Robo3A and Robo3B expression is regulated via alternative promoters and mRNA stability.

BACKGROUND: The transmembrane receptor family Roundabout (Robo) was described to have an essential role in the developing nervous system. Recent studies demonstrated that Robo3 shows an altered expression in rheumatoid arthritis as well as in melanoma. CONTEXT AND PURPOSE OF THE STUDY: Until today no detailed studies of the two Robo3 isoforms (Robo3A and Robo3B) and their roles in rheumatoid arthritis synovial fibroblasts, respectively malignant melanoma are available. To get a better understanding regarding the role of Robo3A and Robo3B in the molecular process of rheumatoid arthritis and melanoma the exact characterization of expression and regulation is object of this study. RESULTS: mRNA and protein expression of the transcriptional variants were analyzed by quantitative RT-PCR respectively western blotting and revealed particularly enhanced expression of Robo3B in rheumatoid arthritis and melanoma. Promoter assays and inhibitor studies also disclosed that there is apparently a cell- and isoform-specific regulation of the Robo3 expression. Finally, dissimilar mRNA stabilities of Robo3A and Robo3B are identified as decisive posttranscriptional gene expression control. CONCLUSION: In summary, this study supported an isotype specific role of Robo3B in disease hinting to different functional roles of each isoform.

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.

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts.

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1ß lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3'UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts.

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1ß lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3'UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.

DCC is expressed in a CD166-positive subpopulation of chondrocytes in human osteoarthritic cartilage and modulates CRE activity.

OBJECTIVE: In a recent study we determined a strong differential expression of DCC in OA compared to normal chondrocytes and a strong impact of the DCC receptor on cellular mobility triggered by its ligand Netrin-1. Migration of chondrocytes or their progenitor cells may play a role in remodeling of cartilage and pathological conditions. The purpose of this study is to identify subsets of chondrocytes expressing DCC and to understand signaling pathways used by DCC in chondrocytes. METHODS: Immunofluorescent histology of human cartilage was used to determine the expression pattern of CD166, DCC and p-CREB. Cell culture of chondrocytes and SW1353, transient transfection, siRNA transfection, EMSA, luciferase assay, quantitative RT-PCR, ELISA, and Western Blotting were used to study signaling down-stream of DCC. RESULTS: DCC expressing chondrocytes are mainly located in the surface layers of OA cartilage. These also express CD166 indicating that DCC expressing chondrocytes are progenitor cells. Interestingly, expression of DCC reduces cAMP levels, CREB DNA-binding activity and CRE activity in chondrocytes, whereas down-regulation of DCC results in induction of CRE signaling. CONCLUSION: In summary, DCC is up-regulated in CD166-positive chondrogenic progenitor cells in OA and induces down-regulation of CREB. These findings indicate that migration of CD166 positive progenitor cells to sites of cartilage damage may be directed by regulation of DCC signaling.

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells.

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on "hanging drop" cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies.

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.