Tissue Inhibitor Of Metalloproteinase-1 Gene Expression Research Articles

Angiotensin-II induces the tissue inhibitor of metalloproteinases-1 through the protein kinase-C signaling pathway in rat liver fibrosis development

It has been shown that tissue inhibitor of metalloproteinases-1 (TIMP-1) plays an important role in the progression of liver fibrosis. TIMP-1 gene expression is regulated by several factors in vivo. Among them, angiotensin-II (AT-II) induces TIMP-1 in endothelial cells (EC) in vitro, however, the interaction between these molecules in liver fibrogenesis has not yet been elucidated. The aim of this study was to examine a possible association between TIMP-1 and AT-II both in vitro and in vivo using the clinically available angiotensin-I converting enzyme (ACE) inhibitor, perindopril (PE), and the AT-II type 1 receptor blocker (ARB), candesartan (CA). In cultured activated hepatic stellate cells (HSC), AT-II increased TIMP-1 in a dose- and time-dependent manner. CA and LY 333531, the protein kinase C (PKC) inhibitor, blocked this augmentation in a dose-dependent manner. In the CCl 4- and pig serum-induced rat liver fibrosis model, a clinically comparable low dose of PE and CA significantly suppressed liver fibrosis development associated with the suppression of TIMP-1 expression in the liver. AT-II induces TIMP-1 via type 1 receptor and PKC as an intracellular signaling pathway in activated HSC. These results suggested that the AT-II-induced TIMP-1 plays an important role in liver fibrosis development.

Increased matrix metalloproteinase 9 activity and mRNA expression in lung ischemia-reperfusion injury

Objectives In lung ischemia-reperfusion injury, neutrophil migration from the vasculature to the interstitial spaces plays a major role in tissue injury. Degradation of the basement membrane, which is composed of extracellular matrix (ECM) molecules, is necessary for neutrophil migration. Matrix metalloproteinases (MMPs) might play a role in ECM degradation in lung ishemia-reperfusion injury. We evaluated the changes in the activity of MMP-2 and MMP-9, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expressions using rat lung transplantation models. Methods We divided animals into 4 groups. Groups I and II served as control groups with intact lungs (Group I) and 24-hour cold-preserved lungs (Group II). Groups III and IV received lung grafts after 24-hour cold preservation. The recipient animals were sacrificed 1 hour (Group III) or 24 hours (Group IV) after transplantation. We evaluated lung injury histologically. We assessed MMP activity using zymography. We assessed MMP-2, MMP-9, and TIMP-1 gene expression using biplex reverse transcriptase-polymerase chain reaction method. Results In Groups III and IV, we noted severe ischemia-reperfusion injury. We noted no significant difference in enzyme activity and gene expression of MMP-2 between Groups I and IV. The MMP-9 activity and gene expression were low during ischemia and increased on reperfusion. TIMP-1 gene expression was low during ischemia and at the early phase of reperfusion, and showed a dramatic increase at the late phase of reperfusion. Conclusions Matrix metalloproteinase 9, but not MMP-2, may play an important role in ischemia-reperfusion injury. TIMP-1 increases at the late phase of reperfusion and may compensate for the activity of MMP-9.

Induction of Dental Pulp Fibroblast Matrix Metalloproteinase–1 and Tissue Inhibitor of Metalloproteinase–1 Gene Expression by Interleukin–1α and Tumor Necrosis Factor–α Through a Prostaglandin–Dependent Pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.

Mechanisms of induction of human tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression by all-trans retinoic acid in combination with basic fibroblast growth factor.

The addition of all-trans retinoic acid (ATRA) in combination with basic fibroblast growth factor (bFGF) to human fibroblasts results in a synergistic induction of tissue inhibitor of metalloproteinases-1 (TIMP-1) protein production. The synergistic stimulation of TIMP-1 protein by ATRA and bFGF increased across 72 h. An incubation of 10 min to 12 h with bFGF alone followed by ATRA gave a similar synergistic induction of TIMP-1 protein to that seen with both agents together. Treatment of cells with ATRA first followed by bFGF was ineffective. Expression of RARbeta mRNA was induced by ATRA alone, but not further induced by ATRA and bFGF; expression of RARgamma mRNA was induced by both ATRA or bFGF alone, and further induced by both reagents together; expression of RXRgamma was repressed by ATRA alone, but not by ATRA in combination with bFGF. Steady-state levels of TIMP-1 mRNA were induced 14 to 40-fold above control by ATRA and bFGF. Treatment with ATRA and bFGF did not alter the stability of TIMP-1 mRNA. The induction of TIMP-1 mRNA by ATRA and bFGF was greatly diminished by cycloheximide and therefore required new protein synthesis. The tyrosine kinase inhibitor genistein caused a dose-dependent inhibition of TIMP-1 protein induction by ATRA and bFGF. A MEK1 inhibitor (PD98059) inhibited both basal and induced levels of TIMP-1. At high concentrations, p38 MAP kinase inhibitors further enhanced the synergistic stimulation of TIMP-1 protein by ATRA and bFGF, but at these concentrations, p42/44 MAP kinase was strongly activated. These data begin to elucidate the mechanisms by which TIMP-1 gene expression can be upregulated.

Upstream Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Element-1, a Novel and Essential Regulatory DNA Motif in the Human TIMP-1 Gene Promoter, Directly Interacts with a 30-kDa Nuclear Protein

Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states.

Control of the tissue inhibitor of metalloproteinases-1 promoter in culture-activated rat hepatic stellate cells: regulation by activator protein-1 DNA binding proteins.

In the injured liver hepatic stellate cells (HSCs) undergo a dramatic phenotypic transformation known as "activation" in which they become myofibroblast-like and express high levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1). HSC activation is accompanied by transactivation of the TIMP-1 promoter. Truncation mutagenesis studies delineated a minimal active promoter consisting of nucleotides -102 to +60 relative to the major start site for transcription. Removal of an AP-1 site located at nucleotides -93 to -87 caused almost a complete loss of promoter activity. Analysis of AP-1 DNA binding activities during culture activation of HSCs initially indicated transient expression of proteins capable of forming a low mobility AP-1 DNA binding complex (LMAP-1). LMAP-1 was maximally induced at 24 hours of culture and then fell to undetectable levels at 120 hours. Western blot studies showed that both c-Fos and c-Jun underwent similar transient inductions. These temporal changes in c-Fos and c-Jun activities were unexpected because TIMP-1 mRNA expression is not detected in HSCs until culture day 3 to 5 and is thereafter sustained at a high level. Previous work in other cell lineages has established a key role for Pea3 binding proteins (Ets-1) in AP-1 mediated transactivation of the TIMP-1 promoter. We show that HSCs express relatively low levels Ets-1 and Ets-2 and show that mutagenesis of the Pea3 DNA binding site in the TIMP-1 promoter has less than a twofold effect on its activity in activated HSCs. Further analysis of AP-1 DNA binding activities in 7- to 14-day culture activated HSCs led to the discovery of high mobility AP-1 complexes (HMAP-1). HMAP-1 DNA binding activities were sequence specific with respect to AP-1 and absent from freshly isolated HSCs. Supershift EMSA and Western blot studies identified JunD, Fra2, and FosB as potential components of the HMAP-1. Mutations of the AP-1 site of the TIMP-1 promoter that prevented formation of HMAP-1 caused a 70% loss of activity in transfected activated HSCs. Taken together the data indicate that sustained upregulation of TIMP-1 gene expression may be at least partially controlled by a novel AP-1 dependent regulation of TIMP-1 promoter activity.

Decreased Expression of Tissue Inhibitor of Metalloproteinase 1 in Stunned Myocardium

Ultrastructural studies of stunned myocardium have shown disorganization and loss of extracellular collagen and increased collagenase activity early after ischemia and reperfusion. The interplay between matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1) regulates the turnover of cardiac extracellular matrix fibrillar collagens. However, the gene expression of MMP-1 and TIMP-1 in stunned myocardium is not known. Here, we determined whether altered expression of MMP-1 and TIMP-1 occurs in globally stunned hearts. An isolated nonworking rabbit heart preparation, perfused with a bovine erythrocyte suspension in modified Krebs solution, was used. Two groups were studied: the stunned group was subjected to 20 min of normothermic global ischemia followed by 120 min of normal reperfusion (n= 8), and the control group underwent 140 min of uninterrupted perfusion (n= 7). The developed pressures at the end of reperfusion for ischemic and control hearts were 67.0 ± 2.73 and 83.1 ± 1.52 mm Hg (P< 0.006) respectively. Ribonuclease protection assays of total left ventricular RNA using riboprobes for MMP-1, TIMP-1, and 18S rRNA were performed. A significant decrease (twofold,P< 0.03) in TIMP-1 gene expression was found in the stunned hearts, while MMP-1 mRNA expression was unchanged. Thus, in early stunning, the decrease in TIMP-1 expression could tip the balance favoring enhanced metalloproteinase activity, promoting collagen turnover, and initiating extracellular matrix remodeling. This may contribute to delayed recovery from myocardial stunning.

Effect of Tenidap on Metalloproteinase Gene Expression in Rheumatoid Arthritis

This study evaluated measures of metalloproteinase gene expression in synovial biopsies as a means of differentiating the activities of two antirheumatic therapies: piroxicam and tenidap. Synovial biopsies and quantitative in situ hybridization for stromelysin (STR), collagenase (COL), tissue inhibitor of metalloproteinase-1 (TIMP), and actin mRNA were performed in a subset of patients with rheumatoid arthritis in a larger doubleblind randomized crossover trial comparing 120 mg/day of tenidap for 6 weeks with 20 mg/day of piroxicam for 6 weeks. There were no consistent differences between tenidap and piroxicam on COL or TIMP mRNA expression, but STR mRNA was significantly lower after tenidap compared with piroxicam (p = 0.037). There were no significant associations between measures of local or systemic clinical activity and STR, COL, or TIMP gene expression. However, changes in STR gene expression were significantly correlated with changes in serum C-reactive protein (p = 0.016). Because tenidap and piroxicam are both potent inhibitors of prostaglandin production, the effect of tenidap on STR gene expression may be due to its additional cytokine modulating activity. Clinical trials with data from synovial biopsies may be useful in evaluating the potential for disease-modifying effects of antirheumatic drugs.

Synergistic Transcriptional Activation of the Tissue Inhibitor of Metalloproteinases-1 Promoter via Functional Interaction of AP-1 and Ets-1 Transcription Factors

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.

Mechanisms of methotrexate action in rheumatoid arthritis. Selective decrease in synovial collagenase gene expression.

To measure the effect of methotrexate (MTX) treatment in rheumatoid arthritis (RA) on the expression of synovial collagenase, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expression in a prospective study. Serial percutaneous synovial biopsies (pretreatment and after 3-4 months) were performed on the knees of 8 patients (7 with RA, 1 with seronegative arthritis) who were beginning oral MTX therapy. Synovial gene expression was determined by quantitative in situ hybridization using computer-assisted image analysis. After therapy, patients had decreased joint counts, morning stiffness, and erythrocyte sedimentation rates. Synovial inflammation in the biopsy tissues was slightly decreased after therapy. In situ hybridization on pretreatment and posttreatment frozen sections was performed to quantify synovial messenger RNA (mRNA) levels. Collagenase gene expression significantly decreased after MTX therapy (P = 0.006) even though cell density in the region was unchanged. TIMP-1 and stromelysin mRNA levels were not changed by MTX therapy. To study the mechanism of MTX action in vitro, MTX-treated and control fibroblast-like synoviocytes were stimulated with interleukin-1 beta (IL-1 beta). MTX did not alter collagenase or TIMP-1 mRNA levels after IL-1 exposure. MTX therapy decreases collagenase gene expression but not TIMP-1 or stromelysin gene expression in the synovium. This action is probably an indirect effect due to an alteration in the synovial cytokine milieu, rather than a direct effect on gene expression.