N-cadherin promoter polymorphisms and risk of osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis. It is characterized by cartilage destruction and bone remodeling, mediated in part by synovial fibroblasts (SFs). Given the functional significance of cadherins in these cells, we aimed at determining the role of genetic variants of N-cadherin (CDH2) in OA of the knee and hip. Six single-nucleotide polymorphisms in the genomic region of the CDH2 gene were genotyped in 312 patients with OA and 259 healthy control subjects. Gene expression of CDH2 was analyzed by qRT-PCR. Liquid chromatography-mass spectrometry was used to identify a transcription factor isolated by DNA pulldown. Its potential for binding to gene variants was examined by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation. Genetic analysis identified a polymorphism located in the CDH2 promoter region to be associated with risk of OA. The minor allele of rs11564299 had a protective effect against OA. Compared to carriers of the major allele, carriers of the minor allele of rs11564299 displayed increased N-cadherin levels in SFs. Based on in silico analysis, the minor allele was predicted to generate a novel transcription factor binding site, Direct-binding assays and mass spectrometric analysis identified hnRNP K as binding selectively to the minor allele. In summary, a CDH2 promoter polymorphism influences the risk of OA, and hnRNP K was found to be involved in the regulation of elevated N-cadherin expression in patients with OA carrying the minor allele of rs11564299.

Role of deleted in colon carcinoma in osteoarthritis and in chondrocyte migration.

OBJECTIVE: The concept of the chondrocyte as a stationary cell surrounded by an apparently impenetrable matrix has been challenged by in vitro observations in recent years. Chondrocyte migration may have a role in remodelling of the cartilage and pathological conditions. Candidate molecules are repellent factors for the regulation of chondrocyte migration, which are expressed in fetal and adult cartilage. We analysed the potential role of the receptor deleted in colon carcinoma (DCC) in chondrocytes, as this may exert attractive activities. METHODS: Gene expression was determined by quantitative RT-PCR and immunohistochemistry, and gene regulation by electro mobility shift assay and chromatin immunoprecipitation. Functional assays on migration and differentiation were done after cell treatment and transfection. RESULTS: DCC was shown to be specifically up-regulated in OA compared with normal chondrocytes in vitro and in vivo. Promoter analysis and transfection studies showed that the up-regulation of DCC in OA chondrocytes may be mediated by the transcription factors Sox9 and AP-2. Netrin-1, the ligand of DCC, was revealed to induce the migration of OA chondrocytes specifically. Expression of DCC in healthy chondrocytes by transient transfection significantly induced cell migration and chemotaxis to Netrin-1. DCC expression had no influence on cell differentiation; however, induction of MMP1 and -3 expression was observed. CONCLUSION: Strong differential expression of DCC in OA compared with normal chondrocytes hints of a possible role of DCC in the pathophysiology of OA. The strong impact of the DCC receptor on cellular mobility of chondrocytes in vitro suggests a major relevance of migratory activities in physiological and pathological conditions of cartilage. However, definite proof of chondrocyte movements in vivo still has to be established.

Fragments of SLIT3 inhibit cellular migration.

The repellent factor family of Slit molecules has been described as having a repulsive function in the developing nervous system on growing axons expressing the Roundabout (Robo) receptors. Recent studies determined the effects of Slit molecules on the migratory and invasive potential of several types of tumor cells but also on synovial fibroblasts (SFs) derived from rheumatoid arthritis (RA) patients. To optimize a potential therapeutic application we aimed at generatingfragments of Slit3 showing the same functional ability as the full-length molecule but having the advantage of a smaller size. Recombinant Slit3 proteins were expressed and analyzed by western blotting. Their activity was defined by functional assays such as migration assays with RASF and melanoma cells. Recombinant Slit3 containing only leucine rich repeat domain 2 (D2), the domain important for Robo binding and the minimal functional unit D2 dNC were both able to inhibit migration of RASFs as effectively as Slit3 with all 4 repeats. Collectively, our data showed that the ability of Slit3 to reduce the migratory activity of synovial cells from patients with RA and melanoma cells can be mimicked by small protein fragments derived from Slit3. Slit3 fragments may be helpful in therapeutic attempts; however, further studies are necessary in order to elucidate their activity in vivo.

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

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.

Functional implication of Netrin expression in malignant melanoma.

BACKGROUND: Malignant melanoma cells are known to have altered expression of genes supporting proliferation and invasion, however, the expression of molecules of the Netrin family of repellent factors has not been analyzed in melanomas until now. RESULTS: Here, we show that Netrin-1 expression is strongly induced in melanoma cells compared to melanocytes in vivo and in vitro controlled at the transcriptional level via ETS-1. In addition, the expression of the netrin receptor UNC5B was induced and that of UNC5C was reduced in the tumor cells. In order to determine the functional relevance of Netrin-1 expression in malignant melanoma, Netrin expression in melanoma cells was reduced by siRNA attempts and primary human melanocytes were treated with recombinant Netrin-1. The cells showed no changes in proliferation or apoptosis, however, a strong reduction of migratory properties was observed in the melanoma cells after reduction of Netrin expression whereas melanocyte migration was strongly induced by treatment with Netrin. CONCLUSION: Our study suggests that Netrin-1 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.