Urokinase expression and binding activity associated with the transforming growth factor beta1-induced migratory and invasive phenotype of mouse epidermal keratinocytes.

Transforming growth factor beta1(TGF-beta1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF-beta1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial-fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous-spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF-beta1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI-1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF-beta1-induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF-beta1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas.     

JNK mediates TGF-beta1-induced epithelial mesenchymal transdifferentiation of mouse transformed keratinocytes

In this study we analyzed the role of the c-Jun N-terminal kinases (JNK) pathway in the TGF-beta1 stimulation of urokinase-type plasminogen activator (uPA), initial stages of epithelial-mesenchymal transdifferentiation (EMT) and cell migration. TGF-beta1 induces JNK phosphorylation, c-Jun transactivation and AP1 activation. The involvement of JNK was evaluated using dominant negative mutants SEK-1 AL, JNK and cJun, depletion of JNK1,2 proteins by treatment of cells with antisense oligonucleotides, as well as the chemical inhibitor SP600125. Our results demonstrated that the JNK pathway is required in the TGF-beta1 enhancement of uPA, fibronectin, E-cadherin delocalization, actin re-organization and vimentin expression, concomitant with the induction of cell migration. These results allow us to suggest a role of JNK in the TGF-beta1 induction of EMT in relation with the stimulation of malignant properties of mouse transformed keratinocytes.     

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-beta(1) in transformed keratinocytes

Transformed PDV keratinocytes respond to TGF-beta(1) by stimulating cell motility and invasiveness concomitantly to enhancement of the urokinase-type plasminogen activator (uPA) expression/secretion. Depletion of extracellular signal-regulated kinase (ERK1, 2) proteins by treatment of PDV cells with antisense oligonucleotides reduced basal uPA production and abolished stimulation of uPA secreted levels and cell motility by TGF-beta(1). PD098059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK), decreased TGF-beta(1)-induced uPA mRNA expression, secreted activity in a dose-dependent manner, and abrogated TGF-beta(1)-stimulated cell motility and invasiveness. PDV-derived dominant-negative RasN17 cell transfectants secreted similar amounts of uPA and exhibited similar invasive abilities as the parental cells or control clones, but were unable to respond to TGF-beta(1) for stimulation of uPA-secreted levels and invasiveness. These results suggest that a Ras/MAPK transduction pathway is involved in the invasive response of transformed keratinocytes to TGF-beta(1).     

Transforming growth factor-beta3 increases the invasiveness of endometrial carcinoma cells through phosphatidylinositol 3-kinase-dependent up-regulation of X-linked inhibitor of apoptosis and protein kinase c-dependent induction of matrix metalloproteinase-9

Tumor cells often acquire intrinsic resistance to the growth inhibitory and pro-apoptotic effects of transforming growth factor-beta (TGF-beta); moreover, TGF-beta can confer invasive properties to established tumor cells. In the present study, we show that TGF-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) trigger proper Smad signaling in human endometrial carcinoma cell lines and efficiently inhibit cellular proliferation. These cells, however, exhibit a high degree of resistance to TGF-beta pro-apoptotic effects; we found that this resistant phenotype would be acquired through up-regulation of X-linked inhibitor of apoptosis protein (XIAP) levels. In addition, using RNA interference and pharmacological inhibitors, we show that TGF-beta increases cellular invasiveness via two distinct signaling pathways in endometrial carcinoma cells: phosphatidylinositol 3-kinase/AKT-dependent up-regulation of XIAP and protein kinase C-dependent induction of matrix-metalloproteinase-9 (MMP-9) expression. Additionally, these findings were correlated with clinical observations showing abundant TGF-beta immunoreactivity in human endometrial carcinoma tumors in vivo, extending from the epithelial compartment to the stroma upon acquisition of an invasive phenotype (gradually from grades I to III). Collectively our results describe for the first time a role for TGF-beta3 in tumor invasiveness.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

MMP-14 mediated MMP-9 expression is involved in TGF-beta1-induced keratinocyte migration

The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP-14 in TGF-beta1-induced keratinocyte migration. TGF-beta1 stimulated cell migration and the expression of MMP-2, -9 in HaCaT human keratinocyte cells. When we lowered MMP-14 mRNA with siRNA, cell migration, and MMP-9 expression decreased. Furthermore, the MMP-14 siRNA also reduced activation of JNK in response to TGF-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.     

Tumor promoter TPA stimulates MMP-9 secretion from human keratinocytes by activation of superoxide-producing NADPH oxidase

Matrix metalloproteinase-9 (MMP-9) is involved in physiological tissue remodelling processes as well as in tumor invasion and metastasis. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases MMP-9 secretion from normal human epidermal keratinocytes (NHEK) in vivo and in vitro. Here we show that the flavoprotein inhibitor diphenyleneiodinium (DPI) and the NADPH oxidase inhibitor apocynin block TPA-induced MMP-9 secretion of NHEK in vitro. Furthermore, N-acetyl-L-cysteine and L-cysteine lowered TPA-induced MMP-9 secretion, suggesting an involvement of reactive oxygen species(ROS). TPA exerts its effect on MMP-9 gene expression and secretion via the superoxide-producing enzyme NADPH oxidase: TPA rapidly stimulates generation of superoxide anion as well as gene expression of two cytosolic NADPH oxidase subunits (p47-phox and p67-phox) after 2 h, which is followed by induction of MMP-9 gene expression after 4 h. Taken together, the novel finding herein is the TPA-induced MMP-9 secretion from normal human epidermal keratinocytes through a NADPH oxidase dependent pathway.     

Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.     

Transforming growth factor (TGF)-beta1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, bid-activated pathway that involves matrix metalloproteinase-12

Fibrosis, apoptosis, and the exaggerated production of transforming growth factor (TGF)-beta(1) are juxtaposed in a variety of pulmonary diseases including the interstitial lung diseases and asthma. In these disorders, the relationships between these responses are not well defined. In addition, the apoptosis pathways that contribute to these responses and the mechanism(s) of their contribution have not been described. We hypothesized that BH3 domain-only protein-induced apoptosis plays an important role in the pathogenesis of TGF-beta(1)-induced pulmonary responses. To test this hypothesis, we characterized the effects of transgenic TGF-beta(1) in mice with wild type (WT) and null Bax loci. To investigate the mechanisms of Bax activation and its effector functions, we also compared the effects of TGF-beta(1) in mice with WT and null Bid and matrix metalloproteinase (MMP)-12 loci, respectively. These studies demonstrate that TGF-beta(1) is a potent stimulator of Bax, Bid, and MMP-12. The studies also demonstrate that Bax and Bid play key roles in the pathogenesis of TGF-beta(1)-induced inflammation, fibrosis, and apoptosis; that TGF-beta(1) stimulates MMP-12, TIMP-1, and cathepsins and inhibits MMP-9 and p21 via Bax- and Bid-dependent mechanisms; and that TGF-beta(1)-stimulated pulmonary fibrosis is ameliorated in MMP-12-deficient animals. Finally, they demonstrate that Bax, Bid, and MMP-12 play similar roles in bleomycin-induced fibrosis, thereby highlighting the importance of this Bid-activated, Bax-mediated pathway and downstream MMP-12 in a variety of fibrogenic settings.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.     

Inhibition of matrix metalloproteinase-9 by interferons and TGF-beta1 through distinct signalings accounts for reduced monocyte invasiveness

Cytokines may provide signals for regulating human monocyte matrix metalloproteinase-9 (MMP-9) activity. In this study, we investigated the roles of interferons (IFN) type I/II and transforming growth factor-beta1 (TGF-beta1) in MMP-9-mediated invasiveness. MMP-9 antibody and inhibitor, IFNs and TGF-beta1 inhibited monocyte transmigration through Matrigel. IFNs and TGF-beta1 downregulated MMP-9 mRNA, protein and activity levels. The inhibitory action of IFNs was associated with the STAT1/IRF-1 pathway since the JAK inhibitor AG490 blocked STAT1 phosphorylation, IRF-1 synthesis and counteracted the blockade of MMP-9 release. TGF-beta1-mediated MMP-9 inhibition appeared STAT1/IRF-1-independent but reversed by the protein tyrosine kinase inhibitor tyrphostin 25. Our data point out the importance of IFNs and TGF-beta1 in the control of monocyte MMP-9-mediated extravasation.     

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.     

Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes

Invasion is an essential cellular response that plays an important role in a number of physiological and pathological processes. Matrix metalloproteinase (MMP) production and cell movement are diverse cellular responses integral to the process of invasion. The complexity of the invasive process suggests the necessity of coordinate activation of more than one signaling pathway in order to activate specific factors responsible for regulating these cellular responses. In this report, we demonstrate that cell movement and MMP-9 production are both directly dependent on the activation of endogenous ERK signaling in hepatocyte growth factor (HGF)-or epidermal growth factor (EGF)-stimulated human epidermal keratinocytes. The kinetic profiles of endogenous MEK and ERK activity suggest that prolonged activation of these signal transducers is an underlying mechanism involved in stimulating cell motility and MMP-9 production. In support of this finding, a transient MEK/ERK signal elicited by keratinocyte growth factor (KGF) or insulin-like growth factor-1 (IGF-1) fails to stimulate these invasion-related responses. Specific inhibition of MEK leads to suppression of ERK activation, marked reduction in steady-state levels of c-Fos, and inhibition of cell movement and MMP-9 production. This occurs despite continued activation of JNK and c-Jun signaling in the presence of MEK-specific inhibition. In contrast, when JNK activity is specifically inhibited in HGF-stimulated cells, AP-1 activity is suppressed but cell motility is not affected. This evidence suggests that while ERK and JNK activity are necessary for AP-1 activation, ERK but not JNK is sufficient in stimulating cell motility. J. Cell. Physiol. 180:271–284, 1999. © 1999 Wiley-Liss, Inc.