A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Abstract

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

FIGURE 1.

HSc025 suppresses collagen gene expression in activated stellate cells. A, chemical structure of HSc025. B, rat primary hepatic stellate cells were untreated or treated with HSc025 for 6 days. Total RNA was extracted from the cells, and quantitative analyses of COL1A1, COL1A2, COL4A1, and fibronectin mRNA expression were performed by real time RT-PCR. Relative expression levels of each gene were normalized against those of GAPDH measured in the same total RNA preparations (mean ± S.D., n = 6).

FIGURE 2.

HSc025 physically interacts with YB-1. A, full-length YB-1, SET, or APRIL was expressed as His₆-tagged proteins. The gel stained with Coomassie Brilliant Blue shows the presence of each protein marked with asterisks on the left. B, approximately equal amounts (Input) of protein were incubated with control or HSc025-immobilized resin in the presence or absence of 1 μm HSc025 for 2 h. Bound proteins were detected by Western blotting with anti-His₆ monoclonal antibody. Input represents one-tenth of the protein used for binding assays. C, approximately equal amounts of full-length of YB-1 or its deletion mutant proteins were incubated with control or HSc025-immobilized resin for 2 h. Bound proteins were detected by Western blotting with anti-His₆ monoclonal antibody. Input represents one-tenth of the protein used for binding assays.

FIGURE 3.

HSc025 physically interferes with the binding between YB-1 and PABP. A, cytoplasmic extracts were prepared from human dermal fibroblasts treated with 10 μm HSc025 for the indicated lengths of time. After immunoprecipitation using anti-YB-1 antibodies, they were separated on a 10% SDS-polyacrylamide gel and visualized by silver staining. Note that the intensity of the one band marked with asterisks on the left was decreased by the treatment of HSc025. This band was excised, analyzed by tandem mass spectrometry, and found to correspond to PABP. B, cytoplasmic extracts from human dermal fibroblasts were prepared and incubated with HSc025 for 1 h at 4 °C. After immunoprecipitation using anti-YB-1 antibodies, they were separated on a 10% SDS-polyacrylamide gel and visualized by silver staining. Note that the band marked with an asterisk on the left, corresponding to PABP, was decreased by the HSc025 treatment. C, full-length of PABP was expressed as GST fusion protein. The gel stained with Coomassie Brilliant Blue shows the presence of GST or GST-fused PABP protein marked with asterisks on the left. D, approximately equal amounts of proteins were immobilized on glutathione-Sepharose beads prior to incubation with full-length or deletion mutant of YB-1 proteins in the presence or absence of 1 μm HSc025. Bound proteins were detected by Western blotting with anti-His₆ monoclonal antibody. Input represents one-tenth of the protein used for GST-pulldown assays.

FIGURE 4.

Displacement of PABP antagonizes fibrotic signaling. A, human dermal fibroblasts were transfected with 2 μg of siRNA and incubated for 48 h. After additional transfection with 2 μg of siRNA for 24 h, whole cell lysates were separated and blotted with PABP antibodies. B, human dermal fibroblasts were transfected with 2 μg of siRNA and incubated for 48 h. After additional transfection with 2 μg of siRNA for 24 h, cytoplasmic and nuclear extracts were separated and blotted with YB-1 antibodies. C, human dermal fibroblasts were transfected with 2 μg of siRNA and incubated for 48 h. After additional transfection with 2 μg of siRNA in the presence or absence of 5 ng/ml TGF-β for 24 h, total RNA was extracted, and a quantitative analysis of COL1A2 mRNA expression was performed by real time RT-PCR. Relative expression levels of COL1A2 were normalized against those of GAPDH measured in the same total RNA preparation (mean ± S.D., n = 6). Cont, control.

FIGURE 5.

Oral administration of HSc025 attenuates liver injury and hepatic fibrosis. Mice were chronically treated with 0.1 ml/kg body weight of CCl₄ once a week for 8 weeks. They were then orally administered 3, 15, or 75 mg/kg/day HSc025 or vehicle for 4 weeks after injection of carbon tetrachloride for 4 weeks. They were killed 72 h after the last CCl₄ injection and subjected to the following: A, determination of relative liver weight that was normalized against body weight; B, measurement of activity of serum ALT; C, H&E staining (original magnification, ×100; bar, 20 μm). D, histological examination. H&E sections were randomly selected and blindly analyzed for the degree of necrosis or hepatocellular vacuolization as measures of hepatic injury. E, Azan-Mallory staining (original magnification, ×100; bar, 20 μm). F, measurement of fibrotic areas. After Azan-Mallory staining, the degree of hepatic fibrosis was semiquantified by measuring the relative areas of fibrosis with the aid of computer software. G, quantification of hydroxyproline in acid-insoluble liver pellets. Hydroxyproline content was determined as described under “Experimental Procedures.” The asterisk signifies that the values are significantly different between the groups. **, p < 0.01; *, p < 0.05. Cont, control.