Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Melatonin promotes angiogenesis during protection and healing of indomethacin‐induced gastric ulcer: role of matrix metaloproteinase‐2

Abstract: Matrix metalloproteinase (MMP)‐2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N‐acetyl‐5‐methoxy tryptamine), an antioxidant and anti‐inflammatory agent, prevents indomethacin‐induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP‐2‐mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin‐induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP‐2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over‐expression of MMP‐2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP‐2. In addition, upregulation of MMP‐2 was parallel to MMP‐14 and inverse to tissue inhibitor of metalloprotease (TIMP)‐2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP‐2 and VEGF over‐expression during protection and healing of gastric ulcers. This study highlights for the first time a phase‐associated regulation of MMP‐2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin‐induced gastropathy.     

Role of melatonin in regulating matrix metalloproteinase-9 via tissue inhibitors of metalloproteinase-1 during protection against endometriosis

Abstract:  Endometriosis is a gynecological disease of women and plausibly regulated by matrix metalloproteinases (MMPs). However, mechanisms of alterations in MMPs during endometriosis remain unclear. Human endometriotic tissues possessing varying degrees of severity were examined for expression of MMPs and tissue inhibitors of metalloproteinase (TIMP)-1. In addition, endometriosis was generated in mice and endometriotic tissues were tested for MMP-9 activity. Results show significant upregulation of secreted and synthesized proMMP-9 activity with duration and severity of endometriosis. Along with upregulation of activity, the expression of proMMP-9 was found increased while TIMP-1 expression followed an inverse trend. The effect of melatonin, a major secretory product of the pineal gland, on endometriosis was examined in preventive and therapeutic models in mice. The results show that melatonin arrested lipid peroxidation and protein oxidation and downregulated proMMP-9 activity and expression in a time and dose-dependent manner while protecting and regressing peritoneal endometriosis. Moreover, the attenuated activity and expression of proMMP-9 were associated with subsequent elevation in the expression of TIMP-1. Our study reveals for the first time the role of melatonin in arresting peritoneal endometriosis in mice and a novel marker, expression ratio of proMMP-9 versus TIMP-1, was identified for assessing severity and progression of endometriosis.     

Expression of immunoreactive matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human normal livers and primary liver tumors

Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion by degrading extracellular matrix proteins. However, little is known about the in situ expression of MMP in human normal livers and primary liver tumors. In this study, we therefore examined the in situ expression of immunoreactive MMP and tissue inhibitors of MMP (TIMP) in 10 normal livers, 11 surgically resected intrahepatic cholangiocarcinomas (CCs), and 6 surgically resected hepatocellular carcinomas (HCCs). In normal livers, MMP and TIMP were infrequently and faintly expressed in bile ducts, but were not expressed in hepatocytes. In the 11 CCs, MMP-1, MMP-2, MMP3, MMP-9, TIMP-1, and TIMP-2 were expressed in tumor cells and/or tumor stroma in 11 (100%), 5 (45%), 8 (73%), 3 (27%), 9 (82%), and 9 (82%), respectively. The expression of MMP and TIMP in tumor cells was located in the cytoplasm with a diffuse or granular pattern; that in the tumor stroma was situated in fibroblasts, leukocytes, and extracellular matrix. Their expression was stronger in CC cases with severe invasion than in CC cases with mild invasion. In contrast, MMP and TIMP were not expressed in any cases of HCC. These results show that intrahepatic bile duct cells may neoexpress or overexpress MMP and TIMP after malignant transformation but that hepatocytes do not, and suggest that MMP and TIMP play an important role in CC cell invasion by degrading extracellular matrix proteins. (Hepatology 1996 Jun;23(6):1341-4)     

Melatonin inhibits matrix metalloproteinase‐9 activity by binding to its active site

The zinc‐dependent matrix metalloproteinases (MMPs) are key enzymes associated with extracellular matrix (ECM) remodeling; they play critical roles under both physiological and pathological conditions. MMP‐9 activity is linked to many pathological processes, including rheumatoid arthritis, atherosclerosis, gastric ulcer, tumor growth, and cancer metastasis. Specific inhibition of MMP‐9 activity may be a promising target for therapy for diseases characterized by dysregulated ECM turnover. Potent MMP‐9 inhibitors including an indole scaffold were recently reported in an X‐ray crystallographic study. Herein, we addressed whether melatonin, a secretory product of pineal gland, has an inhibitory effect on MMP‐9 function. Gelatin zymographic analysis showed a significant reduction in pro‐ and active MMP‐9 activity in vitro in a dose‐ and time‐dependent manner. In addition, a human gastric adenocarcinoma cell line (AGS) exhibited a reduced (~50%) MMP‐9 expression when incubated with melatonin, supporting an inhibitory effect of melatonin on MMP‐9. Atomic‐level interaction between melatonin and MMP‐9 was probed with computational chemistry tools. Melatonin docked into the active site cleft of MMP‐9 and interacted with key catalytic site residues including the three histidines that form the coordination complex with the catalytic zinc as well as proline 421 and alanine 191. We hypothesize that under physiological conditions, tight binding of melatonin in the active site might be involved in reducing the catalytic activity of MMP‐9. This finding could provide a novel approach to physical docking of biomolecules to the catalytic site of MMPs, which inhibits this protease, to arrest MMP‐9‐mediated inflammatory signals.     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

基质金属蛋白酶与动脉粥样硬化斑块

基质金属蛋白酶(MMP)可降解细胞外基质,参与动脉粥样硬化形成和斑块破裂,与斑块稳定性有关.组织金属蛋白酶抑制剂(TIMP)是天然的MMP特异性抑制剂.MMP与TIMP的平衡失调与动脉粥样硬化的形成和发展密切相关.因此,通过调节MMP与TIMP之间的平衡延缓动脉粥样硬化的进展和防治斑块破裂,有可能成为防治心脑血管病的新途径.     

Effect of the anti-tumor necrosis factor-alpha antibody infliximab on the ex vivo mucosal matrix metalloproteinase-proteolytic phenotype in inflammatory bowel disease

BACKGROUND: Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti-TNF-a antibody infliximab. The aims of our study were to determine expression and secretion of MMP-1, -2, -3, -9, and their inhibitors TIMP-1, -2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by infliximab of the proteolytic phenotype. METHODS: Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without infliximab and/or the T-cell activator pokeweed mitogen (PWM). Explants and culture supernatants were analyzed for MMPs, TIMPs, and TNF-alpha protein, activity and/or mRNA levels. All patients were genotyped for functional TNF-alpha, MMP, and TIMP single nucleotide polymorphism (SNP) loci. RESULTS: Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. Dependent on genotype at SNP loci, infliximab downregulated MMP-1, -3, and -9 relative to TIMP-1 and -2 and also decreased MMP-1 and -3 activities, while PWM enhanced these levels, partly counteracted again by infliximab. The expression of MMP-2 relative to TIMP did not change by treatment with infliximab and/or PWM. CONCLUSIONS: The high expression of MMPs in patients with IBD suggests a role for these proteinases in the pathogenesis of this disease. Infliximab seems to induce a genotype-associated matrix protective phenotype, which may contribute to the observed therapeutic efficacy of this drug in IBD, particularly at the mucosal surface.     

基质金属蛋白酶及组织抑制剂在肝癌中的表达及与预后的关系

目的：探讨基质金属蛋白酶（MMPs)及其组织抑制剂（TIMPs）在肝癌中的表达及其意义。方法：应用免疫组化、Northern印迹杂交及图像分析技术对人肝细胞癌（HCC）、肝癌细胞株7721、全反式维甲酸处理的7721细胞（RA－7721）和正常人肝细胞株L－02作MMP－1、2、9和TIMP－2的表达分析。结果;肝癌细胞浆内可表达MMP－1、2和9,但癌内阴性组5年生存率明显高于相应的阳性组（P＜0．05）。体外MMP－9mRNA在7721细胞表达明显高于RA－7721以及L－02细胞,而TIMP－2mRNA的表达与MMP－9相反,MMP－2mRNA在7721细胞中的表达仅略高于L－02细胞。结论：HCC组织内MMP－1和9的表达与患者的预后密切相关;癌细胞高表达MMP－9、低表达TIMP－2,可能是肝癌细胞浸润、转移的主要基础,而MMP-2并无重要作用。     

Melatonin inhibits IL1β-induced MMP9 expression and activity in human umbilical vein endothelial cells by suppressing NF-κB activation

Matrix metalloproteinases (MMPs) have been involved in inflammatory and degradative processes in pathologic conditions. The purpose of this study was to investigate the protective effect of melatonin in human umbilical vein endothelial cell (HUVEC) monolayer permeability and the regulation of MMP9 induced by interleukin 1β (IL1β (IL1B)) in HUVECs. Protection studies were carried out with melatonin, a well-known antioxidant and antiinflammatory molecule. MMP9 expression was increased with IL1β induction in HUVECs. Melatonin showed a barrier-protective role by downregulation of MMP9 and upregulation of tissue inhibitor of metalloproteinase-1 expression in HUVECs. Meanwhile, melatonin also decreased sodium fluorescein permeability and counteracted the downregulation of vascular endothelial cadherin and occludin expression in HUVECs. During inflammatory stimulus, nuclear factor-κB (NF-κB) plays a significant role in regulating MMP genes expression, thus the function of NF-κB in HUVECs' barrier disruption was investigated. IL1β induced nuclear translocation of NF-κB in HUVECs and regulated MMP9 expression. However, NF-κB translocation into the nucleus was inhibited significantly by melatonin. Our results show that melatonin decreases the permeability of monolayer endothelial cell induced by IL1β. At the same time, melatonin decreased the expression and activity of MMP9 by a NF-κB-dependent pathway in HUVECs induced by IL1β.     

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Objective

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen‐degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1‐MMP), in synovial pannus invasiveness.

Methods

The expression and localization of MT1‐MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1‐MMP was analyzed by 3‐dimensional (3‐D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP‐1), TIMP‐2, or GM6001. The effect of adenoviral expression of a dominant‐negative MT1‐MMP construct lacking a catalytic domain was also examined.

Results

MT1‐MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3‐D collagen matrix in an MT1‐MMP–dependent manner. Invasion was blocked by TIMP‐2 and GM6001 but not by TIMP‐1. Invasion was also inhibited by the overexpression of a dominant‐negative MT1‐MMP, which inhibits collagenolytic activity and proMMP‐2 activation by MT1‐MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1‐MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.

Conclusion

MT1‐MMP serves as an essential collagen‐degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1‐MMP–dependent invasion may represent a novel therapeutic strategy for RA.

     

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG– vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N– and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. J. L. Barth, Y. Yu and W. Song contributed equally to this paper.     

Nitric oxide regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-dependent and -independent pathways

Matrix metalloproteinases (MMPs) are of central importance in the proteolytic remodeling of matrix and the generation of biologically active molecules. MMPs are distinguished by a conserved catalytic domain containing a zinc ion, as well as a prodomain that regulates enzyme activation by modulation of a cysteine residue within that domain. Because nitric oxide (NO) and derived reactive nitrogen species target zinc ions and cysteine thiols, we assessed the ability of NO to regulate MMPs. A dose-dependent, biphasic regulatory effect of NO on the activity of MMPs (MMP-9, -1, and -13) secreted from murine macrophages was observed. Low exogenous NO perturbed MMP/tissue inhibitor of metalloproteinase (TIMP)-1 levels by enhancing MMP activity and suppressing the endogenous inhibitor TIMP-1. This was cGMP-dependent, as confirmed by the cGMP analog 8-bromo-cGMP, as well as by the NO-soluble guanylyl cyclase-cGMP signaling inhibitor thrombospondin-1. Exposure of purified latent MMP-9 to exogenous NO demonstrated a concentration-dependent activation and inactivation of the enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentrations similar to that of activated macrophages. Importantly, these results suggest that NO regulation of MMP-9 secreted from macrophages may occur chemically by reactive nitrogen species-mediated protein modification, biologically through soluble guanylyl-cyclase-dependent modulation of the MMP-9/TIMP-1 balance, or proteolytically through regulation of MMP-1 and -13, which can cleave the prodomain of MMP-9. Furthermore, when applied in a wound model, conditioned media exhibiting peak MMP activity increased vascular cell migration that was MMP-9-dependent, suggesting that MMP-9 is a key physiologic mediator of the effects of NO in this model.     

High levels of matrix metalloproteinases regulate proliferation and hormone secretion in pituitary cells

Beside the digestion of the extracellular matrix during tumor invasion and metastasis, more recently, new functions for matrix metalloproteinases (MMPs) have been proposed. We studied the expression and function of these enzymes in pituitary cells. We observed the activities of MMP-2 and MMP-9 together with expression of membrane-type MMP and tissue inhibitor of metalloproteinase-1 in all types of human pituitary adenomas. We found surprisingly high levels of MMP activity and low levels of tissue inhibitor of metalloproteinases, indicating a high level of extracellular matrix-degrading activity in pituitary adenomas. To examine the function of metalloproteinase activity in pituitary cells we used the synthetic MMP inhibitor batimastat. These studies demonstrate that MMPs secreted by pituitary cells can release growth factors anchored to the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. These results define a new additional mechanism for the control of pituitary hormone secretion and indicate new potential therapeutic targets for pituitary adenomas.