Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.     

Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases

Until recently, relaxin (RLX) has been known predominantly for its effects on the reproductive system, where it induces remodelling of the extracellular matrix and up-regulation of matrix metalloproteases (MMPs). In solid cancers, tissue remodelling and MMP activation are essential for invasion and metastasis. We therefore investigated the effect of RLX on invasiveness and MMP expression of human breast cancer cell lines. Upon incubation with porcine RLX, the invasiveness of SK-BR3 cells was significantly increased. Similar effects could be achieved in MCF-7 cells, especially when RLX was combined with epidermal growth factor. Enhanced invasiveness was accompanied by up-regulation of MMP production and could be almost completely blocked by the MMP inhibitor FN 439. Zymography revealed increased secretion of MMP-2, -7 and -9, associated with up-regulated mRNA concentrations of MMP-2, -9, -13 and -14. mRNA expression levels of MMP-1, -3, -7, -8, -10, -11, -12 and of tissue inhibitors of metalloproteases-1, -2, -3 and -4 were either very low or not detectably influenced by RLX. Taken together, RLX enhances in-vitro invasiveness of breast cancer cell lines by induction of MMP expression. It remains to be clarified whether RLX might play a similar role in vivo and promote tumour progression.     

Increased matrix metalloproteinase-9 activity in human ovarian cancer cells cultured with conditioned medium from human peritoneal tissue

Ovarian cancer cells disseminate by attachment to the peritoneal mesothelial cell surface of the abdominal cavity. We therefore investigated the influence of conditioned medium (CM) from human peritoneal tissues and mesothelial cells on the secretion of matrix metalloproteinases (MMPs) by ovarian cancer cells. The molecular weights of MMPs stimulating factors derived from human peritoneal tissues and mesothelial cells were estimated using microconcentrators with various cut-off membranes. Human peritoneal tissues were obtained from 12 surgical patients, and mesothelial cells were isolated from three peritoneal specimens. Exposure to CM from peritoneal tissue caused a concentration-dependent increase of the MMP-2 and MMP-9 bands in CM from NOM1 ovarian cancer cells, as shown by zymography. There was a significant difference in the increase of MMP-2 and MMP-9 (2.46-fold and 7.14-fold, respectively, at 0.4mg/ml protein; P < 0.005). CM from mesothelial cells also significantly increased the secretion of MMP-9 by NOM1 cells. The molecular size of possible MMP-9-stimulating factors secreted by peritoneal tissues and mesothelial cells was above M 100000. Further, CM of peritoneal tissues and mesothelial cells also induced the invasiveness of NOM1 cells. These findings suggest that mesothelial cells may secrete some factors which predominantly induce the MMP-9 production and increase invading cell numbers.     

CD19 signal transduction in normal human B cells: linkage to downstream pathways requires phosphatidylinositol 3-kinase, protein kinase C and Ca2+

CD19 is required for normal antibody responses in mice. We have shown that CD19 enhances the activation of extracellular signal-regulated kinase (ERK) 2 by membrane (m) IgM but otherwise little is known of CD19 signaling in primary human cells. We now ask which pathways link CD19 with ERK2 in human tonsillar B cells. In analyses of signaling intermediates, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin partially suppressed the release of Ca2+ induced by coligation of CD19 and mIgM but the selective PKC inhibitor bisindolylmaleimide I (BIM-I) did not. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, BIM-I and wortmannin each had only a small effect on ERK2 activation induced by surface IgM alone but blocked the enhancement of that activation by CD1 9/mIgM coligation. To analyze the mitogen-activated protein kinase (MAPK) cascade, we measured activation of Raf, MAPK- or ERK kinase (MEK) 1 and ERK2. CD19 consistently enhanced activation of ERK2 and MEK1. However, synergistic activation of Raf was variably observed. In subpopulation analyses, synergistic activation of Raf1 was consistently observed in the IgDlow but not in the IgDhigh cells. Thus, in normal human B cells, PI3K is upstream of the Ca2+ response while PI3K, Ca2+ release and protein kinase C are all required for ERK2 activation, and CD19 enhances the MAPK cascade at multiple levels, depending on the state of differentiation.     

Protein kinase C-mediated in vitro invasion of human glioma cells through extracellular-signal-regulated kinase and ornithine decarboxylase.

We investigated the involvement of protein kinase C (PKC) in the in vitro invasiveness of the A-172, U-87 and U-373 human glioma cell lines, as well as the role of ornithine decarboxylase (ODC) and/or extracellular-signal-regulated kinase (ERK) in the actions of PKC. Thus, cells were treated under serum-free conditions with the PKC activator phorbol 12-myristate 13-acetate (PMA), or with the PKC inhibitors bisindolylmaleimide I (GF 109203X) or calphostin C in the absence or presence of the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO), and/or the mitogen-activated protein kinase/extracellular-signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD 098059). Subsequently, cells were assessed for membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA contents, 72-kD latent, and 59/62-kD activated matrix metalloproteinase 2 (MMP-2) in conditioned media, as well as invasiveness. For these purposes, we used Northern blot analysis, gelatine zymography, and an in vitro filter invasion assay, respectively. Data were related to those found with untreated cells. PKC activity was 2- to 3-fold stimulated by PMA (100 nM for 30 min), and about 2-fold inhibited by calphostin C (40 nM for 2 h) or GF 109203X (5 microM for 20 min). This was accompanied by a similar increase or decrease, respectively, in MT1-MMP mRNA expression, 59/62-kD MMP-2 activity, and in vitro invasion. Inhibition of ODC activity (about 2-fold by 24 h DFMO 5 mM), ERK activation (almost completely by 20 min PD 098059 50 microM), or both these enzymes simultaneously led to a reduction by about half in levels of MT1-MMP mRNA, 59/62-kD MMP-2 activity, and invasion in untreated as well as PMA-stimulated cells. The use of these compounds did not significantly alter the inhibitory effects of GF 109203X or calphostin C. Modulation of PKC and/or ERK activity resulted in corresponding changes in ERK and/or ODC activities, but interference with ODC affected neither ERK nor PKC. Our data suggest a regulatory role for PKC, in co-operation with ERK and ODC, in glioma cell invasion, by modulation of MT1-MMP mRNA expression and MMP-2 activation.     

Production of matrix metalloproteinase-9 by activated human monocytes involves a phosphatidylinositol-3 kinase/Akt/IKKalpha/NF-kappaB pathway

Matrix metalloproteinase-9 (MMP-9) is considered to be an important component in the progression of inflammation. Monocytes/macrophages are prominent at inflammation sites, and activation of these cells by stimulants, such as lipopolysaccharide (LPS) or tumor necrosis factor alpha and granulocyte macrophage-colony stimulating factor, leads to the production of significant amounts of MMP-9. Here, we show that LPS stimulation of monocytes results in MMP-9 production through a phosphatidylinositol-3 kinase (PI-3K)/Akt/inhibitor of kappaB (IkappaB) kinase-alpha (IKKalpha)/nuclear factor (NF)-kappaB pathway. This new role for Akt in signaling leading to MMP-9 production was demonstrated by inhibitor and immunoprecipitation studies. LY294002 or wortmannin, inhibitors of PI-3K, suppressed LPS-induced Akt activity and MMP-9 production. Evidence for the participation of Akt in monocyte MMP-9 synthesis was demonstrated by the inhibition of MMP-9 by SH-5, a specific inhibitor of Akt. The mechanism by which Akt regulates MMP-9 is through the activation of NF-kappaB, as shown by coimmunoprecipitation of the phosphorylated form of IKKalpha and Akt as well as the SH-5 suppression of the dissociation of IkappaB from NF-kappaB and the activation of NF-kappaB p65. The role of NF-kappaB in regulation of MMP-9 was demonstrated further by the inhibition of MMP-9 production by proteasome inhibitors, lactacystin and MG-132, which prevented the ubiquitination and dissociation of IkappaB from NF-kappaB. This is the first demonstration that Akt is involved in the signaling pathway leading to the production of monocyte MMP-9 and provides an additional approach in the regulation of this enzyme in human primary monocytes.     

STAT3 and ERK Signaling Pathways Are Implicated in the Invasion Activity by Oncostatin M through Induction of Matrix Metalloproteinases 2 and 9

PurposeOur previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo.ResultsOSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition.ConclusionBoth STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.     

Role of DDR1 in the gelatinases secretion induced by native type IV collagen in MDA-MB-231 breast cancer cells

Discoidin domain receptors (DDRs) are receptor tyrosine kinases that get activated by collagens in its native triple-helical form. In mammalian cells, DDR family consists of two members, namely DDR1 and DDR2, which mediates migration and proliferation of several cell types. DDR1 is activated by native type IV collagen and overexpressed in human breast cancer. Type IV collagen is the main component of basement membrane (BM), and the ability to degrade and penetrate BM is related with an increased potential for invasion and metastasis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that collectively are capable of degrading all components of the extracellular matrix, including the BM. In breast cancer cells, denatured type IV collagen induces MMP-9 secretion and invasion. However, the role of DDR1 in the regulation of gelatinases (MMP-2 and -9) secretion and invasion in breast cancer cells remains to be studied. We demonstrate here that native type IV collagen induces MMP-2 and -9 secretions and invasion through a DDR1 and Src-dependent pathway, together with an increase of MMP-2 and -9-cell surface levels. MMP-2 and -9 secretions require PKC kinase activity, epidermal growth factor receptor (EGFR) activation, arachidonic acid (AA) production and AA metabolites in MDA-MB-231 breast cancer cells. In summary, our data demonstrate, for the first time, that DDR1 mediates MMP-2 and -9 secretions and invasion induced by native type IV collagen in MDA-MB-231 breast cancer cells.     

Involvement of MMP-7 in invasion of pancreatic cancer cells through activation of the EGFR mediated MEK-ERK signal transduction pathway

AIMS: To clarify the involvement of matrix metalloproteinase-7 (MMP-7) in cell dissociation and the subsequent invasion of pancreatic cancer cells.METHODS: Western blotting, in vitro invasion assay, immunocytochemistry, and immunohistochemistry were performed in pancreatic cancer cell lines or pancreatic cancer tissue.RESULTS: The active form of the MMP-7 protein was expressed exclusively in the conditioned medium of dissociated (PC-1.0 and AsPC-1) pancreatic cancer cells, whereas proMMP-7 protein was only detected in the conditioned medium of non-dissociated (PC-1 and Capan-2) cells. Both intracellular and conditioned medium localised MMP-7 was greatly reduced by treatment with the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the mitogen activated protein kinase kinase (MEK) inhibitor U0126 in pancreatic cancer cells. MMP-7 treatment significantly induced the disruption of tight junction (TJ) structures and subsequent cell dissociation, and activation of the EGFR mediated MEK- ERK (extracellular signal regulated protein kinase) signalling pathway in the non-dissociated pancreatic cancer cells. Moreover, the strong in vitro invasiveness of dissociated cells was inhibited by AG1478 and U0126 treatment, whereas the weak invasiveness of non-dissociated cells was apparently induced by MMP-7 treatment. In addition, MMP-7 expression was stronger at the invasive front than at the centre of human pancreatic tumours.CONCLUSION: MMP-7 is involved in cell dissociation and the subsequent invasion of pancreatic cancer cells. It induces the disruption of TJ structures and forms a positive feedback loop with activation of the EGFR mediated MEK-ERK signalling pathway.     

Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis

Increased expression of the hepatocyte growth factor (HGF) receptor (c-met) and urokinase type plasminogen (uPA) correlated with the development and metastasis of cancers. To investigate the role of HGF/c-met signaling on metastasis in cancer cells stimulated with HGF, we examined the effects of a specific MEK1 inhibitor (PD98059) and a p38 MAP kinase inhibitor (SB203580) on HGF-induced uPA expression in pancreatic cancer cell lines, L3.6PL and IMIM-PC2. Pretreatment of PD98059 decreased HGF-mediated phosphorylation of extracellular receptor kinase (ERK), uPA secretion and expression of matrix metalloproteinases (MMP-2 and MMP-9) in a dose-dependent manner. In contrast, SB203580 pretreatment increased HGF-stimulated ERK phosphorylation, uPA secretion and expression of MMPs. SB203580 also reversed the inhibition of HGF-mediated ERK activation and uPA secretion in the PD98059-pretreated cells. These results suggest that ERK activation by HGF might play important roles in the metastasis of pancreatic cancer and the p38 MAPK pathway also involved in the HGF-mediated uPA secretion and metastasis by regulation of ERK pathway. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oleic acid promotes MMP-9 secretion and invasion in breast cancer cells

Epidemiological and animal studies suggest an association between dietary fatty acids and an increase risk of developing breast cancer. Obesity, which is characterized by hyperlipidemia and an elevation of circulating free fatty acids (FFAs), is also associated with enhanced cancer risk. In breast cancer cells, the FFA oleic acid (OA) induces migration, proliferation, prolong survival, invasion, an increase in cellular Ca²⁺ concentration, MEK1/2, ERK1/2, FAK and Src activation. However, the role of OA on MMP-9 secretion and invasion has not been studied in detail. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 200 μM OA induces an increase on MMP-9 secretion through a PKC, Src, and EGFR-dependent pathway, as revealed by gelatin zymography assays. Furthermore, microtubule network mediates MMP-9 secretion induced by OA. In contrast, OA does not induce an increase on MMP-9 secretion in MCF10A cells, whereas it does not induce MMP-9 secretion in MCF12A mammary non-tumorigenic epithelial cells. In addition, OA induces invasion through an EGFR, Gi/Go proteins, MMPs, PKC and Src-dependent pathway, but it is not able to promote invasion in non-invasive MCF-7 breast cancer cells. In summary, our findings demonstrate that OA promotes an increase on MMP-9 secretion and invasion through a PKC, Src, and EGFR-dependent pathway in breast cancer cells.     

Fibronectin-bound TNF-alpha stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-alpha avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-alpha complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-alpha by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-alpha into ECM-FN. FN-bound TNF-alpha (FN/TNF-alpha) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-alpha activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Cheniotaxis through ECM gels in the presence of soluble or bound TNF-alpha was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-alpha required functional activated beta1 integrins, FN/TNF-alpha-induced MMP-9 secretion was independent of binding to beta1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing nAb to alpha4, alpha5, and beta1 subunits, which blocked cell adhesion; (2) a mAb that stimulated beta1 integrin-mediated adhesion; and (3) binding TNF-alpha to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-alpha-ECM interactions may serve to limit the cytokine's availability and bioactivity to target areas of inflammation.     

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions*

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

Macrophage migration inhibitory factor induces MMP-9 expression in macrophages via the MEK-ERK MAP kinase pathway.

We have shown previously that macrophage migration inhibitory factor (MIF) may play a role in the destabilization of atherosclerotic plaques by activating matrix metalloproteinase protein-9 (MMP-9). The aim of this study is to investigate the signaling mechanism by which MIF induces MMP-9 expression and activation in a murine macrophage line (RAW264.7). MIF was able to activate extracellular signal-regulated kinase 1/2 (ERK1/2), to a less extent JNK, but not p38 mitogen-activated protein (MAP), MAP kinase to induce MMP9 mRNA and protein expression in RAW264.7 murine macrophages. This was confirmed by the findings that addition of an ERK MAP kinase inhibitor (PD98059) but not a p38 inhibitor (SB203589) abolished MIF-induced MMP-9 expression and activation, whereas addition of a JNK inhibitor (SP600125) produced a partially inhibitory effect. The functional role of mitogen-activated protein kinase kinase (MEK)-ERK MAP kinase in MIF-induced MMP-9 expression was further confirmed by overexpressing dominant negative MEK (DN-MEK) and DN-ERK MAP kinases. Interestingly, constitutive expression of a wild-type (WT)-MEK alone was also capable of inducing a low, but significant MMP-9 mRNA and protein expression but did not cause a further increase in MMP-9 in response to MIF. MIF activates the MEK-ERK MAP kinase pathway to induce MMP-9 expression by murine macrophages. Activation of this pathway is necessary for MMP-9 expression and activation in response to MIF stimulation.     

Protein kinase C δ inhibits the production of proteolytic enzymes in murine mammary cells

In previous studies we have determined that protein kinase C (PKC) δ, a widely expressed member of the novel PKC serine-threonine kinases, induces in vitro changes associated with the acquisition of a malignant phenotype in NMuMG murine mammary cells. In this study we show that PKCδ overexpression significantly decreases urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) production, two proteases associated with migratory and invasive capacities. This effect is markedly enhanced by treatment with phorbol 12-myristate 13-acetate (PMA). On the other hand, depletion of PKCδ using RNAi led to a marked increase in both uPA and MMP-9 secretion, suggesting a physiological role for PKCδ in controlling protease secretion. The MEK-1 inhibitor PD98059 reverted the characteristic pattern of proteases secretion and phospho-ERK1/2 up-regulation observed in PKCδ overexpressors, suggesting that the PKCδ effect is mediated by the MEK/ERK pathway. Our results suggest a dual role for PKCδ in murine mammary cell cancer progression. While this kinase clearly promotes mitogenesis and favors malignant transformation, it also down-modulates the secretion of proteases probably limiting metastatic dissemination.     

Regulation of Gelatinases Expression by Cytokines,Endotoxin, and Pharmacological Agents in the Human Osteoarthritic Knee

We examined the amount of gelatinases (matrix metalloproteinase-2 and -9 [MMP-2 and MMP-9] in a series of chondral, meniscal, and synovial cultures of early osteoarthritis (OA) after treatment with or without catabolic cytokines. These included interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), and pharmacological agents, including plasmin/serine proteinase antagonist aprotinin, protein synthesis inhibitor cycloheximide, and protein kinase C (PKC) inhibitors staurosporine, H7, and Gö6976 for investigation of their effects on MMP-2 and -9 production in OA. Gelatin zymography revealed that IL-α, TNF-α, and LPS could elevate MMP-2 secretion in all tissue cultures and also increase MMP-9 production in all synovial and some meniscal cultures. In contrast, aprotinin, cycloheximide, staurosporine, H7, and Gö6976 could suppress MMP-2 secretion in all tissue cultures and also decrease MMP-9 production in all synovial and some meniscal cultures. Our data indicate that catabolic cytokines and LPS may promote tissue destruction and disintegration of extracellular matrix in early OA. Agents that target on the PKC pathway, plasmin/serine proteinase or protein synthesis for MMP-2 and -9 in early OA may inhibit the production of MMPs. These findings might contribute to the design of more efficacious therapies.