2 activation mediated by MT1-MMP may occur, involving TIMP-2 as a receptor; and (ii) the expression of these molecules is not constitutive in this cell type, but may be modulated by FSH and is therefore implicated in spermatogenesis.