Regulation Of Matrix Metalloproteinase-2 Expression Research Articles

Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression

BackgroundExtravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells.MethodsHTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay.ResultsGDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-β type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed.ConclusionsThese findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells.1EyNAiCz9UDZ9srU6xtfDxVideo

Role of Periostin Expression in Non-Small Cell Lung Cancer: Periostin Silencing Inhibits the Migration and Invasion of Lung Cancer Cells via Regulation of MMP-2 Expression.

The involvement of periostin (POSTN) in non-small-cell lung cancer (NSCLC) migration, invasion, and its underlying mechanisms has not been well established. The present study aims to determine epithelial POSTN expression in NSCLC and to assess associations with clinicopathological factors and prognosis as well as to explore the effects of POSTN knockdown on tumor microenvironment and the migration and invasion of lung cancer cells. Immunohistochemistry was used to evaluate epithelial POSTN expression in NSCLC. POSTN mRNA expression in the dissected lung cancer cells was confirmed by laser capture microdissection and real-time PCR. A549 cells were used for transfecting shRNA-POSTN lentiviral particles. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with POSTN-specific short hairpin (sh)RNA. The results demonstrated significantly higher cytoplasmic POSTN expression in the whole NSCLC group compared to non-malignant lung tissue (NMLT). POSTN expression in cancer cells may be considered to be an independent prognostic factor for survival in NSCLC. POSTN knockdown significantly inhibited A549 cell migration and invasion capabilities in vitro. The activity and the expression level of matrix metalloproteinase-2 (MMP-2) were significantly decreased in A549.shRNA compared to control cells. In summary, POSTN may regulate lung cancer cell invasiveness by modulating the expression of MMP-2 and may represent a potential target for novel therapeutic intervention for NSCLC.

The Complement C3a-C3aR Axis Promotes Development of Thoracic Aortic Dissection via Regulation of MMP2 Expression.

Thoracic aortic dissection (TAD), once ruptured, is devastating to patients, and no effective pharmaceutical therapy is available. Anaphylatoxins released by complement activation are involved in a variety of diseases. However, the role of the complement system in TAD is unknown. We found that plasma levels of C3a, C4a, and C5a were significantly increased in patients with TAD. Elevated circulating C3a levels were also detected in the developmental process of mouse TAD, which was induced by β-aminopropionitrile monofumarate (BAPN) treatment, with enhanced expression of C1q and properdin in mouse dissected aortas. These findings indicated activation of classical and alternative complement pathways. Further, expression of C3aR was obviously increased in smooth muscle cells of human and mouse dissected aortas, and knockout of C3aR notably inhibited BAPN-induced formation and rupture of TAD in mice. C3aR antagonist administered pre- and post-BAPN treatment attenuated the development of TAD. We found that C3aR knockout decreased matrix metalloproteinase 2 (MMP2) expression in BAPN-treated mice. Additionally, recombinant C3a stimulation enhanced MMP2 expression and activation in smooth muscle cells that were subjected to mechanical stretch. Finally, we generated MMP2-knockdown mice by in vivo MMP2 short hairpin RNA delivery using recombinant adeno-associated virus and found that MMP2 deficiency significantly reduced the formation of TAD. Therefore, our study suggests that the C3a-C3aR axis contributes to the development of TAD via regulation of MMP2 expression. Targeting the C3a-C3aR axis may represent a strategy for inhibiting the formation of TAD.

In Vitro Effect of NDP Kinase and Its Regulation of MMP-2 Expression in A549 Lung Cancer Cell Line

Nucleoside Diphosphate (NDP) Kinase is an enzyme that has been reported to act as an inhibitor of metastasis or transcription factor in mammals. It also functions as a differentiation-inhibiting factor in leukemic cells that is independent of NDP kinase catalytic activity and dependent on the presence of N-terminal peptide. Present study focused on in vitro effect of Streptomyces sp. NDP kinase and its regulation of Matrix metalloproteinase-2 (MMP-2) expression in A549 cell line. The purified Streptomyces sp. NDP kinase used in this study induced A549 cell death with IC50 value of 15μg/ml. Flow cytometry analysis using Annexin V and Propidium Iodide double staining, the treatment of A549 cells with 10 and 20μg/ml of Streptomyces sp. NDP kinase for 24 h respectively, showed 26.96% and 47.61% of cells undergoing apoptosis respectively, while the percentage of necrotic cells appeared 13.11% in both concentrations. As NDP kinase from human is a potent metastatic suppressor, the effect of Streptomyces sp.-strained NDP kinase on gene expression of MMP-2, which is involved in the induction of cancer metastasis, was studied using real time PCR. NDP kinase significantly down regulates MMP-2 gene expression in concentrationdependent manner. This is the first report of NDP kinase from naturally isolated Streptomyces sp. that regulates MMP-2 gene expression.

Reactive oxygen species released from hypoxic hepatocytes regulates MMP-2 expression in hepatic stellate cells.

Hypoxia is a common environmental stress factor and is associated with fibrogenesis. Matrix metalloproteinase-2 (MMP-2), produced by hepatic stellate cells (HSCs), plays an important role in liver fibrogenesis. However, inconsistent results have been reported on the impact of hypoxia on MMP-2 expression and activity in HSCs. We speculated that cell–cell interaction is involved in the regulation of MMP-2 expression and activity at low oxygen level in vivo. Therefore, in this report we investigated the mechanism by which hypoxic hepatocytes regulates MMP-2 expression in HSCs. Our results showed that the conditioned medium from hypoxia-treated rat hepatocytes strongly induced the expression of MMP-2 mRNA and protein in rat HSC-T6 cells. Reduced glutathione neutralized ROS released from hypoxic hepatocytes, leading to reduced MMP-2 expression in HSC-T6 cells. In addition, phospho-IκB-α protein level was increased in HSC-T6 cells treated with hypoxia conditioned medium, and NF-κB signaling inhibitor inhibited MMP-2 expression in HSC-T6 cells. Taken together, our data suggest that ROS is an important factor released by hypoxic hepatocytes to regulate MMP-2 expression in HSCs, and NF-κB signaling is crucially involved in ROS-induced MMP-2 expression in HSCs. Our findings suggest that strategies aimed at antagonizing the generation of ROS in hypoxic hepatocytes and inhibiting NF-κB signaling in HSCs may represent novel therapeutic options for liver fibrosis.

Regulation of angiogenesis and invasion by human Pituitary tumor transforming gene (PTTG) through increased expression and secretion of matrix metalloproteinase-2 (MMP-2)

BackgroundPituitary tumor transforming gene (PTTG) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Existence of a relationship between PTTG levels and tumor angiogenesis and metastasis has been reported. However, the mechanisms by which PTTG achieve these functions remain unknown. In the present study, we investigated the effect of overexpression of PTTG on secretion and expression of metastasis-related metalloproteinase-2 (MMP-2) in HEK293 cells, cell migration, invasion and tubule formation.ResultsTransient or stable transfection of HEK293 cells with PTTG cDNA showed a significant increase in secretion and expression of MMP-2 measured by zymography, reverse transcriptase (RT/PCR), ELISA, and MMP-2 gene promoter activity. Furthermore, in our studies, we showed that tumor developed in nude mice on injection of HEK293 cells that constitutively express PTTG expressed high levels of both MMP-2 mRNA and protein, and MMP-2 activity. Conditioned medium collected from the HEK293 cells overexpressing PTTG showed a significant increase in cell migration, invasion and tubule formation of human umbilical vein endothelial cells (HUVEC). Pretreatment of conditioned medium with MMP-2-specific antibody significantly decreased these effects, suggesting that PTTG may contribute to tumor angiogenesis and metastasis via activation of proteolysis and increase in invasion through modulation of MMP-2 activity and expression.ConclusionOur results provide novel information that PTTG contributes to cell migration, invasion and angiogenesis by induction of MMP-2 secretion and expression. Furthermore, we showed that tumors developed in nude mice on injection of HEK293 cells that constitutively express PTTG induce expression of MMP-2 and significantly increase its functional activity, suggesting a relationship between PTTG levels and MMP-2 which may play a critical role in regulation of tumor growth, angiogenesis and metastasis. Blocking of function of PTTG or down regulation of its expression in tumors may result in suppression of tumor growth and metastasis, through the down regulation of MMP-2 expression and activity. To our knowledge, this study is the first study demonstrating the modulation of MMP-2 expression and biological activity by PTTG.

K-Ras Regulates the Steady-state Expression of Matrix Metalloproteinase 2 in Fibroblasts

Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not c-K(A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K(B)-Ras through a PI-3 kinase:AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras.

ANALYSIS OF THE GENE EXPRESSION OF SPARC AND ITS PROGNOSTIC VALUE FOR BLADDER CANCER

We analyzed the gene expression of the glycoprotein termed secreted protein, acidic and rich in cysteine (SPARC), also called osteonectin and BM40, in bladder cancer and its relationship with conventional clinical-histopathological manifestations, evaluated its prognostic value for patient outcome and determined the possible mechanism underlying the effect of SPARC on bladder cancer progression. Tissue samples from 63 patients with bladder cancer were used for analysis. Gene expression levels of SPARC and matrix metalloproteinase-2 were analyzed using reverse transcription-polymerase chain reaction. Correlations of the expression of SPARC with histopathological findings or patient outcome and with matrix metalloproteinase-2 were evaluated. Significantly higher expression of SPARC was observed in grades 3 and 2 than in grade 1 tumors (p <0.001 and <0.05, respectively). Stage T2 or greater invasive tumors expressed a significantly higher level of SPARC than stages T1 or less superficial tumors (p <0.0001). Patients in whom the lesions showed high SPARC expression had a significantly worse prognosis than those with low SPARC expression disease (p <0.0001). Even in those with invasive bladder cancer high SPARC expression was associated with significantly worse survival than low expression (p <0.01). Moreover, gene expression of SPARC significantly correlated with matrix metalloproteinase-2 gene expression (p <0.0001), implying that regulation of matrix metalloproteinase-2 expression may be a possible mechanism underlying the effect of SPARC on bladder cancer progression. A significant correlation was detected of the gene expression level of SPARC with histological grade, pathological stage and bladder cancer prognosis. SPARC may have an important role in bladder cancer progression and provide some additional information in patients with bladder cancer.

Phorbol ester tumour promoter mediated altered expression and regulation of matrix metalloproteinase-2 in a H-ras transformed cell line capable of benign tumour formation.

The matrix metalloproteinases (MMPs) are thought to play key roles in tumour formation and malignant progression. The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to the phorbol ester tumour promoter, PMA, in a H-ras transformed cell line, NR3, which is capable of benign tumour formation. PMA treatment of NR3 cells resulted in decreased expression of MMP-2 mRNA levels. Following a lag period, an accompanying change in gelatinolytic activity was also found. These PMA-mediated alterations in MMP-2 mRNA levels were independent of de novo protein synthesis and involved both transcriptional and post-transcriptional events. Most notably, PMA regulates MMP-2 mRNA expression through a mechanism involving message de-stabilization. Additionally, protein kinase C mediated events were found to play a role(s) in the regulation of MMP-2 message expression in NR3 cells. This study demonstrates several novel aspects regarding the regulation of MMP-2 expression in a H-ras transformed cell line and thereby provides further insight into the altered growth regulatory programs associated with H-ras mediated cellular transformation.