Role of Slit2 Expression in Orbital Fibroblast Migration in Graves’ Orbitopathy

Joanna Ma

Introduction. Graves’ disease (GD) is an autoimmune hyperthyroidism disorder that manifests in 25% of patients with inflammation of extraocular tissue, known as Graves’ orbitopathy (GO).1 Aside from the cosmetic distress due to exophthalmos, more severe progressions of GO can lead to permanent vision loss.1,2 The change in tissue volume is due to increased orbital fibroblast (OF) proliferation, which is normally modulated by Slit2, a key signal in normal tissues that prevents monocyte differentiation to fibrocytes.3 TSH receptor antibody (TRAb) is a diagnostic marker for GO, and the AIRE gene is implicated in expression of thyroid proteins by OF.4  A study demonstrated Slit2 is a TSH-inducible regulator of circulating fibrocytes that downregulates promoters of AIRE and thyroid proteins.4 Other studies have elucidated the role of Slit2 in cell migration via modulation of beta-catenin and small GTPases.5,6 These findings suggest modulating Slit2 as a potential therapy for GO. Methods. To determine Slit2 expression in fibrocytes, fibrocytes were cultured from peripheral blood mononuclear cells.4 Slit2 knockdown was conducted via transfecting OF with siRNA.4 Conditioned media were generated by incubating CD34-, CD34+, and mixed OF for 48h, then immune adsorbed with rabbit anti-Slit2 Ab.4 Media were added to fibrocyte cultures and incubated for 3d and analyzed via RT-PCR.4 To determine the role of Slit2 in cell migration, HeLa cells were transfected with anti-Slit2 siRNA.5 Yeast two-hybrid assays were used to assess Arl4A interactions with Robo1.5 Western blotting was used to detect bound beta-catenin proteins.6 Dual luciferase reporter assay was used to determine promoter activity of Slit2 mRNA.6  Results. Levels of AIRE, thyroglobulin, TSHR, and MHC2 are lower in mixed CD34+/CD34- than in circulating CD34+ fibrocytes.4 Residential CD34- OF generate Slit2, attenuating protein expression in CD34+ and modulating induction of inflammatory factors by TSH.4 Slit2 overexpression inhibits transcription of beta-catenin by increasing the beta-catenin/E-cadherin complex, which inactivates beta-catenin.6  Arl4A directly interacts with Robo1, a Slit2 receptor, in a GTP-dependent manner and promotes cell migration by activating Cdc42.5 Binding of Slit2 to Robo1 decreases the interaction between Arl4a and Robo1, promotes association of srGAP1 and Robo1, and decreases activation of Cdc42.5 Conclusions. Modulating Slit2 is pertinent as a potential therapy for GO because of its direct relationship with fibrocyte differentiation.The ability to successfully modulate Slit2 expression in other specific cell types in humans would have major implications in the treatment of diseases that include abnormal cell migration as part of the pathogenesis.

  1. Smith, T. J., & Hegedus, L. (2016). Graves’ Disease. New England Journal of Medicine,375:1552-1565. doi:10.1056/NEJMra1510030
  2. Zahir-Jouzdani, F., Atyabi, F., & Mojtabavi, N. (2017). Interleukin-6 participation in pathology of ocular diseases. Pathophysiology,24(3), 123-131. doi:10.1016/j.pathophys.2017.05.005
  3. Pilling, D., Zheng, Z., Vakil, V., & Gomer, R. (2014). Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis. PNAS,111(51), 18291-18296. doi:10.1073/pnas.1317426112
  4. Fernando R, Diniz Grisolia AB, Lu Y, et al. Slit 2 Modulate the Inflammatory Phenotype of Orbit-Infiltrating Fibrocytes in Graves’ Disease. The Journal of Immunology. 2018;200(12):3942-3949.
  5. Chiang TS, Lin MC, Tsai M, et al. ADP-ribosylation factor-like 4a interacts with Robo1 to promote cell migration by regulating Cdc42 activation. Molecular Biology of the Cell. 2019;30(1):69-81.
  6. Jeon MJ, Lim SH, You MH, et al. The role of Slit2 as a tumor suppressor in thyroid cancer. Molecular and Cellular Endocrinology. 2019;483:87-96.