The active metabolite of leflunomide,A77 1726, inhibits the production of prostaglandin E(2), matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes

OBJECTIVES: To investigate the effects of the active metabolite of leflunomide, A77 1726, on fibroblast-like synoviocytes. In rheumatoid arthritis (RA) synoviocytes participate in tissue destruction by producing metalloproteinases (MMP), prostaglandin E(2) (PGE(2)) and interleukin (IL) 6, which are involved in extracellular matrix degradation, resorption of the mineral phase and osteoclast-mediated bone resorption. METHODS: Human synoviocytes were stimulated with IL-1alpha or tumour necrosis factor alpha (TNF-alpha) in the presence of A77 1726. Culture supernatants were analysed for production of interstitial collagenase (MMP-1), tissue-inhibitor of metalloproteinases 1 (TIMP-1), PGE(2) and IL-6. Total RNA was isolated and analysed for steady-state levels of MMP-1, cyclooxygenase-2 (COX-2) and IL-6 mRNA. RESULTS: A77 1726 inhibited the production of PGE(2) in synoviocytes activated by TNF-alpha and IL-1alpha with median inhibitory concentrations (IC(50)) of 7 and 3 microM respectively. In contrast, MMP-1 and IL-6 production was inhibited at high A77 1726 concentrations (> 10 microM), whereas TIMP-1 was not affected. The inhibition of MMP-1 and IL-6 production was due to the known inhibitory effect of A77 1726 on pyrimidine synthesis, as it was reversed by the addition of uridine. This did not apply to PGE(2) production, which was inhibited via direct action of A77 1726 on COX-2, as shown by the increasing amount of substrate (arachidonic acid) in the culture medium. CONCLUSION: This study shows that some of the beneficial effect of leflunomide in RA patients may be due to the inhibition of PGE(2), IL-6 and MMP-1 production in synoviocytes. This effect, coupled with its multiple inhibitory effects on T lymphocyte functions, might account for the significant reduction in the rate of disease progression in RA patients treated with leflunomide.     

Joint damage and inflammation in c-Jun N-terminal kinase 2 knockout mice with passive murine collagen-induced arthritis

OBJECTIVE: Previous studies have demonstrated that inhibition of c-Jun N-terminal kinase (JNK) decreases joint destruction in the rat adjuvant arthritis model. The present study was undertaken to investigate whether selective loss of JNK-2 function decreases joint destruction in JNK-2 knockout mice, in order to determine the role of this isoform in inflammatory arthritis. METHODS: Passive collagen-induced arthritis (CIA) was induced in Jnk2(-/-) and wild-type mice by administering anti-type II collagen antibodies. Arthritis was assessed daily using a semiquantitative clinical scoring system. Fibroblast-like synoviocytes (FLS) were prepared from Jnk2(-/-) and wild-type mice, and JNK protein expression was determined by Western blot analysis. Matrix metalloproteinase 13 (MMP-13) expression was determined by Northern blot analysis, and activator protein 1 (AP-1) binding activity by electromobility shift assay (EMSA). RESULTS: The JNK protein level in Jnk2(-/-) mice with CIA was 22% of that in wild-type mice with CIA (P < 0.001), and mainly the 46-kd isoform was expressed in the former group. Surprisingly, clinical arthritis was slightly more severe in the Jnk2(-/-) mice. Histologic scores for synovial inflammation were not significantly different. However, Safranin O-stained sections from the Jnk2(-/-) mice exhibited significantly less joint damage. Although joint destruction was decreased in Jnk2(-/-) mice with CIA, EMSA and Northern blot analysis of total joint extracts revealed similar levels of AP-1 binding and MMP-13 expression in Jnk2(-/-) and wild-type mice. The lack of correlation with AP-1 activity and MMP expression was probably because non-FLS cells in the joint may express more JNK-1 than do FLS. CONCLUSION: JNK-2 is a determinant of matrix degradation, but it has little effect on inflammation in arthritis. Complete inhibition of MMP expression and joint destruction will likely require combined JNK-1 and JNK-2 inhibition.     

Sinomenine reduces invasion and migration ability in fibroblast-like synoviocytes cells co-cultured with activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, CD147

CD147 expressed by monocytes, macrophages, and synoviocytes cells can stimulate the production of matrix metalloproteinases (MMPs) associated with the development of rheumatoid arthritis (RA). We investigated the effects of Sinomenine (SIN) on invasion and migration ability and gene expression of CD147, MMP-2, MMP-9 of fibroblast-like synoviocytes cells (FLS) co-cultured with activated human monocytic THP-1 cells (A-THP-1) in vitro. SIN is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. FLS cells were co-cultured with THP-1 cells which were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. Invasion and migration ability of cells was tested by transwell assays. Western blot analysis and zymographic analysis were adopted to detect the expression of CD147 and MMPs, respectively. RT–PCR was used to determine the expression of mRNA of CD147, MMP-2, and MMP-9. The invasion and migration ability of the co-cultured cells was significantly inhibited by SIN in a concentration-dependent fashion, and at the same time, the levels of CD147, MMP-2, MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 and 1.00 mM (P < 0.01). Our results point to a possible mechanism of SIN on treatment of RA is the inhibitory effect of SIN on cell invasion and migration ability, which strongly correlates with repressing the expression of CD147, MMP-2, and MMP-9.     

Rhein, a diacerhein-derived metabolite, modulates the expression of matrix degrading enzymes and the cell proliferation of articular chondrocytes by inhibiting ERK and JNK-AP-1 dependent pathways

OBJECTIVE: To determine the effects of rhein on the expression of matrix metalloproteinases (MMP-1, -3, 13) and ADAMTs 4, 5 (a disintegrin and metalloproteinase with thrombospondin type-I repeat)/aggrecanases-1, -2 in interleukin-1-stimulated bovine articular chondrocytes, and to investigate the signalling pathways involved in the effects of the drug on gene expression and cell proliferation. METHODS: Bovine chondrocytes were treated with 10(-4) M rhein for 18 h, followed by 10 ng/ml IL-1Beta for 30 min (cytoplasmic extracts) or 24 h (RNA extraction and EMSA). mRNA was assessed by RT-PCR for the expression of MMPs and aggrecanases, and the phosphorylation of MAP kinases was studied by Western blotting. NF-kappaB and AP-1 DNA binding were determined by gel retardation assay. The effects of inhibitors of these signalling pathways were compared to those of rhein. The proliferation of human chondrocytes and synoviocytes treated with the drug was also investigated. RESULTS: IL-1Beta-induced stimulation of the MMPs and aggrecanase-1 was markedly inhibited by rhein. The drug reduced IL-1Beta-induced NF-kappaB and AP-1 DNA binding, as well as the phosphorylation of ERK and JNK. Similar effects were produced by the specific inhibitors of these signalling pathways. In addition, rhein reduced the proliferation of both human chondrocytes and synoviocytes. CONCLUSION: Our data indicate that rhein may reduce the deleterious effects of IL-1Beta on osteoarthritic cartilage through its effects on the ERK- and JNK-dependent pathways. Both its anti-catabolic and anti-proliferative properties may explain its value in the treatment of joint diseases.     

Estrogen and progesterone regulation of human fibroblast-like synoviocyte function in vitro: implications in rheumatoid arthritis

OBJECTIVE: Despite increasing evidence regarding the significance of sex hormones in rheumatoid arthritis (RA), their etiopathological role and potential longterm effect on joint destruction remain unclear. We hypothesized that estrogen receptors (ER-alpha) are present in fibroblast-like synoviocytes, and 17beta-estradiol can modulate the production and activity of matrix degrading enzymes produced by these cells. Thus, depending on the endocrine balance, fibroblast-like synoviocyte activity can be suppressed or enhanced, leading to amelioration or exacerbation of the disease process, respectively. METHODS: By utilizing an in vitro cartilage invasion model, in combination with the molecular analyses of hormone receptors, matrix metalloproteinases (MMP) and their respective inhibitors, we investigated the effect of hormones (i.e., estrogen and progesterone) on fibroblast-like synoviocyte phenotypic changes, with particular emphasis on their functional interactions with cartilage. RESULTS: Our studies reveal the presence of functional ER-alpha in fibroblast-like synoviocytes. The findings indicate that estrogen exerts a stimulatory effect, while progesterone has an inhibitory effect on the expression of MMP, their tissue inhibitors (TIMP), and enzymatic activity of MMP produced by these cells. Furthermore, transfection of fibroblast-like synoviocytes with the ER-alpha gene resulted in the increased degradation and invasion of cartilage. CONCLUSION: We identified the presence of functional ER-alpha in fibroblast-like synoviocytes. This renders fibroblast-like synoviocytes as target cells for hormonal regulation. The regulatory effect of estrogen is partly targeted to the MMP and their respective inhibitors associated with fibroblast-like synoviocytes. Such studies provide a link between hormonal status and disease activity in RA and open new venues for future therapeutic intervention to combat this debilitating disease.     

Expression and significance of MMP-7, c-Jun and c-Fos in rats skin photoaging

ObjectiveTo investigate the expression and significance of the MMP-7, c-Jun and c-Fos in rat photoaging skin.MethodsA total of 45 SD rats were randomly divided into control group, model group, natural recovery group, physiological saline injection group and dermal pluripotent stem cells transplantation (DMSCs group), model group, natural recovery group, physiological saline injection group. DMSCs were treated with UV lamp irradiation to establish light aging skin model. Rats were then sacrificed after model prepared, no treatment was processed in the natural recovery group. Saline injections was adopted in saline group, DESCs group was treated with DESCs transplantation. Rats were sacrificed after 4 weeks. The expression of MMP-7, c-Jun and c-Fos were detected using the immunohistochemical method.ResultsIn model group, MMP 7 positive expression was higher than that in the other 4 groups, but without statistically difference (P>0.05); c-Jun, c-Fos expression were higher than that in the control group and DESCs group (P<0.05), there was no significant difference comparing natural recovery group with physiological saline injection group (P>0.05).ConclusionsMMP-7, c-Jun and c-Fos can be used as diagnosis indicators in the early stage of light aging, and they jointly participate in its development. DMSCs transplants is effective in treating light aging skin.     

Role of fibroblast growth factor-2 in the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human intestinal myofibroblasts

BACKGROUND AND AIMS: The coordinated expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) plays a crucial role in tissue remodeling. We investigated the effects of fibroblast growth factor (FGF)-2 on the secretion of MMPs and TIMPs in human intestinal subepithelial myofibroblasts (SEMFs). METHODS: The secretion of MMP-s and TIMPs was determined by ELISA or Western blotting. The mRNA expression of MMPs and TIMPs was assessed by Northern blotting. The activating protein (AP)-1-DNA binding activity was evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: Unstimulated intestinal SEMFs constitutively secreted MMP-2 and TIMP-2. FGF-2 stimulated MMP-1, MMP-3 and TIMP-1 secretion, but did not affect MMP-2 or TIMP-2 secretion. FGF-2 induced AP-1-DNA binding activity, and the c-Jun/AP-1 inhibitor curcumin attenuated the FGF-2-induced MMP-1, -3 and TIMP-1 mRNA expression. Mitogen-activated protein (MAP) kinase inhibitors (U0126 and PD098059) also blocked the MMP-1, -3 and TIMP-1 secretion. Furthermore, FGF-2 dose-dependently induced FGF-2 mRNA expression in these cells. CONCLUSIONS: FGF-2 may be one of important regulatory factors for extracellular matrix turnover via a modulation of MMP and TIMP secretion from SEMFs.     

AP-1 and heat shock protein 27 expression in human astrocytomas

PURPOSE: In previous work we have shown that the expression of heat shock protein 27 (Hsp27) is associated with anaplastic potential of astrocytomas (Anticancer Res 1997, 17:2677-2682). Heat shock protein-coding genes have been found to have a putative AP-1 (activator protein-1)-binding site in their promoter region and the synthesis of these proteins is induced by the same extracellular stimuli that also activate AP-1 (a homo/heterodimer of members of the Jun and Fos families). In order to detect the putative relation of Hsp27 with AP-1 activation in human astrocytomas we examined eighty astrocytic tumors with different grades of malignancy for c-Jun, c-Fos, and Hsp27 expression. METHODS: Six pilocytic astrocytomas (WHO grade I), 15 diffuse fibrillary astrocytomas (WHO grade II), 19 anaplastic astrocytomas (WHO grade III), and 40 glioblastomas multiforme (WHO grade IV), were studied by immunohistochemistry using monoclonal and polyclonal antibodies directed against human Hsp27, c-Fos, and active (phosphorylated) forms of c-Jun (p-c-Jun). Monoclonal antibodies against the phosphorylated forms of the over-expressed MAP kinases JNK (c-Jun N-terminal kinase) (p-JNK) and p38 (p-p38) were also used. RESULTS: Overexpression of p-c-Jun, c-Fos and p-JNK was observed in the majority of glioblastomas (grade IV) whereas only minimal expression was noted in diffuse fibrillary astrocytomas (grade II). Hsp27 expression was observed only in the tumor specimens where c-Jun and c-Fos were co-expressed. AP-1/Hsp27 co-expression was associated with ascending grading of malignancy and it was independent of the proliferation index of the tumors. CONCLUSIONS: Our findings suggest that during malignant progression of astrocytomas there is activation of signal transduction cascade(s) culminating in AP-1 induction.     

Cordycepin inhibits IL-1{beta}-induced MMP-1 and MMP-3 expression in rheumatoid arthritis synovial fibroblasts

Objective. MMP is a key enzyme in the degradation of extracellularmatrices, and its expression plays important roles in inflammatorydiseases. Cordycepin (3'-deoxyadenosine), a bioactive compoundof Cordyceps militaris, has been shown to exhibit many pharmacologicalactivities, such as anti-cancer, anti-inflammatory and anti-infectionactivities. In this study, we aimed at the inhibitory effectof cordycepin on IL-1β-induced MMP-1 and MMP-3 expressionas well as the molecular basis using RA synovial fibroblasts(RASFs). Methods. RASFs were isolated from synovial tissue obtained from12 patients with RA and cultured in monolayer. Expression ofMMP-1 and MMP-3 was evaluated using western blotting and real-timePCR. Chemokines were analysed by ELISA. The phosphorylationof mitogen-activated protein kinase was measured by westernblotting. Electrophoretic mobility shift assay was performedto evaluate binding activities of DNA to nuclear factor-B (NF-B)and activator protein-1 (AP-1). Results. Cordycepin inhibited IL-1β-induced MMP-1 and MMP-3expressions in RASFs in a dose-dependent manner. Among variouschemokines [such as monocyte chemoattractant protein-1 (MCP-1),GRO-, regulated upon activation, normal T-cell expressed andpresumably secreted (RANTES) and epithelial neutrophil activatingpeptide 78 (ENA-78)], cordycepin specifically blocked IL-1β-inducedENA-78 production in RASF. Moreover, cordycepin significantlyinhibited IL-1β-induced p38/JNK and AP-1 activation, butnot extracellular signal-regulated kinase (ERK) and NF-B activation. Conclusions. Cordycepin is a potent inhibitor of IL-1β-inducedchemokine production and MMP expression and strongly blocksthe p38/JNK/AP-1 signalling pathway in RASFs. KEY WORDS: Cordycepin, Interleukin-1β, Matrix metalloproteinase, p38 mitogen-activated protein kinase, Activator protein-1