Tissue Inhibitor Of Metalloproteinase-1 Protein Research Articles

Effect of transforming growth factor β1 on the expression of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and nuclear factor kappa B signalling pathway in the human amniotic cells WISH

To investigate the effect of transforming growth factor β1 (TGF-β1) on the expression of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), nuclear factor kappa B (NF-κB) and the possible signalling pathways in human amniotic cells WISH. The WISH cell line was cultured. WISH cells were added with TGF-β1 of different concentrations (0, 2, 10 and 20 ng/ml, respectively) for 24 hours. Then, reverse transcription (RT) PCR and western blotting were used to analyze the protein and mRNA expression of TIMP-1 and MMP-9; and the expression of NF-κB was analyzed by western blot. (1) The profile of TIMP-1 mRNA (0.413 ± 0.036, 0.623 ± 0.058, 1.392 ± 0.124, 1.387 ± 0.102) in WISH cells elevated when the concentration of TGF-β1 increased (0, 2, 10, 20 ng/ml). In accordance with TIMP-1 mRNA, the expression of TIMP-1 also elevated with the increase of TGF-β1 (0.357 ± 0.031, 0.596 ± 0.048, 1.243 ± 0.097 and 1.359 ± 0.121, respectively). And when 2, 10 or 20 ng/ml of TGF-β1 was added, the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-β1 was added (P < 0.05). (2) In contrast with TIMP-1, MMP-9 mRNA (1.325 ± 0.056, 0.987 ± 0.081, 0.610 ± 0.034, 0.347 ± 0.023) in WISH cells decreased when the concentration of TGF-β1 increased (0, 2, 10, 20 ng/ml). The MMP-9 protein (1.119 ± 0.064, 1.008 ± 0.052, 0.578 ± 0.041, 0.401 ± 0.015) also decreased with the increase of TGF-β1. And when 2, 10 or 20 ng/ml of TGF-β1 was added, the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-β1 was added (P < 0.05). (3) The NF-κB protein (1.423 ± 0.065, 1.116 ± 0.045, 0.796 ± 0.041, 0.359 ± 0.021) was significantly reduced with the increase of TGF-β1 (0, 2, 10, 20 ng/ml;P < 0.05). The mRNA and protein expression of TIMP-1 decreased when TGF-β1 was low in WISH cells, whereas those of MMP-9 elevated when TGF-β1 was low. The unbalance of TIMP-1 and MMP-9 was related to the pathology of the premature rupture of membrane. And the NF-κB singalling pathway might be an important mechanism in the regulation of TIMP-1 and MMP-9 system.

Tissue inhibitor of metalloproteinases-1 protects human neurons from staurosporine and HIV-1-induced apoptosis: mechanisms and relevance to HIV-1-associated dementia.

HIV-1-associated dementia (HAD)-relevant proinflammatory cytokines robustly induce astrocyte tissue inhibitor of metalloproteinases-1 (TIMP-1). As TIMP-1 displays pleotropic functions, we hypothesized that TIMP-1 expression may serve as a neuroprotective response of astrocytes. Previously, we reported that chronically activated astrocytes fail to maintain elevated TIMP-1 expression, and TIMP-1 levels are lower in the brain of HAD patients; a phenomenon that may contribute to central nervous system pathogenesis. Further, the role of TIMP-1 as a neurotrophic factor is incompletely understood. In this study, we report that staurosporine (STS) and HIV-1ADA virus, both led to induction of apoptosis in cultured primary human neurons. Interestingly, cotreatment with TIMP-1 protects neurons from apoptosis and reverses neuronal morphological changes induced by these toxins. Further, the anti-apoptotic effect was not observed with TIMP-2 or -3, but was retained in a mutant of the N-terminal TIMP-1 protein with threonine-2 mutated to glycine (T2G) that is deficient in matrix metalloproteinase (MMP)-1, -2 and -3 inhibitory activity. Therefore, the mechanism is specific to TIMP-1 and partially independent of MMP-inhibition. Additionally, TIMP-1 modulates the Bcl-2 family of proteins and inhibits opening of mitochondrial permeability transition pores induced by HIV-1 or STS. Together, these findings describe a novel function, mechanism and direct role of TIMP-1 in neuroprotection, suggesting its therapeutic potential in HAD and possibly in other neurodegenerative diseases.

High expression of TIMP-1 in human breast cancer tissues is a predictive of resistance to paclitaxel-based chemotherapy

For breast cancer patients with lymph node metastasis, paclitaxel is the first-line chemotherapy drug. Clinical studies showed that some patients with breast cancer were insensitive to paclitaxel, which led to chemotherapy failure. Today, no validated markers exist for the prediction of chemotherapy sensitivity in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis. Epidemiological studies have also associated elevated tumor tissue TIMP-1 levels with a poor response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy. Additionally, our previous study proved that TIMP-1 significantly decreased the sensitivity of breast cancer cells to paclitaxel-induced apoptosis by enhancing degradation of cyclin B1. These data imply that TIMP-1 may be a useful predictive biomarker for chemotherapy resistance. In this retrospective study, we investigated the association between expression levels of TIMP-1 protein in the primary tumor and objective response to paclitaxel-based chemotherapy in 99 patients with breast cancer. With Kaplan-Meier survival analysis, the patients with high TIMP-1 levels were found to have significantly worse 5-year DFS (71.1 %) than the patients with low levels (88.5 %; P = 0.020). Similarly, the patients with high TIMP-1 levels had significantly worse 5-year OS (78.9 %) than patients with low levels (96.7 %; P = 0.004). In Cox's univariate and multivariate analyses, TIMP-1 was prognostic for both DFS and OS. Our data showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to paclitaxel-based chemotherapy, and TIMP-1 might be a potential biomarker for predicting response of breast cancer patients to paclitaxel-based chemotherapy.

Abstract 498: Anti-invasive potential of alginate microcapsule encapsulating TIMP 2-engineered cells against malignant glioma cells

Abstract Objectives. 293T was genetically modified to secrete tissue inhibitor of metalloproteinase-2 (TIMP-2) (abbreviated as 293TIMP2 cells) and 293TIMP2 cells were encapsulated into alginate microcapsules to produce TIMP-2 protien continuously. Since TIMP-2 is known to inhibit matrix metalloproteinase-2 (MMP-2) expression of tumor cells, it could be inhibit invasion of tumor cells through extracellular matrix. The anti-invasive potential of the microcapsules was studied in vitro against U87MG glioma cells. Experimental. The TIMP-2 gene was transfected to 293T cells and the genetically engineered 293TIMP2 cells were encapsulted into alginate microcapsules. The released TIMP-2 protein was examined with Western blot analysis and the anti-invasive potential against U86MG glioma cells was tested with gelatin zymography for matrix metalloproteinase-2 (MMP-2) and a Matrigel assay for invasion. Results. Cell viability within the alginate microcapsules was properly maintained at a cell density of 5×106 cell density. Since polycationic polymers are helpful to maintain mechanical strength of microcapsules with good cell viability, alginate microcapsules were reinforced with chitosan (0.1 % wt/vol). Expression of TIMP-2 protein in cell lysate and the secreted TIMP-2 into conditioned media were confirmed with Western blot analysis. Alginate microcapsules encapsulating 293TIMP2 cells properly released TIMP-2 protein into the medium and the released TIMP-2 protein was effective in inhibiting MMP-2 expression and invasion of U87MG glioma cells. Conclusion. These results indicated that alginate microcapsules encapsulating 293TIMP2 cells are superior candidates for anti-invasive therapy of glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 498. doi:1538-7445.AM2012-498

MicroRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3

Despite improvements in treatment over the past few decades, endometrial cancer remains one of the most common causes of mortality in women and there is an urgent need for the development of targeted therapies. The aim of this study was to confirm the target gene of miR-103 in human endometrial cancer and investigate the biological functions in which miR-103 is involved through the regulation of the expression of its target gene. This study may provide useful data to gain a better understanding of the effect of miR-103 in tumor formation. miR-103 expression levels were measured using real-time quantitative PCR. The effect of miR-103 on tissue inhibitor of metalloproteinase 3 (TIMP-3) expression was assessed in endometrial cancer cell lines with a miR-103 inhibitor to decrease the level of miR-103 expression. Furthermore, the roles of miR-103 in cell growth and invasion were analyzed using miR-103 inhibitor-transfected cells. The level of expression of miR-103 decreased following transfection with the miR-103 inhibitor. miR-103 inhibitor transfection increased the activity of the luciferase reporter assay containing the TIMP-3 3'-untranslated region (UTR) construct and increased the levels of the TIMP-3 protein but not its mRNA in endometrial cancer cell lines. Finally, miR-103 inhibitor-transfected cells exhibited reduced cell growth and invasive characteristics. Our data suggested that miR-103 post-transcriptionally downregulates the expression of the tumor suppressor TIMP-3 and stimulates growth and invasion in endometrial cancer cell lines. This provides a possible therapeutic target that may upregulate TIMP-3 in endometrial cancer.

Transcriptional Basis for Hepatic Fibrosis in Cystic Fibrosis–associated Liver Disease

Liver disease contributes to significant morbidity and mortality in cystic fibrosis (CF). Although all patients with CF express the defective CF transmembrane conductance regulator in cholangiocytes, many develop asymptomatic fibrosing liver disease. Only some develop cirrhosis, with pathogenesis remaining enigmatic. Available noninvasive diagnostic tools do not identify patients at risk before development of advanced fibrosis. We conducted a pilot study to identify genes associated with hepatic injury and fibrosis on liver biopsy that may help elucidate determinants of CF-associated liver disease (CFLD). Liver tissue from children with CFLD with various stages of hepatic fibrosis was compared with pediatric controls using cDNA array analysis. Differential expression of genes of interest was then assessed relative to pediatric control liver and non-CF cholestatic disease control liver from patients with biliary atresia, using both real-time reverse transcription-polymerase chain reaction and immunohistochemistry. cDNA array demonstrated differential expression of numerous genes associated with hepatic fibrogenesis including collagens, matrix metalloproteinases, and chemokines in CFLD versus normal controls, particularly decreased expression in tissue remodeling genes including plasminogen activator inhibitor-1 (PAI-1, up to 25-fold) and tissue inhibitor of metalloproteinase-1 (TIMP-1); this was validated by real-time reverse transcription-polymerase chain reaction (PAI-1, P = 0.004; TIMP-1, P = 0.019). No significant decrease in PAI-1 or TIMP-1 mRNA was observed in biliary atresia versus normal control. Immunohistochemistry confirmed the decreased expression of hepatic PAI-1 and TIMP-1 protein in CFLD versus both normal and biliary atresia disease controls. The coordinated differential expression of these genes associated with liver fibrosis provides evidence for a transcriptional basis for the pathogenesis of CFLD and provides avenues for further study. Clarifying the pathogenesis of CFLD will facilitate techniques for early, precirrhotic detection and targeted interventions.

Prognostic Significance of Tissue Inhibitor of Metalloproteinase-1 in Breast Cancer

Introduction. Despite advances in breast cancer systemic treatment, new prognostic and predictive factors are still needed. Tissue inhibitor of metalloproteinase-1 (TIMP-1), a physiologic inhibitor of matrix metalloproteinases (MMPs), can act in both pro- and antitumoral effects. As role of TIMP-1 in breast cancer is controversial, we aimed to determine the prognostic significance of TIMP-1 in breast cancer. Methods. A single center-based case-control study was applied. Primary breast cancers from women with early stage disease treated with standard adjuvant therapy were analyzed by gene expression microarrays and immunohistochemistry for TIMP-1. Results. At the optimized cut-point, patients with high TIMP-1 RNA levels had a significantly shorter time to relapse, with a hazard ratio (HR) of 1.64 (P = 0.04), but without significant differences in overall survival (HR 1.29, P = 0.37). Although cytoplasmic overexpression of TIMP-1 protein was not correlated with early relapse (HR 1.0, P = 0.92), there was a tendency for short overall survival in patients with high expression (HR 1.41, P = 0.21). Conclusions. Our data indicate that elevated TIMP-1 RNA levels are independently prognostic for early recurrence, and there is a tendency for association of high cytoplasmic TIMP-1 protein levels with short survival in primary breast cancer.

P4-01-17: TIMP-1 Over-Expression Confers Resistance of MCF-7 Breast Cancer Cells to Fulvestrant.

Abstract Background Endocrine resistance represents a major challenge in the management of estrogen receptor (ER) positive breast cancer. Currently no predictive biomarkers for endocrine resistance in ERpositive breast cancer patients are in clinical use. In a clinical study, patients with metastatic breast cancer and high levels of serum Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) had less benefit from endocrine therapy than patients with a lower level of serum TIMP-1 [1]. Therefore, we evaluated the association between TIMP-1 and response to endocrine therapy using an in vitro approach. We have previously presented initial results on TIMP-1 and response to endocrine therapy [2]. Materials and Methods: MCF-7 cells were stably transfected with pcDNA3.1(Hyg)-TIMP-1 plasmid, and a panel of 11 subclones with different expression levels of TIMP-1 was generated. TIMP-1 expression levels were confirmed using enzyme-linked immunosorbent assay (ELISA). Four subclones with high or low TIMP-1 expression were analyzed for the growth response to estrogen, 4-hydroxytamoxifen and fulvestrant. These four subclones were analyzed for protein expression by western blotting. All subclones were analyzed by whole human genome oligo microarrays 4×44K for determination of gene expression levels. Data were analyzed using the limma R/Bioconductor package. Paired-end Solexa sequencing was applied to selected subclones with high and low TIMP-1 levels to identify transcriptomic changes. Results: High expression of TIMP-1 was associated with resistance to fulvestrant, whereas growth response to either estrogen or 4-hydroxytamoxifen was independent of TIMP-1 expression levels. High expression of TIMP-1 protein and mRNA was associated with undetectable levels of progesterone receptor (PgR) protein and mRNA whereas ER protein and mRNA levels were unaffected by TIMP-1. To characterize the potential role of TIMP-1 in estrogen signaling we analyzed the expression of reported estrogen-responsive genes and no general change was observed. We identified genes that correlated positively or negatively to TIMP-1 expression. Among the identified genes was PgR, which is a direct target for ER. Conclusion: Our data suggest that a high expression of TIMP-1 in vitro is associated with resistance to fulvestrant but not to 4-hydroxytamoxifen. Estrogen-regulated genes are not generally affected by changes in TIMP-1 expression levels and therefore TIMP-1 appears to affect endocrine resistance through other mechanisms than globally regulating ER signaling. However, high expression of TIMP-1 is associated with loss of PgR and this may be related to the resistance towards fulvestrant.

P4-09-26: Prognostic Significance of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) in Breast Cancer.

Abstract Background: Despite advances of systemic treatment in breast cancer guided by hormonal status and HER2 amplification, new prognostic and predictive factors are still warranted to optimize treatments among these patients. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), a physiologic inhibitor of Matrix Metalloproteinases (MMPs), can act in both pro- and anti-tumoral effects. The prognostic significance of TIMP-1 in breast cancer is still controversial. This study aims to determine the prognostic significance of TIMP-1 in breast cancer. Material and Methods: One-hundred and seventy-six primary breast cancers from women with early stage disease treated with standard adjuvant therapy were analyzed by gene expression microarrays and immunohistochemistry for TIMP-1. Immunohistochemical analysis was independently reviewed by two pathologists. Results: At the optimized cut-off point, patients with high TIMP-1 RNA levels had a significantly shorter time to relapse, with a hazard ratio (HR) of 1.6 (p = 0.039), but without significant differences in overall survival (HR 1.29, p = 0.37). Although cytoplasmic overexpression of TIMP-1 protein was not correlated with early relapse (HR 1.2, p = 0.35), high expression was associated with shorter overall survival (HR 1.73, p = 0.027). In multivariate analysis, when considering stage, histologic grade, hormonal and HER2 status, TIMP-1 RNA levels remained independently prognostic for early relapse (HR 1.68, p = 0.04). Discussion: Elevated TIMP-1 RNA levels are independently prognostic for early recurrence, whereas protein overexpression of TIMP-1 is correlated with short overall survival in primary breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-26.

Up-regulation of peroxisome proliferator-activated receptor gamma in radiation-induced heart injury in rats

To explore the expression level and the role of peroxisome proliferator-activated receptor gamma (PPAR-γ) in radiation-induced heart injury in a rat model, thirty-two Sprague-Dawley rats were divided into three groups (the control group, the 15-Gy irradiation group and the 18-Gy irradiation group). Experimental animals were exposed to radiation generated by a linear accelerator at the chest and killed after 3months. Heart tissues from these animals were removed for Masson staining, PPAR-γ immunohistochemical staining, Western blot analysis and real-time polymerase chain reaction assay (RT-PCR). In addition, the protein expression of matrix metalloprotein-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor type beta1 (TGF-β1), all of which are associated with fibrosis, was measured. Masson staining revealed significant myocardial fibrosis, degeneration and necrosis in rats exposed to radiation. The results of immunohistochemical staining and Western blot analysis showed that PPAR-γ protein expression in hearts of the irradiation groups was significantly higher than in the control group, especially in myocardium and vascular endothelial (p<0.05). RT-PCR results also showed a parallel increase in PPAR-γ mRNA expression in the heart of the irradiation groups compared with the control group (p<0.05). The expression of MMP-1 protein was not significantly different in three groups (p>0.05). The expression of TIMP-1 and TGF-β1 proteins was, however, higher in two irradiation groups than in the control group (p<0.05). These data demonstrate that PPAR-γ expression is up-regulated on both mRNA and protein levels in heart injured by radiation. PPAR-γ may play an important role in radiation-induced heart injury.

Salivary tissue inhibitor of metalloproteinases-1 localization and glycosylation profile analysis

Salivary tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with pathological conditions in the oral cavity, but the origin of TIMP-1 in saliva remains unknown. Hence, we studied the localization of TIMP-1 in salivary gland tissue and also investigated if TIMP-1 found in blood and saliva is identical. Human salivary gland tissue samples (four parotid gland and four submandibular gland biopsies) were analysed for the presence of TIMP-1 mRNA and protein expression. To assess TIMP-1 glycosylation profiles in blood and saliva, the protein was isolated from plasma and unstimulated and stimulated whole saliva as well as stimulated parotid and submandibular saliva and analysed by MALDI-TOF mass spectrometry. TIMP-1 protein was demonstrated in mucous acinar cells of the submandibular gland and in ductal cells of both the parotid and submandibular gland. However, no TIMP-1 mRNA was detected in any of these cells. The glycosylation profiles of TIMP-1 isolated from whole saliva and saliva from the major glands were highly similar. In contrast, a significant difference was found between the glycoprofiles of salivary TIMP-1 and plasma TIMP-1. Although no clear evidence of TIMP-1 transcription in major salivary glands was demonstrated our results suggest that TIMP-1 in saliva does not originate from plasma.

Effects of butyrate on matrix metalloproteinase-3 expression in intestinal epithelial cell line No.6

Objective To investigate the effects of butyrate on the expression of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in intestinal epithelial cell line No. 6 (IEC-6) cells. Methods IEC-6 cells of logarithmic growth phase were divided into three groups according to different concentrations of sodium butyrate being used. Low-concentration group, the final concentration of sodium butyrate in medium was 2 mmol/L; high-concentration group, the final concentration of sodium butyrate in medium was 14 mmol/L and control group with no sodium butyrate in medium. After cultured for 24 hours, expression of MMP-3 and TIMP-1 mRNA in IEC-6 cells was measured by reverse transcription-polymerase chain reaction. After cultured for 48 hours, Western blot was employed to examine the expression of MMP-3 and TIMP-1 protein in IEC-6 cells. The differences between groups were compared by ANOVA. Results Expression level of MMP-3 mRNA in group treated with butyrate in concentration of 14 mmol/L was 0. 566±0. 140,inactive form protein of MMP-3 in the group was 0. 756±0. 128, and expression level of active form protein was 0. 255±0. 057, which were higher than those of control group (0. 166±0. 041, 0. 312± 0. 057, 0. 042 ± 0. 038) (P＜0.01, respectively). While, there were no significant differences of those indexes between the control group and low-concentration group. In the high and low-concentration and control group, the levels of TIMP-1 mRNA were 0. 427±0. 042, 0. 383±0. 043 and 0. 355±0. 048,the expression of TIMP-1 protein were 0. 576±0. 140, 0. 524±0. 123 and 0. 355±0. 062, there were no singnificant difference among the three groups (F= 1.97 and 3.10, P＞ 0.05). Conclusions High concentration sodium butyrate could induce the expression of MMP-3 in IEC-6 cells, while not at low concentration. Key words: Enterocolitis, necrotizing; Cell line; Matrix metalloproteinase 3; Butyrates

An integrin-binding N-terminal peptide region of TIMP-2 retains potent angio-inhibitory and anti-tumorigenic activity in vivo

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin α3β1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to α3β1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the α3β1-binding domain and the growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43–Ala66, which shows α3β1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to α3β1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that possesses both angio-inhibitory and, using a murine xenograft model of Kaposi's sarcoma, anti-tumorigenic activity in vivo. Thus, both the α3β1-binding and the angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B–C loop).

Serum TIMP-1 Predicts Survival Outcomes of Invasive Breast Carcinoma Patients: A Meta-analysis

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a small secretory glycoprotein with multifunctional activity including anti-apoptosis and the inhibition of matrix metalloproteinase in invasive breast carcinomas. There have been contradictory results as to whether TIMP-1 is a poor or good prognostic factor in breast cancer patients. To address this controversy, we conducted a meta-analysis for the relationship between TIMP-1 levels and prognostic parameters in the breast cancer. The relevant published studies were pooled according to the defined selection criteria. The effect sizes of overall survival and prognostic parameters were calculated by a hazard ratio (HR) or an odds ratio (OR). HRs or ORs were combined using a random-effects model. Survival outcomes between high or elevated and low or normal serum TIMP-1 levels were compared by uni- and multivariate analyses involving 886 and 844 breast cancer patients, respectively. Patients with high or elevated serum TIMP-1 levels had unfavorable survival outcomes compared to patients with low or normal serum TIMP-1 levels in the uni- and multivariate analyses (HR, 1.7 and 2.4; p <0.001 and p= 0.033, respectively). However, no survival difference was evident in the data from tissue TIMP-1 levels by enzyme-linked immunosorbent assay and the expression of tissue TIMP-1 mRNA. The high or positive immunohistochemical expression of tissue TIMP-1 protein was not related to adjusted and unadjusted HRs, lymph node metastasis, and clinical stages. This meta-analysis indicates that serum TIMP-1 levels may be useful for predicting survival outcomes of invasive breast cancer patients.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) deficiency exacerbates carbon tetrachloride-induced liver injury and fibrosis in mice: involvement of hepatocyte STAT3 in TIMP-1 production

BackgroundTissue inhibitor of metalloproteinase 1 (TIMP-1), which is thought to be produced mainly by activated hepatic stellate cells and Kupffer cells in the liver, plays a pivotal role in matrix remodeling during liver injury and repair; while the effect of TIMP-1 on hepatocellular damage remains obscure.ResultsHepatic expression of TIMP-1 mRNA and protein was up-regulated both in acute and chronic liver injury induced by carbon tetrachloride (CCl4). Compared with wild-type mice, TIMP-1 knockout mice were more susceptible to CCl4-induced acute and chronic liver injury, as shown by higher levels of serum alanine aminotransferase (ALT), greater number of apoptotic hepatocytes, and more extended necroinflammatory foci. TIMP-1 knockout mice also displayed greater degree of liver fibrosis after chronic CCl4 injection when compared with wild-type mice. In vitro treatment with TIMP-1 inhibited cycloheximide-induced cell death of primary mouse hepatocytes. Finally, up-regulation of TIMP-1 in the liver and serum after chronic CCl4 treatment was markedly diminished in hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. In vitro treatment with interleukin-6 stimulated TIMP-1 production in primary mouse hepatocytes, but to a lesser extent in STAT3-deficient hepatocytes.ConclusionsTIMP-1 plays an important role in protecting against acute and chronic liver injury and subsequently inhibiting liver fibrosis induced by CCl4. In addition to activated stellate cells and Kupffer cells, hepatocytes are also responsible for TIMP-1 production during liver injury via a STAT3-dependent manner.

TIMP3 regulates migration, invasion and in vivo tumorigenicity of thyroid tumor cells

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.

Anti-sense miRNA-21 oligonucleotide inhibits Tb 3.1 human tongue squamous cell carcinoma growth in vitro

To investigate the effect of micro RNA-21 (miRNA-21) knocking on the Tb3.1 human tongue squamous cell carcinoma growth. Anti-sense miRNA-21 oligonucleotide was delivered with oligofectamine to suppress Tb 3.1 tongue cancer cell growth in vitro. Real-time polymerase chain reaction (PCR) was conducted to detect the miRNA-21 expression after transfection. Methyl thiazolyl tetrazolium (MTT) assay was used to determine Tb 3.1 cell survival rate. Apoptosis were examined by flow-cytometry. Matrigel matrix and transwell assay were used to determine Tb 3.1 cell colony formation and migration ability. Antigen KI-67 (Ki67), B cell lymphoma (Bcl-2), phosphatase and tensin homolog (PTEN), matrirx metalloproteinase 2 (MMP-2, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) protein expression in Tb 3.1 cell were measured by Western blotting. miRNA-21 expression was decreased in miRNA-21 antisense oligonucleotide (ASODN) group. The survival rate of Tb 3.1 cells with AS-miRNA-21 transfection was significantly suppressed (F = 27.02, P = 0.00) and early phase apoptosis (F = 26.641, P = 0.001) induced in Tb 3.1 cell. Ki67, Bcl-2, MMP-2 and MMP-9 protein were down regulated while PTEN and TIMP-1 protein expression was increased. Blocking miRNA-21 expression in Tb3.1 cell could suppress cancer cell growth in vitro and miRNA-21 can serve as a novel target candidate for human tongue cancer gene therapy.

Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Several studies suggested that long-term nitrate therapy may produce negative outcomes in patient mortality and morbidity. A possible mechanism may involve nitrate-mediated activation of various extracellular matrix (ECM) proteases, particularly matrix metalloproteinase-9 (MMP-9), and adhesion molecules in human macrophages, leading to the destabilization of atherosclerotic plaques. We examined the gene and protein regulating effects on THP-1 human macrophages by repeated exposure to therapeutically relevant concentrations of nitroglycerin (NTG) and possible involvement of nuclear factor (NF)-κB signaling mechanism in mediating some of these observed effects. THP-1 human macrophages repeatedly exposed to NTG (at 10 nM, added on days 1, 4 and 7) exhibited extensive alterations in the expression of multiple genes encoding ECM proteases and adhesion molecules. These effects were dissimilar to those produced by a direct nitric oxide donor, diethylenetriamine NONOate. NTG exposure significantly up-regulated NF-κB DNA nuclear binding activity and MMP-9 protein expression, and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression; these effects were abrogated in the presence of the NF-κB inhibitor parthenolide (a chemical inhibitor derived from the feverfew plant). Further, we examined whether our in vitro findings (an elevated MMP-9/TIMP-1 ratio and gelatinase activity) can be translated to in vivo effects, in a rat model. Sprague–Dawley rats exposed continuously to NTG subcutaneously for 8 days via mini-osmotic pumps showed significant induction of plasma MMP-9 dimer concentrations and the expression of a complex of MMP-9 with lipocalin-2 or neutrophil gelatinase associated lipocalin (NGAL). Plasma gelatinase activity was significantly increased by NTG over the entire study period, attaining peak elevation at day 6. Plasma TIMP-1 protein was down-regulated significantly by day 2 and days 4–7 in the NTG-treated rats. Pharmacokinetic monitoring of NTG and its dinitrate metabolites indicated that concentrations were well within therapeutic levels observed in humans. Our studies indicate that clinically relevant concentrations of NTG not only altered ECM matrix by changing the expression of multiple genes that govern cellular integrity, affecting cellular MMP-9/TIMP-1 balance in THP-1 human macrophages possibly via NF-κB activation, but also led to systemic changes in MMP-9/TIMP-1 expression and gelatinase activity in rats. These effects may contribute to extracellular matrix degradation and possible atherosclerotic plaque destabilization.

Human Ovarian Expression and Localization of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period.

Alterations in the interaction between the follicle and the matrix must occur before follicular rupture and ovulation can take place. Such interactions are controlled in part by the delicate balance between the matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We hypothesize that the TIMPs would increase prior to ovulation in order to keep MMP activity in check to provide localized degradation at the apex and prevent wholesale destruction of the follicle wall. In the present study we focused on expression and localization of TIMP3, which is the only TIMP bound to the extracellular matrix, during the periovulatory period in the human. Follicles were collected from patients undergoing elective surgery at different stages of the ovulatory process. Patients were divided into four groups (preovulatory, early ovulatory, late ovulatory, and post-ovulatory phase). Surgery was performed at the preovulatory stage without administering hCG. The remaining women received an injection of 250 µg hCG (Ovitrelle, s.c.) when the diameter of their dominant follicle was ≥14mm and ≤ 20mm on trans-vaginal ultrasound. These patients had surgery during the following time intervals after hCG-injection; early ovulatory phase 12h to ≤18h, late ovulatory phase >18h to ≤ 34h and the postovulatory phase 44h to 70h. For the localization studies, the intact follicle with the apical orientation was embedded and processed for immunohistochemistry (IHC) using a TIMP3 antibody. For the quantitative studies, the follicle was bisected and the granulosa and theca cells separated and collected for real time PCR. Real time PCR analysis for Timp3 mRNA in granulosa and theca cells revealed a significant increase in Timp3 gene expression in granulosa cells from the early to the late ovulatory stage (n=5). Thecal Timp3 expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa cells and theca cell layers. In the preovulatory follicle there was positive staining in the granulosa cells, no staining in the theca. In the early ovulatory stage there was positive staining in both the granulosa and theca interna. The staining was most intense in the granulosa and theca cells; interna and externa of 2 follicles which were in the late ovulatory group. There was staining of TIMP3 in the lumen of some of the blood vessels and it was particularly intense in one early ovulatory follicle that had a large vascular bed surrounding the follicle. Several primordial follicles were seen in the sections and these were positively stained by the TIMP3 antibody. By using human follicles collected throughout the periovulatory period, we have been able to validate our hypothesis that Timp3 gene expression increases as the follicle progresses toward ovulation. We have shown that localization of TIMP3 protein appears first in the granulosa cell layer and then steadily accumulates in the granulosa-thecal layers as ovulation approaches. This localization places TIMP3 in the appropriate cellular layer to control the degradation of the follicular wall by the MMPs. (poster)

Fosinopril and valsartan intervention in gene expression of Klotho, MMP-9, TIMP-1, and PAI-1 in the kidney of spontaneously hypertensive rats.

To determine the role of fosinopril and valsartan intervention in Klotho, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor (PAI-1) gene expression in hypertensive renal interstitial fibrosis (RIF) in the kidney tissue of spontaneously hypertensive rats (SHR). We randomly divided 20 male 22-week-old SHR into 4 groups (5 in each group): a hypertension group (SHR group), a fosinopril group [Fos group, 10 mg/( kg · d) gavage], a valsartan group [Val group, 10 mg/( kg · d) gavage], and a fosinopril plus valsartan group [Fos + Val group, fosinopril 10 mg/( kg · d) + valsartan 50 mg/( kg · d) gavage]. Another five 22-week-old male Wistar Kyoto rats (WKY) were used as controls. Through monitoring the weight of the rats, tail artery pressure, 24-hour urine protein by fosinopril and/or valsartan intervention after the 8-week trial. RT-PCR and Western blot were used to detect the mRNA and protein expression of Klotho, MMP-9, TIMP-1, and PAI-1 in the kidneys. RT-PCR showed that in the SHR group, Klotho mRNA and protein expression were significantly decreased(P<0.01), while mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 were significantly higher compared with the WKY group(P<0.01). With fosinopril and / or valsartan intervention, Klotho mRNA expression in the Fos group (P<0.01), Fos + Val group (P<0.01), Val group (P<0.05), Klotho protein expression in the Fos group(P<0.05), Fos + Val group (P<0.05), Val group (P<0.01), were significantly increased compared with those in the SHR group. The mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 in the Fos group, Val group, and Fos + Val group were significantly lower than those in the SHR group (P<0.01). The expression of Klotho mRNA had negative correlation with the expression of MMP-9 mRNA (r= -0.864, P<0.01), TIMP-1 mRNA (r=-0.725, P<0.01) and PAI-1 mRNA (r=-0.785, P<0.01). The Klotho protein expression had negative correlation with the expression of MMP-9 protein (r=-0.614, P<0.05), TIMP-1 protein (r=-0.579, P<0.05), and PAI-1 protein (r=-0.552, P<0.05). Anti-aging gene Klotho and the genes related with extracellular matrix degradation gene MMP-9, TIMP-1, PAI-1 are involved in hypertensive renal injury. The expression of Klotho and MMP-9, TIMP-1, and PAI-1 is closely correlated. Fosinopril and valsartan which increase the Klotho mRNA and protein expression can alter the expression of Klotho-MMPs/TIMPs, which may be the main mechanism to prevent interstitial fibrosis.