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.

Figure 1

Robo and Slit expression in RASF. The amount of Robo1, Robo2, Robo3, Slit1, Slit2 and Slit3 mRNA expression in rheumatoid arthritis synovial fibroblasts (RASF; n = 5) compared with normal SF (n = 2) in early passages (P3 and P4) was quantified by real-time (RT)-PCR (logarithmic scaling). Robo1 and Robo2 are both expressed in normal SF and RASF, whereas Robo3 is much stronger expressed in RASF compared with normal SF. Slit1, Slit2, and Slit3 are all expressed in normal SF and RASF, with significant reduction of Slit3 expression in RASF (* P < 0.05).

Figure 2

Effect of Robo3 on RASF migration and aggressiveness. (a) Transfection of RASF cells (late passage) with an expression plasmid for Robo3 increased migration of rheumatoid arthritis synovial fibroblasts (RASF; n = 4) in the Boyden Chamber model to about 220% compared with control (* P < 0.05). (b) In vitro assays on cartilage destruction revealed significant induction of free glycosaminoglycans after treatment with Robo3-transfected RASF (late passages; n = 3) compared with mock-transfected cells (** P < 0.01). (c) Normal skin fibroblasts (n = 2) expressing Robo3 after transfection resulted in the release of significantly more free glycosaminoglycans than control transfected cells (* P < 0.05). (d) Matrix metalloproteinase (MMP) 1 (n = 3) and MMP3 (n = 4) mRNA expression was quantified by real-time (RT)-PCR, showing significant induction of MMP1 (* P < 0.05) and high induction of MMP-3 in mRobo3 transfected RASF (late passages) compared with control. (e) MMP1 (n = 3) and MMP3 (n = 4) protein expression from the supernatant released from SF in the GAG assay was quantified via ELISA, showing high induction of MMP-1 and of MMP3 in mRobo3 transfected RASF (late passages) compared to control (* P < 0.05).

Figure 3

Functional analysis of Slit3 effects on RASF. (a) Doubling times of rheumatoid arthritis synovial fibroblasts (RASF; n = 2) were obtained from proliferation assays. RASF incubated with mSlit3 showed decreased growth compared to untreated control (ns, not significant). (b) Recombinant mouse Slit3 (0.1 μg/ml) decreased migration of RASF in early passages (P3 and P4; n = 4) to about 55% compared with control both in upper and lower chamber (** P < 0.01). (c) In later passages (P5 and P6), Slit3 treatment has lost its inhibitory effect on migration of RASF compared with control (n = 4). (d) Slit3 incubation (0.1 μg/ml) had no effect on migration of normal SF (n = 2) in early passages (P3 and P4) compared with untreated control. (e) Relative comparison of migratory ability revealed high induction in RASF compared with normal SF (* P < 0.05).

Figure 4

Robo and Slit expression in RASF in three different passages. (a) Robo1, Robo2, Robo3, Slit1, Slit2, and Slit3 mRNA expression in rheumatoid arthritis synovial fibroblasts (RASF; n = 5) was quantified by real-time (RT)-PCR starting from passage 3 (P3) over passage 5 (P5) to passage 10 (P10; logarithmic scale). Robo1 and Robo2 expression decrease slightly in RASF over the passages. Robo3 mRNA levels dropped dramatically in P5 and P10 compared with P3. No considerable differences in mRNA expression were measured with Slit1, Slit2, and Slit3 over the passages. (b) Protein expression of Robo3 in RASF of P3 and P5 was analyzed by FACS using a Robo3 specific antibody. RASF in early passages showed a shift (I) compared with negative control, whereas cells of later passages (II) did not reveal Robo3 expression. (c) In vitro assays on cartilage destruction revealed significant reduction of free glycosaminoglycans (GAGs) after coculture with RASF of late passages compared with early passages.