Slit2-Robo2 signaling modulates the fibrogenic activity and migration of hepatic stellate cells.

Slit/Robo signaling was originally identified as a repulsive guidance cue in regulating axon branching and neuronal migration. Hepatic stellate cells (HSCs) are the key fibrogenic cells in the liver, which are migratory when activated, and express neural crest markers. The aim of the present study was to investigate the functional significance of Slit/Robo signaling in liver fibrogenesis and in HSCs. By transcriptomic analysis it was found that axon guidance signaling pathways were significantly upregulated in both diethylnitrosamine (DEN) and thioacetamide (TAA)-induced experimental liver fibrosis. The up-regulation of the ligand Slit2 and membrane receptor Robo2 genes within this pathway was further validated in TAA-induced fibrotic livers. By immunofluorescence staining, Robo2 was localized in fibrotic septa of fibrotic liver and on the surface of HSCs. By Western blot analysis, recombinant Slit2 (rSlit2) was found to promote fibrogenic protein expression in JS1 cells, an immortalized mouse HSC line, while activating PI3K/Akt signaling pathway. This effect was abrogated by LY294002, a PI3K/Akt pathway inhibitor. In addition, rSlit2 stimulation markedly inhibited JS1 cells migration in transwell migration assays, which was abrogated by small interfering RNA (siRNA) knockdown of Robo2 in the cells. The present study provides evidence that Slit2/Robo2 signaling mediates the pathogenesis of hepatic fibrogenesis and regulates HSCs biology, thus providing potential markers for HSCs, and therapeutic and diagnostic target toward liver fibrosis.