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.

Zeng Z ，Wu Y ，Cao Y ，Yuan Z ，Zhang Y ，Zhang DY ，Hasegawa D ，Friedman SL ，Guo J ... -  《-》

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-β1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Chang J ，Lan T ，Li C ，Ji X ，Zheng L ，Gou H ，Ou Y ，Wu T ，Qi C ，Zhang Q ，Li J ，Gu Q ，Wen D ，Cao L ，Qiao L ，Ding Y ，Wang L ... -  《-》

Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells.

Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.

Li C ，Yang G ，Lin L ，Xuan Y ，Yan S ，Ji X ，Song F ，Lu M ，Lan T ... -  《-》

SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion.

Fan X ，Yang H ，Kumar S ，Tumelty KE ，Pisarek-Horowitz A ，Rasouly HM ，Sharma R ，Chan S ，Tyminski E ，Shamashkin M ，Belghasem M ，Henderson JM ，Coyle AJ ，Salant DJ ，Berasi SP ，Lu W ... -  《-》

Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) exists in two forms: a transmembrane form and a soluble form (sTREM-1). The levels of sTREM-1 are elevated in supernatants of activated HSCs. However, the role of sTREM-1 in HSC activation and liver fibrosis remains undefined. Previous studies have primarily focused on the transmembrane form of TREM-1; we innovatively observed the function of sTREM-1 as a ligand in liver fibrosis and screened its receptor. Here, recombinant sTREM-1 was used as a stimulator which induced HSC activation and further aggravated liver fibrosis. Then, screening for sTREM-1 interacting membrane receptors was performed using pull-down assay followed by mass spectrometry, and the membrane receptor roundabout guidance receptor 2 (Robo2) was identified as a candidate receptor for sTREM-1. The interaction between sTREM-1 and Robo2 was verified by pull-down and immunofluorescence. The role of Robo2 on sTREM-1-induced HSC activation and its downstream signal pathways was assessed by knockdown of Robo2 in LX-2 cells. Furthermore, HSC-specific knockdown of Robo2 was achieved in a mouse model of liver fibrosis by using a recombinant adeno-associated virus (AAV) vector to confirm the role of the receptor, and we proved that Robo2 knockdown inhibited the activation of HSC and liver fibrosis, which also led to the inactivation of Smad2/3 and PI3K/Akt pathways in sTREM-1-induced HSC activation and liver fibrosis. In conclusion, sTREM-1 acts as a new ligand of Robo2; the binding of sTREM-1 to Robo2 initiates the activation of the downstream Smad2/3 and PI3K/Akt signalling pathways, thereby promoting HSC activation and liver fibrosis.

Liu T ，Chen S ，Xie X ，Liu H ，Wang Y ，Qi S ，Shi L ，Zhou X ，Zhang J ，Wang S ，Wang Y ，Chen S ，Dou S ，Jiang X ，Cui R ，Jiang H ... -  《-》