EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis

Objectives: Eph/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). Methods: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from SSc patients and healthy controls, which was followed by in vivo analysis of fibroblast-specific EphB2 deficient mice. Results: Expression of EphB2 is upregulated in SSc skin tissue and explanted SSc dermal fibroblasts compared to healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-ß (TGFß) cytokines upregulate EphB2 in dermal fibroblasts via non-canonical TGFß/SMAD signaling and silencing EphB2 in dermal fibroblasts is sufficient to dampen TGFß-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. Conclusion: Our data implicate EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc. Overall design: Human dermal fibroblasts (70% confluency) were transfected with EPHB2 Mission®-esiRNA (Sigma Aldrich, USA) delivered into the cells using the lipofectamine® RNAiMAX (Invitrogen, USA) according to the manufacturer's instruction. Mission®-esiRNA complexes targeting eGFP were used as nontargeting control. After 48H incubation, transfected NHDF were stimulated with recombinant human TGF-ß1 at 10 ng/ml for another 24H. Cells were harvested, total RNA was extracted using RNA Stat60® (Tel-Test Inc, USA) following standard phenol-chloroform procedure, and further purified using the NucleoSpin RNA-II purification kit (Machery-Nagel) following the manufacturer's instructions.