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Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis.

ABSTRACT: INTRODUCTION: The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. However, until today no data are available on whether these repellent factors are involved in the regulation of synovial fibroblast (SF) activity in rheumatoid arthritis (RA). METHODS: mRNA expression in primary synovial fibroblasts was quantified by quantitative reverse transcription PCR and protein expression was measured by fluorescence activated cell sorting (FACS) analysis. Different functional assays were performed with rheumatoid arthritis synovial fibroblasts (RASF): proliferation, migration and a novel in-vitro cartilage destruction assay. RESULTS: First, we found increased expression of Robo3 expression in RASF compared to normal SF. Interestingly, analysis of data from a recently published genome-wide association study suggests a contribution of ROBO3 gene polymorphisms to susceptibility of RA. Functional assays performed with RASF revealed induction of migration and cartilage destruction by Robo3 and increased matrix metalloproteinase (MMP)1 and MMP3 expression. Treatment of RASF in early passages with Slit3 led to inhibition of migration whereas RASF in later passages, having reduced Robo3 expression in cell culture, were not inhibited by Slit3 treatment. Here, reduction of Robo3 expression from passage 3 to 10 might reflect an important step in losing repulsive activity of Slit3. CONCLUSIONS: Taken together, our data showed that deregulation of the Robo3 receptor in synovial fibroblasts in RA correlates with aggressiveness of the fibroblasts. Slit3 reduces the migratory activity of synovial cells from patients with RA, potentially by repulsion of the cells in analogy to the neuronal system. Further studies will be necessary to prove Slit activity in vivo.

SUBMITTER: Denk AE

PROVIDER: S-EPMC2888193 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis.

Denk Alexandra E AE Kaufmann Simone S Stark Klaus K Schedel Jörg J Lowin Torsten T Schubert Thomas T Bosserhoff Anja K AK

Arthritis research & therapy 20100318 2

<h4>Introduction</h4>The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. However, until today no data are available on whether these repellent factors are involved in the regulation of synovial fibroblast (SF) activity in rheumatoid arthritis (RA).<h4>Methods</h4>mRNA expression in primary synovial fibroblasts was quantified by quantitative reverse transcription PCR and protei ...[more]

PMID: 20298552

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Project description:BackgroundAntcin K, an extract of Antrodia cinnamomea (a medicinal mushroom endemic to Taiwan commonly used in Chinese medicine preparations), inhibits proinflammatory cytokine production and angiogenesis in human rheumatoid arthritis synovial fibroblasts (RASFs), major players in RA disease. Antcin K also inhibits disease activity in mice with collagen-induced arthritis (CIA). Up until now, the effects of Antcin K upon cell adhesion molecules (CAMs) were unknown.MethodsRA and healthy synovial tissue samples (n = 10 in each group) were retrieved from the Gene Expression Omnibus (GEO) database (accession code: GDS5401) to compare CAM and monocyte marker expressions. In addition, synovial tissue samples from six RA patients and six patients undergoing arthroscopy for trauma/joint derangement (healthy controls) were subjected to immunohistochemical (IHC) analysis. mRNA and protein expression levels were analyzed in RASFs using RT-qPCR (Reverse transcription-quantitative polymerase chain reaction) and Western blot. RASFs were incubated with Antcin K and examined for monocyte adherence by fluorescence microscopy. Ankle joint tissue specimens from a CIA mouse model and healthy controls were stained with hematoxylin and eosin (H&E) and Safranin-O/Fast Green to examine histological changes and evidence of bone loss. IHC analysis determined levels of vascular cell adhesion molecule 1 (VCAM-1) and CD11b in CIA ankle tissue and clinical synovial tissue.ResultsLevels of VCAM-1 expression were higher in the GEO database specimens and the study's clinical samples of RA synovial tissue compared with the healthy specimens. Antcin K dose-dependently inhibited VCAM-1 expression and monocyte adhesion in RASFs. Antcin K also significantly inhibited levels of VCAM-1 and monocyte CD11b expression in CIA tissue. These effects appeared to be mediated by MEK1/2-ERK, p38, and AP-1 signaling.ConclusionsAntcin K seems promising for the treatment of RA and deserves further investigations.

Project description:Management of pain in the treatment of rheumatoid arthritis (RA) is a priority that is not fully addressed by the conventional therapies. In the present study, we evaluated the efficacy of cannabinoid receptor 2 (CB2) agonist JWH-015 using RA synovial fibroblasts (RASFs) obtained from patients diagnosed with RA and in a rat adjuvant-induced arthritis (AIA) model of RA. Pretreatment of human RASFs with JWH-015 (10-20 μM) markedly inhibited the ability of pro-inflammatory cytokine interleukin-1β (IL-1β) to induce production of IL-6 and IL-8 and cellular expression of inflammatory cyclooxygenase-2 (COX-2). JWH-015 was effective in reducing IL-1β-induced phosphorylation of TAK1 (Thr184/187) and JNK/SAPK in human RASFs. While the knockdown of CB2 in RASFs using siRNA method reduced IL-1β-induced inflammation, JWH-015 was still effective in eliciting its anti-inflammatory effects despite the absence of CB2, suggesting the role of non-canonical or an off-target receptor. Computational studies using molecular docking and molecular dynamics simulations showed that JWH-105 favorably binds to glucocorticoid receptor (GR) with the binding pose and interactions similar to its well-known ligand dexamethasone. Furthermore, knockdown of GR using siRNA abrogated JWH-015's ability to reduce IL-1β-induced IL-6 and IL-8 production. In vivo, administration of JWH-015 (5 mg/kg, daily i.p. for 7 days at the onset of arthritis) significantly ameliorated AIA in rats. Pain assessment studies using von Frey method showed a marked antinociception in AIA rats treated with JWH-015. In addition, JWH-015 treatment inhibited bone destruction as evident from micro-CT scanning and bone analysis on the harvested joints and modulated serum RANKL and OPG levels. Overall, our findings suggest that CB2 agonist JWH-015 elicits anti-inflammatory effects partly through GR. This compound could further be tested as an adjunct therapy for the management of pain and tissue destruction as a non-opioid for RA.

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Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis.

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Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis.

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