Induced MMP-9 Expression Research Articles

Abstract 2089: MMP7-mediated cleavage of nucleolin at the Asp255 induces MMP9 expression to promote tumor malignancy

Abstract Nucleolin, expressed in various cellular compartments, participates in DNA transcription, ribosomal biogenesis, regulation of RNA stability. It is evidence that the posttranslational cleavage of nucleolin determine the fate of cells, apoptosis or proliferation. However, the contributions of nucleolin accumulation and cleavage to tumor development are still not clear. Herein we found that nucleolin was dramatically increased in lung cancer and correlated with poor prognosis. The overexpressed nucleolin was cleaved to the smaller fragment (55 kDa), identified in human lung cancer and mouse models of lung cancer. To study the activated mechanism found that EGF-mediated EGFR and KRAS activations could enhance nucleolin expression through increasing Sp1-mediated transcription. Furthermore, to clarify the cleavage residue and cutting enzyme found that nucleolin could be cleaved at 255th Asp by MMP7 (255-710 a.a., TNCL). By using overexpression of TNCL indicated that TNCL enhanced proliferation and metastasis in vitro and in vivo. To further clarify the mechanism of TNCL-enhanced tumor malignancy show that TNCL significantly increase several oncogenes expression including MMP9, Alk and CD74 through associating with 3′-UTR to enhance mRNA stability. Taken together, MMP7 can cleave nucleolin to increase the oncogenes expression, leading lung cancer formation Citation Format: Jan-Jong Hung, Wen-Chang Chang. MMP7-mediated cleavage of nucleolin at the Asp255 induces MMP9 expression to promote tumor malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2014-2089

Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

Fibronectin Affects Transient MMP2 Gene Expression through DNA Demethylation Changes in Non-Invasive Breast Cancer Cell Lines

Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.

Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells.

Thyroid hormone (3,5,3'-triiodothyronine, T3; L-thyroxine, T4) enhances cancer cell proliferation, invasion and angiogenesis via a discrete receptor located near the RGD recognition site on αvβ3 integrin. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation interfere with binding of T3/T4 to the integrin. This integrin is overexpressed in multiple myeloma (MM) and other cancers. MM cells interact with αvβ3 integrin to support growth and invasion. Matrix metalloproteinases (MMPs) are a family of enzymes active in tissue remodeling and cancer. The association between integrins and MMPs secretion and action is well established. In the current study, we examined the effects of thyroid hormone on myeloma cell adhesion, migration and MMP activity. We show that T3 and T4 increased myeloma adhesion to fibronectin and induced αvβ3 clustering. In addition, the hormones induced MMP-9 expression and activation via αvβ3 and MAPK induction. Bortezomib, a standard myeloma treatment, caused a decrease in activity/quantity of MMPs and thyroid hormone opposed this effect. RGD peptide and tetrac impaired the production of MMP-9 in cell lines and in primary BM cells from myeloma patients. In conclusion, thyroid hormone-dependent regulation via αvβ3 of myeloma cell adhesion and MMP-9 production may play a role in myeloma migration and progression.

Extracellular matrix protein laminin induces matrix metalloproteinase-9 in human breast cancer cell line mcf-7.

Studies on interaction of tumor cells with extracellular matrix (ECM) components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human breast cancer cell line MCF-7-laminin (LM) interaction on MMPs and the underlying signaling pathways. Culturing of MCF-7 cells on LM coated surface upregulated MMP-9 expression as well as reduced tissue inhibitor of metalloproteinases-1 (TIMP-1) expression. LM induced MMP-9 expression is abrogated by the blockade of α2 integrin. Inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI3K), extracellular signal regulated kinase (ERK) and nuclear factor-kappaB (NF-κB) in LM induced signaling. LM treatment also enhanced phosphorylation of FAK (focal adhesion kinase), PI3K, ERK; nuclear translocation of ERK, pERK, NF-κB and cell migration. Our findings indicate that, binding of MCF-7 cells to LM, possibly via α2β1 integrin, induces signaling involving FAK, PI3K, ERK, NF-κB followed by upregulation of MMP-9 and cell migration.

Cdc42 Inhibits ERK-Mediated Collagenase-1 (MMP-1) Expression in Collagen-Activated Human Keratinocytes

Following injury, keratinocytes switch gene expression programs from one that promotes differentiation to one that supports migration. A common feature of human wounds and ulcerations of any form is the expression of MMP-1 (collagenase-1) by leading-edge basal keratinocytes migrating across the dermal or provisional matrix. Induction of MMP-1 occurs by signaling from the α2β1 integrin in contact with dermal fibrillar type I collagen, and the activity of MMP-1 is required for human keratinocytes to migrate on collagen. Thus, MMP-1 serves a critical role in repair of damaged human skin. Here, we evaluated the mechanisms controlling MMP-1 expression in primary human keratinocytes from neonatal foreskin and adult female skin. Our results demonstrate that shortly following contact with type I collagen, ERK and p38 MAPK were markedly activated, whereas JNK phosphorylation remained at basal levels. ERK inhibition dramatically blocked collagen-stimulated MMP-1 expression in keratinocytes. In contrast, inhibiting p38 or JNK pathways had no effect on MMP-1 production. Moreover, investigating the role of Rho GTPases revealed that Cdc42 attenuates MMP-1 expression by suppressing ERK activity. Thus, our data indicates that injured keratinocytes induce MMP-1 expression through ERK activation, and this process is negatively regulated by Cdc42 activity.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

MMP7-mediated cleavage of nucleolin at Asp255 induces MMP9 expression to promote tumor malignancy

Nucleolin (NCL) participates in DNA transcription, ribosomal biogenesis and the regulation of RNA stability. However, the contribution of NCL to tumor development is still not clear. Herein, we found that NCL expression correlated with poor prognosis in lung cancer patients. Overexpressed NCL was predominantly cleaved to C-terminal truncated NCL (TNCL). In lung cancer formation, activation of the epidermal growth factor receptor pathway induced NCL expression, and also the expression of matrix metalloproteinase (MMP) 7, which then cleaved NCL at Asp255 to generate TNCL of 55 kDa. TNCL increased the expression of several oncogenes, including MMP9, anaplastic lymphoma kinase (ALK), HIF1a and CBLB, and decreased the expression of tumor suppressors including BRD4, PCM1, TFG and KLF6 by modulating mRNA stability through binding to the 3'-untranslated regions of their transcripts, thus ultimately enhancing metastasis activity. In conclusion, this study identified a novel role of the cleavage form of NCL generated by MMP7 in stabilizing MMP9 mRNA. We also provide a new insight that MMP7 not only cleaves the extracellular matrix to promote tumor invasion but also cleaves NCL, which augment oncogenesis. Blocking NCL cleavage may provide a useful new strategy for lung cancer therapy.

Low-level laser therapy promotes proliferation and invasion of oral squamous cell carcinoma cells

Low-level laser therapy (LLLT) has been shown to be effective in promoting cell proliferation. There is speculation that the biostimulatory effect of LLLT causes undesirable enhancement of tumor growth in neoplastic diseases since malignant cells are more susceptible to proliferative stimuli. This study evaluated the effects of LLLT on proliferation, invasion, and expression of cyclin D1, E-cadherin, β-catenin, and MMP-9 in a tongue squamous carcinoma cell line (SCC25). Cells were irradiated with a diode laser (660 nm) using two energy densities (0.5 and 1.0 J/cm(2)). The proliferative potential was assessed by cell growth curves and cell cycle analysis, whereas the invasion of cells was evaluated using a Matrigel cell invasion assay. Expression of cyclin D1, E-cadherin, β-catenin, and MMP-9 was analyzed by immunofluorescence and flow cytometry and associated with the biological activities studied. LLLT induced significantly the proliferation of SCC25 cells at 1.0 J/cm(2), which was accomplished by an increase in the expression of cyclin D1 and nuclear β-catenin. At 1.0 J/cm(2), LLLT significantly reduced E-cadherin and induced MMP-9 expression, promoting SCC25 invasion. The results of this study demonstrated that LLLT exerts a stimulatory effect on proliferation and invasion of SCC25 cells, which was associated with alterations on expression of proteins studied.

Interleukin-11 induces the expression of matrix metalloproteinase 13 in gastric cancer SCH cells partly via the PI3K-AKT and JAK-STAT3 pathways

Interleukin (IL)-11 is expressed in the majority of gastric carcinomas and has been associated with an aggressive phenotype and poor prognosis of gastric adenocarcinoma. Matrix metalloproteinase (MMP)-13 has been detected in numerous invasive malignant tumor types and exhibits a broad spectrum of activities on connective tissue components. In this study, we investigated whether IL-11 affects the expression of MMP-13 in human gastric cancer cells, as well as the underlying mechanism. Using western blot assays, we investigated the effect of recombinant human (rh) IL-11 on the expression of MMP-13 in gastric carcinoma cell lines. Using the PI3K inhibitor wortmannin and RNA interference to target the STAT3 gene, we investigated the effects of PI3K inhibition and/or STAT3 depletion on the expression of the MMP-13 protein. Results showed that IL-11 induced MMP-13 expression in a time- and concentration-dependent manner in SCH cells. IL-11 activated PI3K-AKT and JAK-STAT3 signal transduction. Wortmannin and depletion of STAT3 by means of small interfering RNA (siRNA) synergistically reduced the expression of MMP-13. These findings suggested that IL-11 induces the expression of MMP-13 in gastric cancer SCH cells partly via the PI3K-AKT and JAK-STAT3 pathways.

P27KIP1 is involved in ERK1/2-mediated MMP-9 expression via the activation of NF-κB binding in the IL-7-induced migration and invasion of 5637 cells

Interleukin-7 (IL-7) plays a pivotal role in the development and survival of lymphocytes, but its role in cancer cell responses remains unexplained. In this study, IL-7 treatment resulted in a significant induction in the wound-healing migration and Matrigel invasion of the 5637 bladder cancer cells, but it did not result in cell proliferation. In addition, IL-7 treatment strongly induced MMP-9 expression, and increased the binding activation of NF-κB and AP-1 motifs, the important transcription factors that regulate MMP-9 expression. Moreover, the treatment of 5637 cells with IL-7 stimulated the phosphorylation of ERK1/2. U0126, an ERK1/2-specific inhibitor, blocked IL-7-induced cell migration and invasion, and also suppressed the expression of MMP-9 in the presence of IL-7. Inhibition of the ERK1/2 function consistently reversed the binding activity of NF-κB without altering AP-1 activation in IL-7-stimulated cells. Among the cell cycle regulators examined, only the expression of the cell cycle inhibitor p27KIP1 was induced by IL-7. Moreover, the inhibition of p27KIP1 by small interfering RNA (siRNA) abolished the migration, invasion and phosphorylation of ERK1/2, the expression of MMP-9, and the binding activity of the NF-κB motif in IL-7-stimulated 5637 cells. These results demonstrated that the cell cycle inhibitor p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via activation of the NF-κB binding motif, which leads to the migration and invasion of bladder cancer cells induced by IL-7. These novel results could help explain the migration and invasion of bladder tumor cells.

Toll‐like receptor 9 expression in mucoepidermoid salivary gland carcinoma may associate with good prognosis

Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. Although several biomarkers have been evaluated, histological grade remains the most valuable prognostic marker. Toll-like receptor 9 (TLR9) is an immune receptor recognizing microbial DNA. Its expression associates with prognosis or cancer properties in several cancers. This study examined the role of TLR9 in MEC. Sixty patients with salivary gland MEC were collected from two Finnish university hospitals, and tumor samples were stained for TLR9. Salivary gland high-grade MEC cell line (UT-MUC-1) was cultured to assess TLR9 and MMP-13 expression. The function of TLR9 was studied in vitro using traditional Matrigel(®) invasion assay and novel human myoma organotypic model. Cancer-specific survival was related with tumor grade (P = 0.01), and there were no deaths in patients with low-grade MEC. TLR9 was expressed in 56 of 60 (93%) tumors. High TLR9 expression indicated better survival in the patient series (P = 0.002) and showed a trend for association with lower disease stage (P = 0.06) and higher differentiation grade (P = 0.068). In multivariate analysis, TLR9 expression was prognostically insignificant due to heavy correlation to disease stage and higher gradus. Treating UT-MUC-1 cells with TLR9 ligand CpG in vitro induced MMP-13 expression and invasion in Matrigel(®) invasion assay, whereas decreased invasion was seen in myoma organotypic model. Functional TLR9 is present in salivary MEC, and high level of expression may indicate good prognosis. However, more studies are needed to evaluate biological consequences of TLR9 interaction in tumor cells.

Dexamethasone inhibits interleukin-1β-induced matrix metalloproteinase-9 expression in cochlear cells.

ObjectivesTo investigate the effect of interleukin (IL)-1β on matrix metalloproteinase (MMP)-9 expression in cochlea and regulation of IL-1β-mediated MMP-9 expression by dexamethasone and the molecular and signaling mechanisms involved.MethodsHouse ear institute-organ of Corti 1 (HEI-OC1) cells were used and exposed to IL-1β with/without dexamethasone. Glucocorticoid receptor antagonist, RU486, was used to see the role of dexamethasone. PD98059 (an extracellular signal-regulated kinases [ERKs] inhibitor), SB203580 (a p38 mitogen-activated protein kinases [MAPK] inhibitor), SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor) were also used to see the role of MAPKs signaling pathway(s) in IL-1β-induced MMP-9 expression in HEI-OC1 cells. Reverse transcription-polymerase chain reaction and gelatin zymography were used to measure mRNA expression level of MMP-9 and activity of MMP-9, respectively.ResultsTreatment with IL-1β-induced the expression of MMP-9 in a dose- and time-dependent manner. IL-1β (1 ng/mL)-induced MMP-9 expression was inhibited by dexamethasone. Interestingly, p38 MAPK inhibitor, SB203580, significantly inhibited IL-1β-induced MMP-9 mRNA and MMP-9 activity. However, inhibition of JNKs and ERKs had no effect on the IL-1β-induced MMP-9 expression.ConclusionThese results suggest that the pro-inflammatory cytokine IL-1β strongly induces MMP-9 expression via activation of p38 MAPK signaling pathway in HEI-OC1 cells and the induction was inhibited by dexamethasone.

IL-6–Mediated Induction of Matrix Metalloproteinase-9 Is Modulated by JAK-Dependent IL-10 Expression in Macrophages

The mechanisms by which IL-6 contributes to the pathogenesis of chronic inflammatory diseases and cancer are not fully understood. We previously reported that cyclooxygenase-2 (Cox-2)-dependent PGE2 synthesis regulates macrophage matrix metalloproteinase (MMP)-9 expression, an endopeptidase that participates in diverse pathologic processes. In these studies, we determined whether IL-6 regulates the Cox-2→PGE2→MMP-9 pathway in murine macrophages. IL-6 coinduced Cox-2 and microsomal PGE synthase-1, and inhibited the expression of 15-hydroxyprostaglandin dehydrogenase, leading to increased levels of PGE2. In addition, IL-6 induced MMP-9 expression, suggesting that the observed proteinase expression was regulated by the synthesis of PGE2. However, inhibition of PGE2 synthesis partially suppressed IL-6-mediated induction of MMP-9. In the canonical model of IL-6-induced signaling, JAK activation triggers STAT and MAPK(erk1/2)-signaling pathways. Therefore, the ability of structurally diverse JAK inhibitors to block IL-6-induced MMP-9 expression was examined. Inhibition of JAK blocked IL-6-induced phosphorylation of STAT3, but failed to block the phosphorylation of MAPK(erk1/2), and unexpectedly enhanced MMP-9 expression. In contrast, MEK-1 inhibition blocked IL-6-induced phosphorylation of MAPK(erk1/2) and MMP-9 expression without affecting the phosphorylation of STAT3. Thus, IL-6-induced MMP-9 expression is dependent on the activation of MAPK(erk1/2) and is restrained by a JAK-dependent gene product. Using pharmacologic and genetic approaches, we identified JAK-dependent induction of IL-10 as a potent feedback mechanism controlling IL-6-induced MMP-9 expression. Together, these data reveal that IL-6 induces MMP-9 expression in macrophages via Cox-2-dependent and -independent mechanisms, and identifies a potential mechanism linking IL-6 to the pathogenesis of chronic inflammatory diseases and cancer.

진귤 과피 추출물의 MMP-1 발현조절 효과

Flavonoids are one of the major components found in the peels of citrus fruits. Present evidence has suggested that polymethoxyflavonoids, including nobiletin and tangeretin isolated from Citrus sunki, have many biological properties, such as anti-inflammatory, anti-oxidant, and anti-obesity capabilities. Here, we investigated the effect of Citrus sunki peel extract and its possible mechanisms on oxidative stress-induced MMP-1 expression, a major marker of skin photoaging. H₂ O₂ induced MMP-1 expression in a dose- and time-dependent manner. Extract of Citrus sunki peel (1-25 μg/ml) dose-dependently decreased MMP-1 mRNA levels. When H₂ O₂ was combined with Citrus sunki peel extract, the phosphorylation of ERK was further decreased compared to a single treatment with H₂ O₂ alone. Moreover, U0216, an MEK inhibitor, markedly prevented the production of MMP-1. These data suggest that Citrus sunki peel extract has demonstrated protective activity against oxidative damage on MMP-1 expression, and ERK MAP kinase may be involved.

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]

VEGF Secreted by Hypoxic Müller Cells Induces MMP-2 Expression and Activity in Endothelial Cells to Promote Retinal Neovascularization in Proliferative Diabetic Retinopathy

In proliferative diabetic retinopathy (PDR), retinal ischemia promotes neovascularization (NV), which can lead to profound vision loss in diabetic patients. Treatment for PDR, panretinal photocoagulation, is inherently destructive and has significant visual consequences. Therapies targeting vascular endothelial growth factor (VEGF) have transformed the treatment of diabetic eye disease but have proven inadequate for treating NV, prompting exploration for additional therapeutic options for PDR patients. In this regard, extracellular proteolysis is an early and sustained activity strictly required for NV. Extracellular proteolysis in NV is facilitated by the dysregulated activity of matrix metalloproteinases (MMPs). Here, we set out to better understand the regulation of MMPs by ischemia in PDR. We demonstrate that accumulation of hypoxia-inducible factor-1α in Müller cells induces the expression of VEGF, which, in turn, promotes increased MMP-2 expression and activity in neighboring endothelial cells (ECs). MMP-2 expression was detected in ECs in retinal NV tissue from PDR patients, whereas MMP-2 protein levels were elevated in the aqueous of PDR patients compared with controls. Our findings demonstrate a complex interplay among hypoxic Müller cells, secreted angiogenic factors, and neighboring ECs in the regulation of MMP-2 in retinal NV and identify MMP-2 as a target for the treatment of PDR.

C-Src/Jak2/PDGFR/PKCδ-Dependent MMP-9 Induction Is Required for Thrombin-Stimulated Rat Brain Astrocytes Migration

Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) were not completely understood. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 in RBA-1 cells and cells migration which were attenuated by pretreatment with the inhibitor of receptor tyrosine kinase (Genistein), c-Src (PP1), Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), PKCs (Ro318220), PKCδ (Rottlerin), or NF-κB (Bay11-7082) and transfection with siRNA of c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, or IKKβ. In addition, thrombin-stimulated c-Src, Jak2, or PDGFR phosphorylation was inhibited by a thrombin inhibitor (PPACK), PP1, AG490, or AG1296. Thrombin further stimulated c-Src and PDGFR complex formation in RBA-1 cells. Thrombin also stimulated Akt and PKCδ phosphorylation and PKCδ translocation which were reduced by PPACK, PP1, AG490, AG1296, or LY294002. We further observed that thrombin markedly stimulated ATF2 or IκBα phosphorylation and NF-κB p65 translocation which were inhibited by Rottlerin or LY294002. Finally, thrombin stimulated in vivo binding of p65 to the MMP-9 promoter, which was reduced by pretreatment with Rottlerin or LY294002. These results concluded that in RBA-1 cells, thrombin activated a c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ pathway, which in turn triggered ATF2 and NF-κB activation and ultimately induced MMP-9 expression associated with cell migration.

The DNA hypomethylating agent, 5‐aza‐2′‐deoxycytidine, enhances tumor cell invasion through a transcription‐dependent modulation of MMP‐1 expression in human fibrosarcoma cells

In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5-aza-2'-deoxycytidine (5-azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP-1, -2, and -9 in human HT1080 fibrosarcoma cells. We demonstrate that 5-azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad-spectrum and specific MMP inhibitors, we establish that MMP-1, but not MMP-2 and -9, plays a key role in 5-azadC-enhanced cell invasion. We show that 5-azadC induces MMP-1 expression through a transcriptional mechanism without affecting MMP-1 promoter methylation status. Finally, we demonstrate that 5-azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP-1 promoter, resulting in chromatin remodeling associated to 5-azadC-induced MMP-1 expression. All together, our data indicate that the hypomethylating agent 5-azadC modulates, mainly via Sp1 recruitment, MMP-1 expression resulting in an increased invasive potential of HT1080 cells.

Solar-simulated radiation and heat treatment induced metalloproteinase-1 expression in cultured dermal fibroblasts via distinct pathways: Implications on reduction of sun-associated aging

BackgroundSun exposure is an important environmental factor affecting human beings. Most knowledge regarding solar aging focused on light radiation (photoaging), and little emphasis has been placed on heat, a factor that is also closely associated with sun exposure. ObjectiveThis study was launched to evaluate the effects of simulated solar radiation (SSR) and environmental heat on skin fibroblasts in terms of dermal aging. MethodsCultured human dermal fibroblasts were treated with moderate amount of SSR (200J/cm2) and heat (+2°C). The metalloproteinase-1 (MMP-1) expression was used as a surrogate marker for dermal aging and the involved regulatory mechanisms were explored. ResultsBoth treatment conditions did not affect viability but significantly increased the expressions of MMP-1. In parallel, both treatments increased the intracellular levels of reactive oxygen species (ROS), but the increase induced by SSR is much greater than heat. In contrast, transient receptor potential vanilloid 1 (TRPV-1), the sensor of environmental heat, was upregulated by heat but not SSR treatment. Pretreating fibroblasts with antioxidant abrogated the SSR-induced MMP-1 but has limited effect on heat-induced MMP-1. On the other hand, TRPV-1 antagonist pretreatment reduced heat-induced MMP-1 in fibroblasts but not their SSR-treated counterparts. ConclusionBoth SSR and heat induced MMP-1 expression in dermal fibroblasts but through different pathways. As current strategies for reducing sun-related aging focused on filtering of light and use of antioxidants, future strategies design to reduce solar aging should also incorporate heat-induced aging into consideration.