Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and G?6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.     

Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion.

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.     

Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

The expression of matrix metalloproteinases (MMPs) produced by cancer cells has been associated with the high potential of metastasis in several human carcinomas, including breast cancer. Several pieces of evidence demonstrate that protein tyrosine phosphatases (PTP) have functions that promote cell migration and metastasis in breast cancer. We analyzed whether PTP inhibitor might control breast cancer invasion through MMP expression. Herein, we investigate the effect of 4-hydroxy-3,3-dimethyl-2H benzo[g]indole-2,5(3H)-dione (BVT948), a novel PTP inhibitor, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. The expression of MMP-9 and cell invasion increased after TPA treatment, whereas TPA-induced MMP-9 expression and cell invasion were decreased by BVT948 pretreatment. Also, BVT948 suppressed NF-κB activation in TPA-treated MCF-7 cells. However, BVT948 didn’t block TPA-induced AP-1 activation in MCF-7 cells. Our results suggest that the PTP inhibitor blocks breast cancer invasion via suppression of the expression of MMP-9. [BMB Reports 2013; 46(11): 533-538]     

Effect of prostaglandin E2 on PMA-induced macrophage differentiation

Major trauma such as severe bums and extensive surgery could result in accelerated macrophage differentiation and hyperactivation causing an excessive release of proinflammatory cytokines and prostaglandin E2 (PGE2) with consequent severe impairment of immunologic reactivity. HL-60 cells stimulated with phorbol 12-myristate 13-acetate (PMA) have been used as a model to asses the PGE2 role in the macrophage differentiation observed after major trauma. Cell adhesion, matrix metalloproteinase-9 (MMP-9) and tumor necrosis factor-alpha (TNF-alpha) production were measured after 24 h of PMA treatment in the presence of PGE2 (1 nM - 1 microM). PGE2 increased both the PMA-induced cell adhesion and MMP-9 production via EP2/EP4 receptors while it had no effect on the induced TNF-alpha release. The cAMP/PKA pathway, usually linked to EP2/EP4 activation, was not involved in the phenomenon, suggesting that an alternative signalling pathway could be linked to a PKC-activated enzyme. In fact PGE2 activity was partially inhibited by Wortmannin, a phosphoinositide-3 kinase (PI-3K) inhibitor indicating that PGE2 act as a co-factor able to increase macrophage differentiation in vitro via a PI-3K dependent pathway that could be also involved in the immunosuppression observed in the aftermath of trauma.     

Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells

In the current study, we examined the function of N-myc downstream-regulated gene 2 (NDRG2) expression in breast cancer cells, especially focusing on the role of bone morphogenetic protein-4 (BMP-4) induced by NDRG2. NDRG2 expression in MDA-MB-231 cells inhibited the mRNA expression of several matrix metalloproteinases (MMPs) and the gelatinolytic activity of MMP-9. Interestingly, a specific induction of active BMP-4 was exclusively observed in MDA-MB-231-NDRG2 cells but not in MDA-MB-231-mock cells. Neutralization of BMP-4 in MDA-MB-231-NDRG2 cells resulted in the rescue of MMP-9 mRNA expression and migration capacity. In addition, treatment with recombinant BMP-4 dramatically suppressed MMP-9 mRNA expression, gelatinolytic MMP-9 activity, migration, and invasion capacity both in MDA-MB-231 and PMA-treated MCF-7 cells. Collectively, our data show that BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity.     

Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: relationship with enhanced expression of transforming growth-factor-beta 1.

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.     

Prostaglandin E2 downregulates TNF-alpha-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.     

Silibinin prevents TPA-induced MMP-9 expression and VEGF secretion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast cancer cells

Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression are pivotal steps in cancer metastasis. Herein, we investigated the effect of silibinin, a major constituent (flavanolignan) of the fruits of Silybum marianum, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and VEGF expression in MCF-7 human breast cancer cells. The expression of MMP-9 and VEGF in response to TPA was increased, whereas TPA-induced MMP-9 and VEGF expression was decreased by silibinin. To investigate the regulatory mechanism of silibinin on TPA-induced MMP-9 and VEGF expression, we pretreated cells with various inhibitors, such as UO126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor). Interestingly, TPA-induced MMP-9 expression was significantly inhibited by UO126, but not by SP600125 and SB203580. In addition, we pretreated cells with 100 μM silibinin prior to TPA treatment. TPA-induced MEK and ERK phosphorylation was significantly decreased by silibinin in MCF7 cells. TPA-induced VEGF expression was also suppressed by UO126. On the other hand, we found that adenoviral constitutive active-MEK (Ad-CA-MEK) significantly increased MMP-9 and VEGF expression. Taken together, we suggest that the inhibition of TPA-induced MMP-9 and VEGF expression by silibinin is mediated by the suppression of the Raf/MEK/ERK pathway in MCF-7 breast cancer cells.     

Differential regulation of metalloproteinase production, proliferation and chemotaxis of human lung fibroblasts by PDGF, interleukin-1beta and TNF-alpha

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor

Elevated levels of PGE(2) have been reported in synovial fluid and cartilage from patients with osteoarthritis (OA). However, the functions of PGE(2) in cartilage metabolism have not previously been studied in detail. To do so, we cultured cartilage explants, obtained from patients undergoing knee replacement surgery for advanced OA, with PGE(2) (0.1-10 muM). PGE(2) inhibited proteoglycan synthesis in a dose-dependent manner (maximum 25% inhibition (p < 0.01)). PGE(2) also induced collagen degradation, in a manner inhibitable by the matrix metalloproteinase (MMP) inhibitor ilomastat. PGE(2) inhibited spontaneous MMP-1, but augmented MMP-13 secretion by OA cartilage explant cultures. PCR analysis of OA chondrocytes treated with PGE(2) with or without IL-1 revealed that IL-1-induced MMP-13 expression was augmented by PGE(2) and significantly inhibited by the cycolooygenase 2 selective inhibitor celecoxib. Conversely, MMP-1 expression was inhibited by PGE(2), while celecoxib enhanced both spontaneous and IL-1-induced expression. IL-1 induction of aggrecanase 5 (ADAMTS-5), but not ADAMTS-4, was also enhanced by PGE(2) (10 muM) and reversed by celecoxib (2 muM). Quantitative PCR screening of nondiseased and end-stage human knee OA articular cartilage specimens revealed that the PGE(2) receptor EP4 was up-regulated in OA cartilage. Moreover, blocking the EP4 receptor (EP4 antagonist, AH23848) mimicked celecoxib by inhibiting MMP-13, ADAMST-5 expression, and proteoglycan degradation. These results suggest that PGE(2) inhibits proteoglycan synthesis and stimulates matrix degradation in OA chondrocytes via the EP4 receptor. Targeting EP4, rather than cyclooxygenase 2, could represent a future strategy for OA disease modification.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

Matrix metalloproteinase-dependent microsomal prostaglandin E synthase-1 expression in macrophages: role of TNF-α and the EP4 prostanoid receptor

Matrix metalloproteinase (MMP)-9 contributes to the pathogenesis of chronic inflammatory diseases and cancer. Thus, identifying targetable components of signaling pathways that regulate MMP-9 expression may have broad therapeutic implications. Our previous studies revealed a nexus between metalloproteinases and prostanoids whereby MMP-1 and MMP-3, commonly found in inflammatory and neoplastic foci, stimulate macrophage MMP-9 expression via the release of TNF-α and subsequent induction of cyclooxygenase-2 and PGE(2) engagement of EP4 receptor. In the current study, we determined whether MMP-induced cyclooxygenase-2 expression was coupled to the expression of prostaglandin E synthase family members. We found that MMP-1- and MMP-3-dependent release of TNF-α induced rapid and transient expression of early growth response protein 1 in macrophages followed by sustained elevation in microsomal prostaglandin synthase 1 (mPGES-1) expression. Metalloproteinase-induced PGE(2) levels and MMP-9 expression were markedly attenuated in macrophages in which mPGES-1 was silenced, thereby identifying mPGES-1 as a therapeutic target in the regulation of MMP-9 expression. Finally, the induction of mPGES-1 was regulated, in part, through a positive feedback loop dependent on PGE(2) binding to EP4. Thus, in addition to inhibiting macrophage MMP-9 expression, EP4 antagonists emerge as potential therapy to reduce mPGES-1 expression and PGE(2) levels in inflammatory and neoplastic settings.     

Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages

COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1-4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors.