Peripheral blood monocytes will be obtained after the blood withdrawal and synovial fibroblasts after the mechanical and enzymatical modifications of the synovial tissue obtained during the joint surgery. Extracellular roles will be studied by stimulatory experiments with an additional inhibition of the RAGE and intracellular roles by means of gene silencing using small interfering (si) RNA. Nucleofector device Amaxa will be used for cell transfection. Gene expression of apoptotic molecules (bax, bcl-family, XIAP, smac), pro-inflammatory cytokines (TNF, IL-1, IL-6 and MCP-1) and matrix degrading enzymes (MMP-1, MMP-3 and MMP-13) will be analyzed using real-time PCR. The protein synthesis will be confirmed by means of ELISA and Western-blot. Activationof the programmed cell death pathway will be measured using fluorimetric Caspase-3 activity assay.

Periferní krevní monocyty budou získány stočením krve po krevním odběru a synoviální fibroblasty po mechanické a enzymatické úpravě synoviální tkáně získané během transplantace kloubu. Extracelulární působení bude studováno pomocí stimulačních experimentů s využitím inhibice receptoru RAGE a intracelulární úloha pomocí utlumení genové exprese za použití malého inhibitoru (si) RNA s využitím nukleofekčního přístroje Amaxa. Genová exprese apoptotických molekul (bax, bcl rodiny, XIAP, smac), prozánětlivýchcytokinů (TNF, IL-1, IL-6 a MCP-1) a matrix degradujících enzymů (MMP-1, MMP-3 a MMP-13) bude analyzována pomocí real-time PCR. Syntéza proteinů bude potvrzena pomocí metod ELISA a Western-blot. Aktivace dráhy vedoucí k programované smrti buněk bude analyzována pomocí fluorimetrické eseje měřící aktivitu kaspázy-3.

• MeSH
• apoptóza MeSH
• cytokiny MeSH
• malá interferující RNA MeSH
• matrixové metaloproteinasy MeSH
• polymerázová řetězová reakce MeSH
• proteiny S100 MeSH
• revmatoidní artritida patofyziologie   MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• revmatologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

The metastasis-associated protein S100A4 belongs to the large family of S100 calcium-binding proteins that appear to play regulatory roles in diverse biological activities. Moreover, a prognostic role of S100A4 has been suggested for patients with several types of cancer. Cancer promoting properties for S100A4 have been demonstrated, particularly through its regulation of cell motility, proliferation and apoptosis, as well as by stimulation of angiogenesis and remodelling of the extracellular matrix. Increased expression of S100A4 mRNA has been detected in proliferating synovial fibroblasts in rheumatoid arthritis. Furthermore, strong upregulation of the S100A4 protein in rheumatoid arthritis synovial tissue compared with osteoarthritis and control tissues has been demonstrated recently, especially at sites of joint invasion. Several immune and vascular cells were also identified to be producing S100A4 within the synovium. The local upregulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein existing in the bioactive oligomeric form in patients with rheumatoid arthritis. Consistent with data from cancer studies, the extracellular S100A4 oligomer appears to be involved in regulation of several matrix-degrading enzymes and modulation of the transcriptional activation function of the tumour suppressor protein p53 in rheumatoid arthritis synovial fibroblasts. Taken together, one can speculate that increased S100A4 protein in circulation and locally at sites of inflammation, particularly at sites of joint destruction, might be linked to the process of aggressive fibroblast behaviour contributing to the pathogenesis of chronic autoinflammatory diseases such as rheumatoid arthritis.

• MeSH
• fibroblasty metabolismus   MeSH
• financování organizované MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové proteiny fyziologie   MeSH
• proteiny S100 fyziologie   metabolismus   MeSH
• revmatoidní artritida metabolismus   patologie   MeSH
• synoviální membrána metabolismus   patologie   MeSH
• zánět metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

S100A4 patří do proteinové rodiny „S100“, která zahrnuje více než 25 nízkomolekulárních proteinů podílejících se na regulaci řady buněčných procesů. S100A4 se účastní regulace buněčné motility, proliferace, apoptózy, angiogeneze a remodelace extracelulární matrix. Funkce S100A4 je spojována s metastazováním a tento protein je navíc považován za významný prognostický marker některých nádorových onemocnění. Jeho přítomnost byla nově zjištěna také v proliferujících synoviálních fibroblastech u revmatoidní artritidy (RA). Zvýšená exprese S100A4 proteinu byla potvrzena také v synoviální tkáni pacientů s RA ve srovnání s kontrolní a artrotickou synoviální tkání. Protein byl v synoviální tkáni exprimován i v místech destrukce kloubní chrupavky. S100A4 je tvořen nejen synoviálními fibroblasty, ale i buňkami imunitního systému a endoteliálními buňkami cév. U RA byla zjištěna zvýšená koncentrace bioaktivního oligomeru S100A4 nejen lokálně v zánětlivé tkáni, ale také v plazmě a synoviální tekutině. U synoviálních fibroblastů byl podobně jako u tumorů pozorován vliv oligomeru S100A4 na produkci enzymů degradujících matrix – metaloproteináz a vliv na biologickou funkci tumor supresorového proteinu p53. Ze současných znalostí lze předpokládat, že zvýšené hladiny S100A4 mohou ovlivňovat agresivní chování fibroblastů a celkově tak mohou přispívat k patogenezi chronických autoimunitních zánětlivých onemocnění.

S100A4 protein belongs to the large group of calcium binding protein family that consists of more than 25 low-weight molecules and appears to play regulatory roles in diverse biological activities. The S100A4 protein participates in the regulation of cell motility, proliferation and apoptosis, angiogenesis and remodeling of extracellular matrix. Functional role of S100A4 is associated with metastasis and the protein is suggested as a prognostic marker for various types of cancers. Increased expression of S100A4 mRNA was recently detected in proliferating synovial fibroblasts in rheumatoid arthritis (RA). Up-regulation of S100A4 protein in RA synovial tissue compared with osteoarthritic and control tissues have been also demonstrated. Moreover, the protein has been found at the sites of joint invasion. S100A4 is produced not only by synovial fibroblasts but also by several immune and vascular cells. Increased amount of bioactive S100A4 oligomer was determined not only locally at the inflammatory synovial tissue but also in plasma and synovial fluid. Consistently with the data in cancer studies, extracellular S100A4 oligomer can stimulate production of several matrix degrading enzymes – metaloproteinases and modulate biological function of the tumor suppressor protein p53 in synovial fibroblasts. Taken together, one can speculate that increased S100A4 protein might be implicated in the process of aggressive fibroblast behavior contributing to the pathogenesis of chronic autoimmune inflammatory rheumatic diseases.

• MeSH
• biologické markery MeSH
• lidé MeSH
• proteiny S100 MeSH
• revmatoidní artritida patofyziologie   MeSH
• zánět parazitologie   MeSH
• Check Tag
• lidé MeSH

OBJECTIVE: To examine the involvement of the metastasis-inducing protein S100A4 (Mts-1) in the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue, synovial fluid, and plasma were obtained from RA and osteoarthritis (OA) patients who were undergoing joint surgery. Immunohistochemical and immunofluorescence analyses and enzyme-linked immunosorbent assays were used to determine the locations and concentrations of S100A4. The conformational structure of S100A4 in plasma and synovial fluid was determined after fractionation by size-exclusion chromatography, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis. Expression of various S100 proteins in RA synovium was determined by immunofluorescence and double-staining using specific anti-S100 antibodies. RESULTS: We found an up-regulation of S100A4 in cells infiltrating RA synovial tissue. Most cell types identified by cell-specific markers (fibroblasts, immune cells, and vascular cells) contributed to the production of S100A4 in RA synovial tissue. The pattern of S100A4 expression differed significantly from that of the proinflammatory proteins S100A9 and S100A12, which were restricted to phagocytes and granulocytes. The up-regulation of S100A4 in RA synovial tissue was consistent with the high concentrations of the protein in RA versus OA plasma (mean 1,100 versus 211 ng/ml) and synovial fluid (mean 1,980 versus 247 ng/ml). Moreover, we found that S100A4 in RA plasma and synovial fluid was present in bioactive multimeric (M-S100A4) conformations, whereas in OA, the majority of extracellular S100A4 was detected as the less active dimeric form. Consistent with our observations in tumor models, extracellular S100A4 stabilized the p53 tumor suppressor in RA synovial fibroblast-like cells and affected the regulation of p53 target genes, including Bcl-2, p21(WAF), and Hdm-2, as well as matrix metalloproteinases. CONCLUSION: Overexpression of S100A4 in RA synovial tissue and its release as M-S100A4 can influence p53 function and modulate the expression of several genes that are potentially implicated in the disease process. Thus, S100A4 might play an important role in the pathogenesis of RA and might represent a new target for the treatment of RA.

• MeSH
• apoptóza MeSH
• kalgranulin B genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• proteiny S100 fyziologie   genetika   chemie   metabolismus   MeSH
• revmatoidní artritida metabolismus   patofyziologie   MeSH
• studie případů a kontrol MeSH
• synoviální membrána metabolismus   patologie   MeSH
• upregulace * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

OBJECTIVES: To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients. METHODS: Plasma levels of the S100A4 protein were analysed in 40 anti-TNF-alpha naive patients with active RA. Of the 40 patients, 25 were treated with adalimumab and monitored over time. The conformational form of S100A4 was analysed using size-exclusion gel chromatography. TNF-alpha mRNA expression and protein synthesis were analysed by RT-PCR and ELISA, respectively. RESULTS: Baseline levels of S100A4 were significantly correlated with disease activity in RA patients (r = 0.41; P < 0.01). After 12 weeks of treatment with adalimumab, there was an obvious shift in the conformations of S100A4 from the multimeric to the dimeric forms, whereas the total levels of the S100A4 protein remained unchanged. This suggests that the bioactive (multimer) S100A4 may decline in response to successful treatment with adalimumab. In addition, we showed significant up-regulation of TNF-alpha mRNA (P < 0.01), and protein release to the cell culture medium of monocytes stimulated with the S100A4 multimer compared with those treated with the dimer and to the unstimulated monocytes (P < 0.001). CONCLUSIONS: This is the first study to show that the levels of the S100A4 protein are correlated with RA disease activity. Furthermore, only the bioactive form, but not the total amount of S100A4, decreases after successful TNF-alpha blocking therapy in patients with RA. These data support an important role for the S100A4 multimer in the pathogenesis of RA.

• MeSH
• antirevmatika terapeutické užití   MeSH
• biologické markery krev   MeSH
• dospělí MeSH
• humanizované monoklonální protilátky MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• monocyty imunologie   účinky léků   MeSH
• monoklonální protilátky terapeutické užití   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• proteiny S100 farmakologie   krev   MeSH
• revmatoidní artritida farmakoterapie   krev   MeSH
• stupeň závažnosti nemoci MeSH
• TNF-alfa antagonisté a inhibitory   biosyntéza   genetika   MeSH
• upregulace imunologie   účinky léků   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH