Abstract
IntroductionThe 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).MethodsmRNA 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.ResultsFirst, 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.ConclusionsTaken 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.
Highlights
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
Robo and Slit expression in rheumatoid arthritis synovial fibroblasts (RASF) compared with normal synovial fibroblast (SF) First, we characterized the expression pattern of the receptors Robo1, Robo2, Robo3, and their ligands Slit1, Slit2, and Slit3 in RASF compared with normal SF in early passages (P3 and P4; Figure 1)
Robo1 and Robo2 were almost expressed in normal SF and RASF, whereas Robo3 mRNA was strongly enhanced in RASF compared with SF of healthy donors
Summary
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. 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). As loss of integrity of compartment borders in the joint between cartilage and SF is a key event in RA, we were interested in analyzing molecular cues, such as the repellent factors, that might be deregulated in RA patients and thereby contribute to destruction of joint borders. Repellent factors of the Roundabout (Robo)- and Slitfamily are primarily known to be involved in regulating cell-cell interactions and cell-matrix interactions of migrating cells during embryonic development [4] and by mediating axon guidance through attraction or repulsion of growth cones [5,6,7]. The Robo-/Slit-system has been described to mediate cell adhesion of fibroblasts [8] and to induce tumor angiogenesis [6]
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