Membrane Type 1-matrix Metalloproteinase mRNA Research Articles

Local protein synthesis of neuronal MT1-MMP for agrin-induced presynaptic development.

Upon the stimulation of extracellular cues, a significant number of proteins are synthesized distally along the axon. Although local protein synthesis is crucial for various stages throughout neuronal development, its involvement in presynaptic differentiation at developing neuromuscular junctions remains unknown. By using axon severing and microfluidic chamber assays, we first showed that treatment of a protein synthesis inhibitor, cycloheximide, inhibits agrin-induced presynaptic differentiation in cultured Xenopus spinal neurons. Newly synthesized proteins are prominently detected, as revealed by the staining of click-reactive cell-permeable puromycin analog O-propargyl-puromycin, at agrin bead-neurite contacts involving the mTOR/4E-BP1 pathway. Next, live-cell time-lapse imaging demonstrated the local capturing and immobilization of ribonucleoprotein granules upon agrin bead stimulation. Given that our recent study reported the roles of membrane-type 1 matrix metalloproteinase (MT1-MMP) in agrin-induced presynaptic differentiation, here we further showed that MT1-MMP mRNA is spatially enriched and locally translated at sites induced by agrin beads. Taken together, this study reveals an essential role for axonal MT1-MMP translation, on top of the well-recognized long-range transport of MT1-MMP proteins synthesized from neuronal cell bodies, in mediating agrin-induced presynaptic differentiation.

Serum Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) mRNA Protected by Exosomes as a Potential Biomarker for Gastric Cancer.

BackgroundOur previous research revealed that membrane type 1-matrix metalloproteinase (MT1-MMP) is overexpressed and plays a crucial role in gastric cancer (GC) progression. Exosomes are important for GC carcinogenesis, and the exosomal contents are ideal biomarkers. However, the expression of exosomal MT1-MMP mRNA in serum and its potential significance in GC remains unclear.Material/MethodsThe expression of exosomal MT1-MMP mRNA in serum of patients with GC, chronic gastritis, or atypical hyperplasia, and healthy controls was detected using real-time quantitative RT-PCR. Serum CEA, CA19-9, and CA72-4 were also measured by electrochemiluminescence assay.ResultsExosomes were isolated and identified in serum, and serum exosomal MT1-MMP mRNA was found to be higher in patients with GC compared with healthy controls and patients with chronic gastritis or atypical hyperplasia (all P<0.05). Serum exosomal MT1-MMP mRNA was significantly correlated with tumor diameter, differentiation, Borrmann type, invasion depth, lymphatic metastasis, distal metastasis, and TNM stage. The AUC of exosomal MT1-MMP mRNA was 0.788 (95% CI: 0.714–0.850) with 63.9% sensitivity and 87.1% specificity, and was higher than that of CEA (0.655 (95% CI: 0.573–0.730)). The combination of 2 markers gave an AUC of 0.821 (95% CI: 0.750–0.878), which was better than with the individual marker. The sensitivity, specificity, and positive and negative predictive values were 75.6%, 83.9%, 94.7%, and 47.3%, respectively. Moreover, the multiple logistic regression model showed that tumor diameter, differentiation, invasion depth, and exosomal MT1-MMP mRNA were the risk factors for lymphatic metastasis in GC.ConclusionsOur results characterized serum exosomal MT1-MMP mRNA in GC, providing a foundation for discovering serum exosomes-targeted biomarkers for GC diagnosis.

Exosomal MT1-MMP mRNA in Serum As a Potential Biomarker for Gastric Cancer

Background: Our previous results reveal that membrane type 1-matrix metalloproteinase (MT1-MMP) is overexpressed and plays a crucial roles in gastric cancer (GC) progression. Exosomes are important for GC carcinogenesis, and the exosomal contents are used as ideal biomarkers. However, the expression of exosomal MT1-MMP mRNA in serum and its potential significance in GC remains unclear. Methods: The expression of exosomal MT1-MMP mRNA in serum of GC, chronic gastritis, atypical hyperplasia, and healthy controls were detected using real-time quantitative RT-PCR. Meanwhile, serum CEA, CA19-9 and CA72-4 were also measured by electrochemiluminescence assay. Findings: Exosomes were isolated and identified in serum. Our results revealed that exosomal MT1-MMP mRNA in serum was elevated in GC, compared with healthy controls, chronic gastritis, atypical hyperplasia, respectively (all P<0.05). It was significantly correlated with tumor diameter, differentiation, borrmann type, invasion depth, lymphatic metastasis, distal metastasis, TNM stage. AUC of exosomal MT1-MMP mRNA was 0.788 (95%CI: 0.714-0.850) with sensitivity 63.9% and specificity 87.1%, and higher than that of CEA (0.655 (95%CI: 0.573-0.730)). The combination of two markers gave an AUC of 0.821 (95%CI: 0.750-0.878), better than the individual marker. The sensitivity, specificity, positive and negative predictive value were 75.6%, 83.9%, 94.7%, 47.3%, respectively. Moreover, the multiple logistic regression model showed that tumor diameter, differentiation, invasion depth and exosomal MT1-MMP mRNA were the risk factors of lymphatic metastasis in GC. Interpretation: As a pilot investigation to characterizing the serum exosomal MT1-MMP mRNA in GC, our study provided a foundation for discovering serum exosomes-targeted biomarkers for GC diagnosis. Funding Statement: National Natural Science Foundation of China (81572070, 31500741). Natural Science Foundation of Shandong Province (BS2014SW022, ZR2016HM37). Key Research and Development Project of Shandong Province (2016GSF201124, 2015GSF118152). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Our study was approved by Medical Ethics Committee of Qilu Hospital of Shandong University (KYLL-2015-097). The participants signed written informed consents.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Effects of Prolactin and Testosterone on MT1‐MMP mRNA Expression and Enzymatic Activity of MMP‐2 and MMP‐9 in the Rat Prostate

Endocrine studies have shown prolactin (PRL) and testosterone (T) regulate prostate function. Both hormones are involved in the diseases of this gland as hyperplasia, dysplasia, and metastatic cancer. Since it has been reported metalloproteinases (MMPs) are underlying processes of metastasis and these are regulated by PRL and T. By histological studies we have detected PRL and T induce hyperplasia in many areas of the rat prostate within one month of treatment. In this work, we evaluated membrane type‐1 matrix metalloproteinase (MT1‐MMP) mRNA and enzymatic activity of MMP‐2 and MMP‐9 MMPs at the ventral (PV) and dorsolateral (PDL) prostate of the rat in response to treatments induced with PRL and T for one month. Our preliminary results show not differences of MT1‐MMP mRNA expression in all groups at PV and PDL. Although there is a trend of increased MT1‐MMP mRNA in groups T and PRL+T at PV, probably in response to an increase of androgen receptor. MMP‐2 and MMP‐9 were found. In all groups, inactive MMP‐2 was detected in all conditions and active MMP‐2 in the majority, although there were not differences in all groups. Inactive MMP‐9 was detected in almost all groups and active form was not detected, but there is a trend of increase of enzymatic activity in T group. In conclusion, we detected MT1‐MMP, MMP‐2 and MMP‐9 MMPs are involved in hyperplasia and T increases expression and enzymatic activity respectively of MMPs. Acknowledgements: CONACYT fellowship No. 290978 y Cuerpo Académico de Neuroquímica UV‐CA‐304.

Membrane Type-1 Matrix Metalloproteinase Expression in Acute Myeloid Leukemia and Its Upregulation by Tumor Necrosis Factor-α

Membrane type-1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion, as well as trafficking of normal hematopoietic cells, and acts as a physiologic activator of proMMP-2. In this study we examined MT1-MMP expression in primary acute myeloid leukemia (AML) cells. Because tumor necrosis factor (TNF)-α is known to be elevated in AML, we also investigated the effect of TNF-α on MT1-MMP expression. We found (i) MT1-MMP mRNA expression in 41 out of 43 primary AML samples tested; (ii) activation of proMMP-2 in co-cultures of AML cells with normal bone marrow stromal cells; and (iii) inhibition of proMMP-2 activation and trans-Matrigel migration of AML cells by gene silencing using MT1-MMP siRNA. Moreover, recombinant human TNF-α upregulated MT1-MMP expression in AML cells resulting in enhanced proMMP-2 activation and trans-Matrigel migration. Thus, AML cells express MT1-MMP and TNF-α enhances it leading to increased MMP-2 activation and most likely contributing to the invasive phenotype. We suggest that MT1-MMP, together with TNF-α, should be investigated as potential therapeutic targets in AML.

Effects of laminar shear stress on expression of MT1‐MMP in rat bone marrow‐derived mesenchymal stem cells

ObjectiveTo investigate the effect of laminar shear stress on the expression of membrane type‐1 matrix metalloproteinase (MT1‐MMP) in rat bone marrow‐derived mesenchymal stem cells (rMSCs).MethodsrMSCs were isolated from SD rat lower extremities and loaded by laminar shear stress with the parallel‐plate flow chamber system, and then the expression of MT1‐MMP mRNA of rMSCs was examined by real‐time PCR.ResultsShear stress with different magnitude of 5, 15, 30 dynes/cm2 all induced MT1‐MMP mRNA expression, especially high shear stress, 30 dynes/cm2, which was time‐dependent. The expression of MT1‐MMP mRNA was inhibited by SB202190, an inhibitor of p38, but enhanced by wortmannin, an inhibitor of PI3K/Akt.ConclusionShear stress can activate the expression of MT1‐MMP. The amount of MT1‐MMP expression is closely related to stimulating time and the strengths of shear stress, and p38MAPK and PI3K/Akt signal pathway may play a critical role during the process.

E0083 Membrane type 1 matrix metalloproteinase activation is enhanced by low shear stress through integrin pathway in ApoE-/- mice

ObjectiveLow shear stress and matrix metalloproteinase are involved in atherogenesis and plaque stability. The aim of this study wad to explore the relation and possible mechanism of membrane type-1 matrix...

Relation of MT1‐MMP gene expression to outcomes in colorectal cancer

Matrix metalloproteinases are members of a large family of endopeptidases that participate in the extracellular-matrix degradation that accompanies cancer cell invasion, metastasis and angiogenesis. The membrane-type 1 matrix metalloproteinase (MT1-MMP) gene has been reported in various cancers and is associated with tumor invasion and metastasis. This study examined the relation of the relative expression of MT1-MMP gene to clinicopathological factors and outcomes in patients with colorectal cancer (CRC). We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 202 patients with untreated CRC. The relative expression levels of MT1-MMP mRNA in cancer and in normal adjacent mucosa were measured by quantitative real-time reverse-transcriptase polymerase chain reaction. MT1-MMP gene expression was higher in cancer tissue than in adjacent normal mucosa. The level of MT1-MMP gene expression was not related to any clinicopathological factor. Overall survival at 5 years differed significantly between patients with high MT1-MMP gene expression and those with low expression. Overexpression of the MT1-MMP gene is considered a useful independent predictor of outcomes in patients with CRC.

Sympathetic Neurons Express and Secrete MMP-2 and MT1-MMP to Control Nerve Sprouting via Pro-NGF Conversion

Recently, we have shown that high frequency electrical field stimulation (HFES) of sympathetic neurons (SN) induces nerve sprouting by up-regulation of nerve growth factor (NGF) which targets the tyrosine kinase A receptor (TrkA) in an autocrine/paracrine manner. There is increasing evidence that matrix metalloproteinase-2 (MMP-2) is not only involved in extracellular matrix (ECM) turnover but may also exert beneficial effects during neuronal growth. Therefore, this study aimed to investigate the regulation and function of MMP-2 and its major activator membrane type 1-matrix metalloproteinase (MT1-MMP) as well its inhibitor TIMP-1 in SN under conditions of HFES. Moreover, we analyzed molecular mechanisms of the beneficial effect of losartan, an angiotensin II type I receptor (AT-1)blocker on HFES-induced nerve sprouting. Cell cultures of SN from the superior cervical ganglia (SCG) of neonatal rats were electrically stimulated for 48 h with a frequency of 5 or 50 Hz. HFES increased MMP-2 and MT1-MMP mRNA and protein expression, whereas TIMP-1 expression remained unchanged. Under conditions of HFES, we observed a shift from pro- to active-MMP-2 indicating an increase in MMP-2 enzyme activity. Specific pharmacological MMP-2 inhibition contributed to an increase in pro-NGF amount in the cell culture supernatant and significantly reduced HFES-induced neurite outgrowth. Losartan abolished HFES-induced nerve sprouting in a significant manner by preventing HFES-induced NGF, MMP-2, and MT1-MMP up-regulation. In summary, specific MMP-2 blockade prevents sympathetic nerve sprouting (SNS) by inhibition of pro-NGF conversion while losartan abolishes HFES-induced SNS by reducing total NGF, MMP-2 and MT1-MMP expression.

Alterations in mRNA and protein levels of metalloproteinases-2, -9, and -14 and tissue inhibitor of metalloproteinase-2 responses to traumatic skeletal muscle injury

This study characterizes the temporal relationship of membrane type-1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in skeletal muscle following injury. Tibialis anterior (TA) muscles from 60 mice were exposed and injured by applying a cold steel probe (-79 degrees C) to the muscle for 10 s. Thereafter, TA muscles from uninjured and injured legs were collected at 3, 10, 24, 48, and 72 h postinjury for analysis of local MT1-MMP, TIMP-2, and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) mRNA and protein content via quantitative RT-PCR, immunoblotting, zymography, and immunofluorescence. All data are expressed as fold change of injured leg vs. uninjured leg. MT1-MMP mRNA levels were decreased significantly at 48 and 72 h postinjury by approximately 9- and 21-fold, respectively (P < 0.01). Both TIMP-2 and MMP-2 mRNA expression significantly decreased in the injured leg by approximately 4- to 10-fold at 10-72 h postinjury (P < 0.01). MMP-9 mRNA expression was significantly increased at 10, 24, and 48 h postinjury by 6- (P < 0.05), 25-, and 12-fold (P < 0.01), respectively. Protein content of latent (63 kDa) MT1-MMP was decreased at 48 and 72 h postinjury by approximately 2-fold (P < 0.01). Content of the soluble (50 kDa) fragment of MT1-MMP was significantly increased by approximately 17-, 25-, and 67-fold at 24 (P < 0.05), 48, and 72 h (P < 0.01) postinjury, respectively. TIMP-2 protein levels diminished from 3 to 48 h postinjury by 1.5-fold to 1.8-fold (P < 0.01), before returning to baseline levels at 72 h postinjury. Zymography revealed visual increases in gelatinase activity in molecular weight regions corresponding to MMP-9 and MMP-2. In conclusion, skeletal muscle injury initiates a sequence of events in the MT1-MMP proteolytic cascade resulting in elevated levels of the soluble (50 kDa) fragment of MT1-MMP, which could enhance pericellular extracellular matrix remodeling.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

The E-cadherin-repressed hNanos1 gene induces tumor cell invasion by upregulating MT1-MMP expression

In this study, we examined the role of the E-cadherin-repressed gene human Nanos1 (hNanos1) in tumor invasion process. First, our in vivo study revealed that hNanos1 mRNAs were overexpressed in invasive lung carcinomas. Moreover, hNanos1 was co-localized with MT1-MMP (membrane type 1-matrix metalloproteinase) in E-cadherin-negative invasive lung tumor clusters. Using an inducible Tet-on system, we showed that induction of hNanos1 expression in DLD1 cells increased their migratory and invasive abilities in a three-dimensional migration and in a modified Boyden chamber assay. Accordingly, we demonstrated that hNanos1 upregulated MT1-MMP expression at the mRNA and protein levels. Inversely, using an RNA interference strategy to inhibit hNanos1 expression in invasive Hs578T, BT549 and BZR cancer cells, we observed a downregulation of MT1-MMP mRNA and protein and concomitantly a decrease of the invasive capacities of tumor cells in a modified Boyden chamber assay. Taken together, our results demonstrate that hNanos1, by regulating MT1-MMP expression, plays an important role in the acquisition of invasive properties by epithelial tumor cells.

Inhibitory effect of antisense RNA on the gene expression of membrane-type 1 matrix metalloproteinase and invasiveness of human gastric carcinoma cells

AIM:To investigate the influence on the gene expression of membrane-type 1 matrix metalloproteinase(MT1-MMP)and invasiveness of human gastric carcinoma cell line BGC823 by antisense RNA. METHODS:The eukaryotic expression vector carrying MT1-MMP antisense RNA was con- structed with recombinant technology and then transfected into human gastric cancer cell line BGC823.The changes of MT1-MMP mRNA ex- pression,cell proliferation,activation of gelatin- ase A and cell invasion ability were examined by reverse transcription-polymerase chain reaction (RT-PCR),MTT assay,zymography and Tran- swell invasion assay,respectively. RESULTS:The eukaryotic expression vector (named pasMMP14)containing MT1-MMP an- tisense RNA was successfully constructed and transfected into BGC823 cells.The expression of MT1-MMP mRNA was down-regulated with an inhibitory rate of 36%,in comparison with that in negative control group.At the 48th hour after transfection with pasMMP14,the activity of gelatinase A was dramatically inhibited.Af- ter 72 hours,cell proliferation was significantly decreased as compared with that in pcDNA3.0 group and normal control group(t=2.358,P0.01;t=2.727,P0.01).Transwell invasion assay showed that the invasive property was greatly suppressed in pasMMP14-transfected group as compared with that in pcDNA3.0 group and normal control group(t=5.744,P0.01;t=5.695,P0.01). CONCLUSION:Antisense RNA can evidently inhibit the gene expression of MT1-MMP and invasiveness of gastric cancer cells,suggesting that MT1-MMP gene may be a molecular target of anti-invasion therapy for gastric cancer.

Expression of MT1-MMP during deciduous tooth resorption in odontoclasts

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

Altered expression of Fas/Fas ligand/caspase 8 and membrane type 1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats

One of the characteristics of polycystic ovary syndrome (PCOS) is the presence of cystic follicles in various stages of growth and atresia, the latter of which is known to be the result of apoptosis and tissue remodeling. To further investigate the process of follicular atresia, we compared ovarian expression and localization of Fas, Fas ligand (FasL), casapse-8 and membrane-type1 matrix metalloproteinase (MT1-MMP) in rats treated with dehydroepiandrosterone (DHEA) as a model of PCOS, and in control rats. We found that the numbers of TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive follicles were significantly higher in ovaries from PCOS rats than in those from control rats (P < 0.05), as were ovarian levels of FasL mRNA and protein, processed caspase-8 protein and MT1-MMP mRNA. Correspondingly, we also observed an increase in the level of MTI-MMP catalytic activity and a decrease in the level of pro-caspase-8 protein. In addition, immunohistochemical analyses showed that MT1-MMP and FasL co-localize with TUNEL-positive apoptotic granulosa cells within atretic follicles of PCOS ovaries. Our results suggest that under the PCOS-like conditions induced by DHEA, the Fas/FasL/Caspase-8 (death receptor dependent) pathway is pivotal for follicular atresia, and that increased levels of MT1-MMP likely play an important role in tissue remodeling during structural luteolysis.

JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP.

Expression of membrane type-1 matrix metalloproteinase, MT1-MMP in human breast cancer and its impact on invasiveness of breast cancer cells

MT1-MMP (membrane type-1 matrix metalloproteinase), otherwise known as MMP14 is a proteolytic enzyme known to be involved in degradating extracellular matrix and assist progression of cancer invasion and progression. We investigated the impact of targeting the expression of MT1-MMP in breast cancer and its clinical relevance. Human breast cancer cell line MDA-MB-231 was used. Expression of MT1-MMP in the breast cancer cell line was manipulated by way of retroviral ribozyme transgene. The in vitro invasion, growth and cell migration were determined on cell lines transfected with either the transgene or control plasmid. Protein and message levels of MMP14 was also assessed using immunohistochemistry and real-time quantitative analysis, and correlated with clinical and pathological information of the patients. Retroviral ribozyme transgene to human MT1-MMP successfully knocked down the levels of MT1-MMP mRNA from MDA-MB-231 cells. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus, HGF, compared with control and wild-type cells. The invasion index for MT1-MMP knockdown cells were 13+/-3.1 (without HGF) and 16.4+/-2.3 (with HGF, p=0.14), and the index for transfection control cells 25.3+/-4.3 (without HGF) and 40.4+/-4.1 (with HGF, p=0.0049). Transfection with the transgenes did not change the rate of cell growth. In clinical breast cancer, MT1-MMP staining was both membranous and cytoplasmic. Tumour cells displayed stronger staining compared with normal mammary epithelial cells. Tumour tissues had a marginally higher levels of the MMP14 transcript (8.6+/-1.9), compared with normal tissues (4.7+/-1.4), p=0.13. No significant difference was observed between node positive and node negative tumours (9.0+/-2.2 vs 8.7+/-3.1, p=0.24). Marginally higher levels of the MMP14 transcript were seen in tumours which developed metastasis and local recurrence. However, tumours from patients who died of breast cancer related causes had significantly higher levels of the transcript, compared with tumours from patients who remained disease-free 10 years after initial surgery (12.2+/-2.5 vs 6.3+/-1.2, p=0.0091). MT1-MMP is a proteolytic enzyme that is pivotal in controlling the invasiveness of breast cancer cells. It is highly expressed in aggressive breast tumours and is associated with clinical outcome. The enzyme is a potential therapeutic target in breast cancer.

Phenylethyl isothiocyanate and its N-acetylcysteine conjugate suppress the metastasis of SK-Hep1 human hepatoma cells

Phenylethyl isothiocyanate (PEITC), a hydrolysis compound of gluconasturtiin, is metabolized to N-acetylcysteine (NAC)-PEITC in the body after the consumption of cruciferous vegetables. We observed an inhibitory effect of PEITC and its metabolite NAC-PEITC on cancer cell proliferation, adhesion, invasion, migration and metastasis in SK-Hep1 human hepatoma cells. PEITC and NAC-PEITC suppressed SK-Hep1 cell proliferation in a dose-dependent manner, and exposure to 10 μM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. NAC-PEITC inhibited cancer cell adhesion, invasion and migration to a similar or to an even larger degree than PEITC. The expression of matrix metalloproteinase (MMP) 2, MMP-9 and membrane type 1 matrix metalloproteinase (MT1-MMP) is a known risk factor for metastatic disease. Gelatin zymography analysis revealed a significant downregulation of MMP-2/MMP-9 protein expression in SK-Hep1 cells treated with 0.1–5 μM PEITC or NAC-PEITC. PEITC and NAC-PEITC treatment caused dose-dependent decreases in MMP-2/MMP-9 and MT1-MMP mRNA levels, as determined by reverse transcription polymerase chain reaction. PEITC and NAC-PEITC also increased the mRNA levels of tissue inhibitors of matrix metalloproteinase (TIMPs) 1 and 2. Our data suggest that this inhibition is mediated by downregulation of MMP and upregulation of TIMPs.

Differential transcriptional activation of matrix metalloproteinase-2 and membrane type-1 matrix metalloproteinase by experimental deep venous thrombosis and thrombin

Resolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT. DVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (beta-galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for beta-galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined. MMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. beta-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 +/- 8.6 vs 69.445 +/- 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 +/- 7.3 vs 49.2 +/- 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 +/- 0.023 vs 0.63 +/- 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 +/- 0.009 vs 0.059 +/- 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05). Resolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process. Postthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome.