Activation of Slit2-Robo1 signaling promotes liver fibrosis.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.
Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China; Department of Oncology, Affiliated Heping Hospital of Changzhi Medical College, Changzhi, China.
Jianlan
J
Chang
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Tian
T
Lan
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Changzheng
C
Li
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Xiaoqian
X
Ji
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Lingyun
L
Zheng
Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.
Hongju
H
Gou
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Yitao
Y
Ou
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Teng
T
Wu
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Cuiling
C
Qi
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Qianqian
Q
Zhang
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Jiangchao
J
Li
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Quliang
Q
Gu
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Dingwen
D
Wen
Department of Developmental Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.
Liu
L
Cao
Storr Liver Centre, the Westmead Millennium Institute for Medical Research, the University of Sydney, NSW 2145, Australia. Electronic address: liang.qiao@sydney.edu.au.
Liang
L
Qiao
Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China. Electronic address: dyq@fimmu.com.
Yanqing
Y
Ding
Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou 510006, China. Electronic address: wanglijing62@163.com.
Lijing
L
Wang
D000818:Animals; D002251:Carbon Tetrachloride / Q000633:toxicity; D016022:Case-Control Studies; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D055166:Hepatic Stellate Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000209:etiology* / Q000378:metabolism* / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
D002251:Carbon Tetrachloride; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein; C498888:Robo2 protein, mouse
Collagen I; Liver fibrosis; PI3K/AKT pathway; Robo1/2(+/−) mice; Slit2-Robo1 signaling; Slit2-Tg mice; Smad2/3 pathway; α-SMA