Upregulation Of TIMP-1 Expression Research Articles

Inhibitory effect of cathepsin K inhibitor (ODN-MK-0822) on invasion, migration and adhesion of human breast cancer cells in vitro.

Approximately 90% of patients with advanced breast cancer develop bone metastases; an event that results in severe decrease of quality of life and a drastic deterioration in prognosis. Therefore, to increase the survival of breast cancer patients,the development of new therapeutic strategies to impair metastatic process and skeletal complications is critical. Previous studies on the role of cathepsin K (CTSK) in metastatic spreading led to several strategies for inhibition of this molecule such as MIV-711 (Medivir), balicatib and odanacatib (ODN) which were on trial in the past. The present study intended to assess the anti-metastatic efficacy of ODN in breast cancer cells. Human breast cancer cell lines MDA-MB-231 were treated with different concentrations of ODN and performed invasion, adhesion and migration assays and, RT-PCR and western blot to evaluate the effect of ODN on the metastatic potential of breast cancer cells. ODN markedly decreased wound healing cell migration,invasion and adhesion at a dose dependent manner. ODN inhibits cell invasion by decreasing the matrix metalloproteinase (MMP-9) with the upregulation of TIMP-1 expression. ODN effectively inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal Kinase (JNK), and blocked the expression of β-integrins and FAK proteins. ODN also significantly inhibited PI3K downstream targets Rac1, Cdc42, paxillin and Src which are critical for cell adhesion, migration and cytoskeletal reorganization. ODN exerts anti-metastatic action through inhibition of signaling pathway for MMP-9, PI3K and MAPK. This indicates potential therapeutic effects of ODN in the treatment of metastatic breast cancer.

Platelet-derived growth factor-D intensifies fibrogenesis through upregulation of TIMP-1 expression and signalling through both platelet-derived growth factor receptors type α and β

Platelet-derived growth factor-D intensifies fibrogenesis through upregulation of TIMP-1 expression and signalling through both platelet-derived growth factor receptors type α and β

In Vivo Characterization of Doxycycline Effects on Tear Metalloproteinases in Patients with Chronic Blepharitis

Matrix metalloproteinases (MMPs) have a role in the pathogenesis of rosacea-associated chronic blepharitis. Doxycycline is largely used as a treatment for recalcitrant chronic blepharitis. It has been shown in vitro that doxycycline inhibits MMPs activation. The aim of this study was to investigate in vivo the effect of doxycycline in modulating MMPs in patients with chronic idiopathic blepharitis. Eight patients (6 male, 2 female; mean age 45.7+/-17.5 years) were included in the study. Doxycycline (100 mg) was administered orally, twice a day, for 2 weeks and once a day for an additional 2 weeks. Clinical signs and symptoms were evaluated and scored (0-3) at baseline and after 4 weeks. Total sign (TSS) and total symptom (TSyS) scores were calculated. Tear samples and conjunctival impression cytologies were collected at baseline and after 4 weeks of treatment to evaluate MMP-9 and TIMP-1 expression and activity. An improvement in TSS (4.5+/-1.1 vs 2.7+/-1.5) and TSyS (6.6+/-1.3 vs. 3.1+/-1.9) was observed after 4 weeks, with significant amelioration of hyperemia, marginal blepharitis, and superficial punctuate keratopathy. Zymography revealed a decrease of MMP-9 activity after 4 weeks. MMP-9 mRNA and protein levels did not change, while an upregulation of TIMP-1 expression was observed. This study suggests that 4-week treatment with doxycycline significantly improved symptoms and signs in patients with chronic blepharitis in association with a decrease in MMP-9 activity. Upregulation of TIMP-1 is proposed as a possible mechanism of MMP-9 inactivation.

Nitric oxide (NO), methylation and TIMP-1 expression in BL6 melanoma cells transfected with MHC class I genes.

We have previously found that transfection of BL6-8 melanoma cells with the H-2K, but not H-2D/L genes resulted in loss of their metastatic ability that was associated with decrease in their invasiveness and up-regulation of TIMP-1 expression. In the present study using the methylation-specific PCR (MSP) we found that lack of TIMP-1 expression in BL6-8 is associated with methylation in the TIMP-1 5' regulatory area. In the H-2Kb transfected CL8-1 melanoma cells up-regulation of TIMP-1 was in parallel with loss of TIMP-1 gene methylation. Treatment of BL6-8 with 5-azacytidine or with an inhibitor of histone deacetylase trichostatin A resulted in up-regulation of TIMP-1 expression. These results indicate that methylation and histone deacetylation play an important role in transcription repression of TIMP-1 in BL6 melanoma cells. Some data showed that nitric oxide (NO) could affect methylation and expression of various gene. Therefore we analyzed NO production in B16 melanoma cell lines with different expression of TIMP-1. We have found that B16F10 and BL6-8 melanoma cells do not express TIMP-1 and do not produce nitric oxide (NO) even after stimulation with IFN-gamma and LPS. However, BL6-8 cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld gene expressed TIMP-1 and produced NO constitutevely. NO production in these cells was further stimulated by IFN-gamma and LPS. Northern blot analysis showed that expression of iNOS was paralleled with TIMP-1 expression in the tested melanoma cells. However, NO produced by SNAP or inhibition of NO production by NMA did not affect TIMP-1 expression in the tested melanoma cells. Thus, TIMP-1 expression and NO production in BL6 melanoma cells transfected with MHC class I gene coincides but it remains unclear whether NO is responsible for the change in TIMP-1 methylation and expression.

Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma

Background: Bronchial asthma is characterized by airway wall remodeling. Matrix metalloproteinases (MMPs) are members of a family of proteolytic enzymes that degrade the extracellular matrix and that restrain the effects of their tissue inhibitors (TIMPs). Treatment with inhaled corticosteroids may prevent airway remodeling in asthma. However, the effects of corticosteroid treatment on MMPs and TIMPs in asthma are unknown. Objective: We examined the effects of inhaled beclomethasone dipropionate (BDP) on the expression of MMP-9 and TIMP-1 and subepithelial collagen deposition in bronchial biopsy specimens from 30 subjects with asthma. Methods: Inhaled BDP, 800 μg daily, or placebo was administered for 6 months in a double-blind, parallel-group study, and bronchial biopsies were performed before and after treatment. Biopsy specimens were examined for extent of collagen type III in the subepithelial basement membrane by means of immunohistochemistry, and expression of both epithelial and submucosal MMP-9 and TIMP-1 was quantitated. Numbers of inflammatory cells were also determined. Results: We observed significant decreases in collagen type III deposition ( P < .01) and the expression of submucosal MMP-9 ( P < .01) and a significant increase in the expression of submucosal TIMP-1 ( P < .05) in the BDP group. Significant correlations were found between the subepithelial collagen type III deposition and epithelial ( r s = 0.37, P < .05) and submucosal expression of MMP-9 ( r s = 0.47, P < .01). Additionally, the number of many inflammatory cells and myofibroblasts in airway mucosa were significantly decreased in the BDP group. Conclusion: Our findings suggest that corticosteroid treatment of asthma can reduce subepithelial collagen deposition by downregulation of MMP-9 expression and upregulation of TIMP-1 expression. (J Allergy Clin Immunol 1999;104:356-63.)

Distinct mechanisms regulate TIMP-1 expression at different stages of phorbol ester-mediated differentiation of U937 cells.

Upon exposure to 12-O-tetradecanoylphorbol 13-acetate (PMA), promonocyte-like U937 cells differentiate into macrophage-like cells and begin to express certain metalloproteinases and TIMP-1. We report here that distinct mechanisms regulate TIMP-1 production in PMA-treated U937 cells. TIMP-1 protein and steady-state mRNA levels increased about 10-fold in PMA-differentiated cells compared to undifferentiated cells. TIMP-1 transcription increased about 2.5-fold, but this stimulation was not detected until at least 48 h post-PMA. In contrast, the half-life for TIMP-1 mRNA increased about 3-fold and was detected at 8 h post-PMA. Using in vitro translation assays, we found that TIMP-1 mRNA from PMA-differentiated cells translated about 5-fold less efficiently than that from basal cells, suggesting structural differences in TIMP-1 mRNA in basal and differentiated U937 cells. Although primer extension and RNase protection analyses showed 5' heterogeneity of TIMP-1 transcripts, all forms were equally stimulated in response to PMA-mediated differentiation. The poly(A) tail length of TIMP-1 mRNA, however, was longer in PMA-treated cells. Our findings suggested that up-regulation of TIMP-1 expression in PMA treated U937 cells is mediated early by enhanced TIMP-1 mRNA stability, possibly related to increased poly(A) tail length, and later by an increase in transcription rate.

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes.

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2Kb or H-2Kd gene-transfected melanoma cells was associated with reduced adherence to endothelial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demonstrated reduced ability to invade Matrigel that paralleled up-regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2Dd/Ld genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2Kb transfected cells was associated with alterations in cell surface carbohydrates with appearance of alpha-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes.

Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential.

Malignant astrocytomas are highly invasive tumors which infiltrate diffusely into regions of normal brain. The degradation of the extracellular matrix (ECM) by matrix metalloproteinases is thought to be one of the most important steps in the process of tumor invasion. However, the activity of most matrix metalloproteinases (MMPs) can be modulated by simultaneously secreted inhibitors (tissue inhibitors of metalloproteinases, TIMPs). We have previously shown that an imbalance between the levels of MMPs and TIMPs may be essential in the determination of the invasiveness of certain human malignant astrocytoma cell lines. To determine if the up-regulation of TIMP genes and gene products could modulate the invasiveness of human malignant astrocytoma cells, in the present study we have transfected a highly invasive astrocytoma cell line, SF-188, with an expression vector carrying a full-length TIMP-1 cDNA. The parental SF-188 astrocytoma cell line overexpresses the 72-kDa and 92-kDa type IV collagenases with little expression of TIMPs-1 and -2. Following transfection with TIMP-1, SF-188 astrocytoma clones expressed the 0.9 kb TIMP-1 message by northern analysis, and produced a 21 kDa metalloproteinase inhibitor by reverse zymography. The stable TIMP-1 SF-188 transformants demonstrated morphological changes and diminished growth rates in soft agar when compared to controls. The invasion of successfully TIMP-1 transfected astrocytoma cells across matrigel-coated filters was significantly decreased over controls. These results suggest that upregulation of TIMP-1 expression in SF-188 astrocytoma cells has decreased their in vitro invasive potential.