Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells
Sheng Cao, … , Debabrata Mukhopadhyay, Vijay H. Shah
Sheng Cao, … , Debabrata Mukhopadhyay, Vijay H. Shah
Published June 23, 2010
Citation Information: J Clin Invest. 2010;120(7):2379-2394. https://doi.org/10.1172/JCI41203.
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Research Article Hepatology

Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

Abstract

PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor β (PDGFRβ) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-β–dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRβ-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions.

Authors

Sheng Cao, Usman Yaqoob, Amitava Das, Uday Shergill, Kumaravelu Jagavelu, Robert C. Huebert, Chittaranjan Routray, Soha Abdelmoneim, Meher Vasdev, Edward Leof, Michael Charlton, Ryan J. Watts, Debabrata Mukhopadhyay, Vijay H. Shah

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Figure 7

NRP-1 promotes Rac1 activity through c-Abl kinase activity.

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NRP-1 promotes Rac1 activity through c-Abl kinase activity. (A) Rac acti...
(A) Rac activity assay of c-Abl/Arg–/– MEFs was performed using GST-PBD pulldown assay after administration of PDGF (10 ng/ml) at different time points (0–10 minutes). Each group was analyzed by Western blot using Rac1 Ab. c-Abl/Arg–/– MEFs displayed impaired Rac1 activity. (B) siRNA knockdown of c-Abl was examined in LX2 cells incubated with vehicle or PDGF (10 ng/ml) for 5 minutes. Rac activity assay was performed from cell lysates and revealed reduced activity in cells transfected with c-Abl siRNA. Total Rac, c-Abl, and actin blots were performed on parallel, identically loaded membranes at the same time as GST pulldowns or autoradiography. (C) NRP-1 was overexpressed in wild-type or c-Abl/Arg–/– MEFs, and Rac1 activity was measured. Rac activity was markedly diminished in c-Abl/Arg–/– MEFs despite NRP-1 overexpression. (D) LX2 cells or MEFs (wild-type, Nrp1–/–, c-Abl/Arg–/–) were assessed for c-Abl activity in the presence or absence of PDGF. PDGF stimulation increased c-Abl activity in LX2 cells (left panel), and PDGF-induced c-Abl activity was impaired in MEFs isolated from Nrp1–/– MEFs (middle panel). Overexpression of NRP-1 enhanced c-Abl activity in the presence of PDGF (right panel). (E) Association of c-Abl and NRP-1 was assessed by coimmnuoprecipitation assay using c-Abl Ab. Binding of c-Abl and NRP-1 was enhanced in LX2 cells overexpressing NRP-1 (left panel). PDGF promoted binding of c-Abl and NRP-1 (middle panel). PDGF-induced binding was not detected in MEFs isolated from c-Abl/Arg–/– or Nrp1–/– MEFs (right panel). (C–E) Samples were run on the same membrane; noncontiguous lanes are denoted by white lines.