Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.     

Herbal medicine Sho-saiko-to (TJ-9) increases expression matrix metalloproteinases (MMPs) with reduced expression of tissue inhibitor of metalloproteinases (TIMPs) in rat stellate cell

We have reported that Sho-saiko-to (TJ-9) prevents liver fibrosis in vivo. To gain further insights into the effect of TJ-9, the matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) balance was examined. Hepatic stellate cells (HSCs) were isolated from male Wistar rats and cultured with TJ-9 (0-1000 microg/ml) on uncoated plastic dishes for 4 days. To elucidate the effects on the MMPs/TIMPs balance by TJ-9, quantitative analysis of type IV collagen-degrading activity, gelatin zymography and reverse zymography were carried out. Northern blot analysis was performed to determine the expression of MMP-2, 13 and TIMP-1 mRNAs. TJ-9 treatment resulted in dose-dependent upregulation of MMP-2, 13 mRNA and downregulation of TIMP-1 mRNA up to 500 microg/ml. Gelatin zymography, reverse zymography and quantitative analysis of type IV collagen-degrading activity confirmed that TJ-9 increased MMP-2 activity and prevented TIMP-1, 2 activities in a dose-dependent manner. SB203580 diminished the reduction of mRNA as well as the activity of TIMP-1 by TJ-9 and induction of mRNA as well as the activity of MMP-2. These results show that TJ-9 increased MMP-2, 13 activity with reduced TIMP-1, 2 activities on HSCs possibly via P38 pathway.     

基质金属蛋白酶及其抑制因子与扩张型心肌病的关系

目的研究基质金属蛋白酶-2(MMP-2)及金属蛋白酶组织抑制因子(TIMP-1)在扩张型心肌病(DCM)中的变化,探讨血管紧张素转换酶抑制剂(ACEI)对心肌纤维化的影响及其调控机制。方法雄性SD大鼠腹腔注射阿霉素(2 mg/kg,每周1次,连续8周)建立扩张型心肌病模型。将达到DCM诊断标准的大鼠分3组:①H组(卡托普利高剂量干预组,50mg/Kg.d);②L组(卡托普利低剂量干预组,25mg/Kg.d);③C组(DCM对照组)。HE染色和苦味酸天狼星红染色观察各组大鼠心肌细胞和间质胶原变化,RT-PCR法检测心肌MMP-2及TIMP-1的表达。结果与正常对照组比较,DCM组心肌细胞坏死明显、胶原纤维和胶原容积分数增加(P<0.05);而H组和L组的胶原纤维较C组减少(P<0.05)。DCM组心肌MMP-2 mRNA表达较正常对照组显著增加(P<0.01),H组和L组较C组降低(P<0.05)。DCM组TIMP-1mRNA的表达较正常对照组降低(P<0.05),H组较C组有所增加(P<0.05)。结论 MMP-2及TIMP-1与心肌细胞外基质的重塑密切相关,ACEI有降解MMP-2的作用,可以减轻心肌间质的...     

Increased Expression of Retinal TIMP3 mRNA in Simplex Retinitis Pigmentosa Is Localized to Photoreceptor-Retaining Regions

Abstract: The human tissue inhibitor of metalloproteinases-3 (TIMP3) gene is the most recently characterized member of a family of genes whose products are implicated in extracellular matrix (ECM) remodelling. We previously described an increase in expression of TIMP3 mRNA in retinas affected by the progressive photoreceptor degenerative disease, simplex retinitis pigmentosa (RP). To gain further insight into the association between TIMP3 overexpression and retinal degeneration, we have analyzed the cellular localization of TIMP3 mRNA in control and simplex RP retinas using in situ hybridization. No TIMP3 mRNA expression was detectable in control neural retina. In RP-affected retinas, overexpression of TIMP3 mRNA was observed in photoreceptor inner segments and in the ganglion cell layer only in those regions retaining relatively nondystrophic retinal architecture. Modulation of TIMP3 expression in these regions, possibly in association with matrix metalloproteinases, may reflect remodelling of the retinal ECM and concomitant reorganization of neuronal connectivity.     

Expression of dihydrotestosterone synthases and androgen receptor in sheep oviduct ampulla and its regulation by estradiol and progesterone

Oviduct ampulla plays an important role in steroid hormone-regulated sperm-oocyte binding in female animals. Although studies have shown that androgen receptor are expressed in many species oviduct, the interaction among androgen receptor (AR), estradiol (E2) and progesterone (P4) in the sheep oviduct have rarely been reported. In this study, we evaluated the localization of two isoforms of dihydrotestosterone (DHT) sythetase enzymes 5α-reductase (5α‐red1, 5α‐red2) and AR in sheep oviduct ampulla by immunohistochemistry and immunofluorescence. Results showed that they were all distributed in oviduct epithelium layer. In epithelial cells, 5α‐red1, 5α‐red2 were expressed in cytoplast and nuclear, but AR were stained in nuclear. We also investigated their expression pattern in the sheep oviduct ampulla at different development stages of follicles (Large follicles stage; hemorrhagium, luteum and albicans of corpus stage) by molecular experiments. We found that 5α‐red1, 5α‐red2 and AR mRNA abundance and protein were expressed highest in corpus albicans stage and lowest in corpus hemorrhagium stage. In vitro, when sheep oviduct ampulla epithelial cells (SOAECs) were cultured and treated with different concentrations of E2/P4 (10^?9–10^?6 M), we found that E2 inhibited the expression of AR mRNA and protein, while P4 promoted this expression. In addition, when the SOAECs were treated with E2 (10^?8 M) and/or its non-selective inhibitor ICI182780 (10^?7 M) as well as with P4 (10^?6 M) and/or its non-specific inhibitor RU486 (10^?5 M), we found that E2 and P4 inhibited and promoted the expression of AR mRNA and proteins, respectively, via their nuclear receptor pathways. This study provides a basic insight for the further research of oviduct epithelium physiological function closely related to androgen.     

Inhibition of fibroblast-induced angiogenic phenotype of cultured endothelial cells by the overexpression of tissue inhibitor of metalloproteinase (TIMP)-3

In this study, we examined the effect of overexpression of tissue inhibitor of metalloproteinase (TIMP)-3 on the angiogenic phenotype expressed by vascular endothelial cells (ECs). ECs were infected with a recombinant adenovirus carrying the TIMP-3 gene at various multiplicities of infection, and TIMP-3 expression by transfected cells was confirmed by Western blotting and reverse zymography. At transfection doses of 6.25, 12.5, 25, 50 and 100 multiplicity of infection, EC migration was reduced to 66, 45, 25, 17 and 5%, respectively, of that of the control. At the multiplicity of infection of 20, capillary tube length was reduced by 80% compared to that of the control. Thus, expression of TIMP-3 by ECs effectively inhibited EC migration and tube formation. Overexpression of TIMP-3 by ECs may be considered a gene therapy strategy for the treatment of pathological angiogenesis such as cancer and diabetic retinopathy.     

基质金属蛋白酶和组织金属蛋白酶抑制剂在肾细胞癌组织中的表达

目的:基质金属蛋白酶及组织金属蛋白酶抑制剂在肾细胞癌转移中占有重要的作用,研究肾细胞癌组织中MMP-2、MMP-9、TIMP-1和TIMP-2的表达情况,为肾癌转移的治疗提供理论依据。方法:选取36例肾细胞癌肾组织标本,从相同的肾细胞癌组织及癌旁肾组织获得对照样本,均进行根治性肾切除手术切除。肿瘤分期按TNM分期标准。为了统计评估,肿瘤1期和2期为低级,3期以上为高级。RT-PCR检测肿瘤和正常组织中的MMP-2、MMP-9、TIMP-1和TIMP-2的表达。结果:不同样本MMPs和TIMPs表达水平各不相同。肾细胞癌组织中MMP-2、MMP-9、TIMP-1、TIMP-2在肾细胞癌中的表达明显高于正常肾组织(P0.05)。在肾细胞癌的肿瘤分期方面,MMP-2与MMP-9和肿瘤的分期显著相关,随着肿瘤分期的增加,MMP-2与MMP-9的表达明显升高(P0.05),而TIMP-1与TIMP-2与肿瘤的分期无关。结论:肾细胞癌组织中TIMP-2、MMP-2,MMP-9,TIMP-1的mRNA表达显著高于正常肾组织,抑制MMPS的表达将成为治疗肾细胞癌转移的新的方向。     

Systemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery

Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.     

Balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the cervical mucus plug estimated by determination of free non-complexed TIMP

Background  

The cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy. The CMP contains high concentrations of matrix metalloproteinase 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). This indicates a potential to degrade extracellular matrix components depending on the balance between free non-complexed inhibitors and active enzymes.     

Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4

Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo.     

四周尾部悬吊大鼠颈总动脉MMP9和TIMP1的表达及活性改变

目的:探讨四周尾部悬吊模拟失重大鼠颈总动脉基质金属蛋白酶(matrix metalloprotein9,MMP9)和金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase1,TIMP1)的基因、蛋白表达及酶活性变化。方法:采用4周(week,wk)尾部悬吊大鼠模拟失重影响,通过透射电镜检测颈总动脉壁基质含量,实时定量聚合酶链式反应检测MMP9和TIMP1的mRNA表达,Western blot和免疫组织化学染色检测其蛋白表达和分布,明胶酶谱法测定MMP9活性水平。结果:与对照组相比,悬吊组大鼠细胞外基质面积较对照组显著增加(P0.05),胶原蛋白含量显著增加(P0.05);悬吊组大鼠颈总动脉MMP9的m RNA表达量无明显改变,而其蛋白表达量和酶活性均显著降低(P0.05);TIMP1 mRNA和蛋白表达量则显著升高(P0.05)。结论:模拟失重使大鼠颈总动脉MMP9水平降低,TIMP1水平升高,可能与其管壁基质增生和胶原蛋白含量增加有关。     

基质金属蛋白酶及其抑制剂在乳腺癌中的表达

目的:研究基质金属蛋白酶2(Matrix Metalloproteinase-2,MMP-2),基质金属蛋白酶7(MMP-7),基质金属蛋白酶9(MMP-9),膜型基质金属蛋白酶(Membrane Type-1 Matrix Metalloproteinase,MT1-MMP),金属蛋白酶组织抑制剂1(Tissue Inhibitor of Metalloproteinase,TIMP-1),金属蛋白酶组织抑制剂2(TIMP-2)在乳腺癌组织中mRNA的表达,及与临床病理变量之间的关联。方法:采用150例乳腺癌患者的组织样本。使用半定量逆转录-聚合酶链反应(RT-PCR)法来测定肿瘤组织和正常乳腺组织中MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2的mRNA表达。结果:MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2在乳腺癌中的mRNA表达显著高于正常组织。结论:MMP-2,MMP-7,MMP-9,和MTI-MMP的表达增加和临床病理参数之间的关联,可以用来预测乳腺癌的侵害行为。     

Analysis of coding sequences for tissue inhibitor of metalloproteinases 1 (TIMP1) and 2 (TIMP2) in patients with aneurysms.

Aneurysms are characterized by dilation, i.e. expansion and thinning of all the arterial wall layers, which is accompanied by remodeling of the connective tissue. Genes involved in the regulation of tissue remodeling are therefore candidate genes. We analyzed TIMP1 and TIMP2 coding sequences in 12 individuals with abdominal aortic aneurysms (AAA), one individual with AAA and intracranial aneurysms (IA), four individuals with IA and two clinically unaffected individuals. We identified two nucleotide variants in both the TIMP1 and the TIMP2 coding sequences. All differences occurred in the third base positions of codons and were neutral polymorphisms. A significant difference was observed in the frequency of TIMP2 nt 573 polymorphism between 168 alleles from AAA patients and 102 control alleles.     

Matrix metalloproteinases regulate mesonephric cell migration in developing XY gonads which correlates with the inhibition of tissue inhibitor of metalloproteinase-3 by Sry

In the mouse, the sex determining gene Sry, on the Y chromosome, controls testis differentiation during embryogenesis. Following Sry expression, indifferent XY gonads increase their size relative to XX gonads and form cord-like structures with the adjacent mesonephros, providing XY gonad somatic cells. This mesonephric cell migration is known to depend on Sry, but the molecular mechanism of mesonephric cell migration remains unknown. In this study, it was shown that cells expressing Sry induced proliferation of mesonephric cells migrating into male gonads, and inhibited expression of the tissue inhibitor of metalloproteinases (TIMP)-3 gene, which is the endogenous inhibitor of matrix metalloproteinases (MMP). In addition, the mesonephric cell migration was blocked by a chemically synthesized inhibitor of MMP in a gonad/mesonephros organ co-culture system with enhanced green fluorescent protein transgenic embryos. The findings indicate that MMP may play a critical role in mesonephric cell migration, and the function of MMP may be regulated by a Sry-TIMP-3 cascade. These findings are an important clue for the elucidation of testicular formation in developing gonads.     

基质金属蛋白酶-2、-9及其组织抑制剂-1在大鼠肺组织中的增龄变化

目的研究基质金属蛋白酶（MMPs）-2、-9及组织型基质金属蛋白酶抑制剂（TIMPs）-1在大鼠肺组织中的增龄性变化规律。方法不同月龄（1月龄、3月龄、12月龄、18月龄、24月龄）雄性清洁级（SD）大鼠47只分为5组,按月龄取实验大鼠的右下叶肺组织,用流式细胞术检测MMP-2、MMP-9及TIMP-1的表达水平,进行定量分析。结果 MMP-2、MMP-9和TIMP-1均随着月龄的增长呈先降后升波谷型曲线变化;3月龄、12月龄和18月龄MMP-2、MMP-9和TIMP-1均较1月龄明显下降,且以12月龄为最低值;24月龄MMP-2、MMP-9和TIMP-1均较1月龄上升。MMP-2/TIMP-1比值和MMP-9/TIMP-1比值均随着月龄的增长呈先升后降波峰型曲线变化;12月龄和18月龄均较1月龄明显升高,且以18月龄为最高点;3月龄的MMP-2/TIMP-1较1月龄有所上升,3月龄的MMP-9/TIMP-1与1月龄持平;24月龄的MMP-2/TIMP-1和MMP-9/TIMP-1较1月龄也有不同程度上升。结论大鼠肺组织中MMPs及其抑制剂TIMPs随月龄的增长,呈特征性变化规律。     

Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila

In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.     

Matrix metalloproteinases and gastrointestinal cancers: Impacts of dietary antioxidants

The process of carcinogenesis is tightly regulated by antioxidant enzymes and matrix degrading enzymes, namely, matrix metalloproteinases(MMPs). Degradation of extracellular matrix(ECM) proteins like collagen, proteoglycan, laminin, elastin and fibronectin is considered to be the prerequisite for tumor invasion and metastasis. MMPs can degrade essentially all of the ECM components and, most MMPs also substantially contribute to angiogenesis, differentiation, proliferation and apoptosis. Hence, MMPs are important regulators of tumor growth both at the primary site and in distant metastases; thus the enzymes are considered as important targets for cancer therapy. The implications of MMPs in cancers are no longer mysterious; however, the mechanism of action is yet to be explained. Herein, our major interest is to clarify how MMPs are tied up with gastrointestinal cancers. Gastrointestinal cancer is a variety of cancer types, including the cancers of gastrointestinal tract and organs, i.e., esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum and anus. The activity of MMPs is regulated by its endogenous inhibitor tissue inhibitor of metallopro-teinase(TIMP) which bind MMPs with a 1:1 stoichiometry. In addition, RECK(reversion including cysteinerich protein with kazal motifs) is a membrane bound glycoprotein that inhibits MMP-2,-9 and-14. Moreover, α2-macroglobulin mediates the uptake of several MMPs thereby inhibit their activity. Cancerous conditions increase intrinsic reactive oxygen species(ROS) through mitochondrial dysfunction leading to altered protease/anti-protease balance. ROS, an index of oxidative stress is also involved in tumorigenesis by activation of different MAP kinase pathways including MMP induction. Oxidative stress is involved in cancer by changing the activity and expression of regulatory proteins especially MMPs. Epidemiological studies have shown that high intake of fruits that rich in antioxidants is associated with a lower cancer incidence. Evidence indicates that some antioxidants inhibit the growth of malignant cells by inducing apoptosis and inhibiting the activity of MMPs. This review is discussed in six subchapters, as follows.