The 44-mer protects mice from CCl₄-induced hepatic fibrosis.

<p>Mice were treated with CCl₄ twice per week for 5 weeks. (A) Histopathological detection of collagen in the liver by Sirius red-staining. (Original magnification, ×200). Representative images from five mice in each subgroup are shown. (B) Estimation of the area of hepatic fibrosis by Sirius red staining. Data were assessed by analyzing 10 Sirius red-stained liver sections per animal with a computerized image system. *<i>P <</i>0.001 <i>versus</i> the CCl₄+control peptide-treated group. (C and D) Whole liver protein lysates at week 5 post-CCl₄ treatment were extracted for western blot analysis with the antibodies indicated. Representative blots and densitometric analysis are shown from five mice in each subgroup. *<i>P <</i>0.005 <i>versus</i> CCl₄+control peptide-treated group. **<i>P <</i>0.01 <i>versus</i> CCl₄+control peptide-treated group.</p

Pigment Epithelium Derived Factor Peptide Protects Murine Hepatocytes from Carbon Tetrachloride-Induced Injury

<div><p>Fibrogenesis is induced by repeated injury to the liver and reactive regeneration and leads eventually to liver cirrhosis. Pigment epithelium derived factor (PEDF) has been shown to prevent liver fibrosis induced by carbon tetrachloride (CCl₄). A 44 amino acid domain of PEDF (44-mer) was found to have a protective effect against various insults to several cell types. In this study, we investigated the capability of synthetic 44-mer to protect against liver injury in mice and in primary cultured hepatocytes. Acute liver injury, induced by CCl₄, was evident from histological changes, such as cell necrosis, inflammation and apoptosis, and a concomitant reduction of glutathione (GSH) and GSH redox enzyme activities in the liver. Intraperitoneal injection of the 44-mer into CCl₄-treated mice abolished the induction of AST and ALT and markedly reduced histological signs of liver injury. The 44-mer treatment can reduce hepatic oxidative stress as evident from lower levels of lipid hydroperoxide, and higher levels of GSH. CCl₄ caused a reduction of Bcl-xL, PEDF and PPARγ, which was markedly restored by the 44-mer treatment. Consequently, the 44-mer suppressed liver fibrosis induced by repeated CCl₄ injury. Furthermore, our observations in primary culture of rat hepatocytes showed that PEDF and the 44-mer protected primary rat hepatocytes against apoptosis induced by serum deprivation and TGF-β1. PEDF/44-mer induced cell protective STAT3 phosphorylation. Pharmacological STAT3 inhibition prevented the antiapoptotic action of PEDF/44-mer. Among several PEDF receptor candidates that may be responsible for hepatocyte protection, we demonstrated that PNPLA2 was essential for PEDF/44-mer-mediated STAT3 phosphorylation and antiapoptotic activity by using siRNA to selectively knockdown PNPLA2. In conclusion, the PEDF 44-mer protects hepatocytes from single and repeated CCl₄ injury. This protective effect may stem from strengthening the counter oxidative stress capacity and induction of hepatoprotective factors.</p></div

Effect of STAT3 inhibitor and PEDF receptor siRNA on hepatocyte apoptosis induced by serum deprivation.

<p>(A) Inhibitor of STAT3 prevents the induction of PEDF by the 44-mer. Hepatocytes were pretreated with STAT3 inhibitor or ERK inhibitor (PD98059) or PPARγ antagonist (GW9662) for 2 h and then treated with the 44-mer for 24 h. To examine the role of PEDF receptor on antiapoptotic effect of the 44-mer, hepatocytes were transfected with the indicated siRNA. Two days later, the hepatocytes were starved of serum and exposed to the 44-mer for further 24 h. Subsequently, apoptosis was determined by TUNEL assay. Graphs represent means ± SE (n = 4). *<i>P</i><0.001 versus 44-mer-treated cell. **<i>P</i> < 0.05 relative to the cells pretreated with control siRNA/44-mer. (B) The 44-mer induces STAT3 phosphorylation. Hepatocytes were treated with the 44-mer for the indicated time periods and phosphorylated STAT3 were detected by western blot analysis. Representative immunoblots and densitometric analysis are shown as the mean ± SE (n = 3). *<i>P</i> < 0.001 versus untreated cells (time 0). (C-E) PNPLA2 siRNA reduces the PEDF/44-mer-induced STAT3 phosphorylation. Hepatocytes were transfected with control siRNA (lanes 2 and 3) or PEDF receptor specific siRNA (PNPLA2, LRP6 or LR; lanes 4 and 5) as indicated. Mock indicates cells treated with transfection reagent. PEDF receptor levels were normalized to the β-actin. Numbers below the blots refer to a densitometric measure expressed as a fold of mock control. To examine p-STAT3 levels, hepatocytes were treated with PEDF or 44-mer for 10 min and then lysed. Total and phosphorylated STAT3 protein levels were examined by western blotting. p-STAT3 was normalized to the STAT3. Graphs represent mean ± SE of 3 different experiments. *<i>P</i> < 0.05 versus Cont siRNA/PEDF-treated cells. **<i>P</i> < 0.05 versus Cont siRNA/44-mer-treated cells.</p

PEDF and the 44-mer protect primary rat hepatocyte against apoptosis induced by serum deprivation and TGF-β1.

<p>Hepatocytes were either left cultured in serum-free (SF) medium or treated with PEDF peptides, TGF-β1 or a combination of PEDF peptide and TGF-β1 for 24 h. (A) Apoptosis was determined by TUNEL staining (<i>green dots</i>) and doubly stained with Hoechst 33258 (<i>blue dots</i>). A representative of four independent experiments is shown. (B) The percentage of cell death was quantified by dividing the number of TUNEL-positive cells to a population of 2000 counted cells per condition. Control: hepatocytes cultured in medium with 10% FBS. Graphs represent means ± SE (n = 4). *<i>P</i><0.001 versus cell cultured in 10% FBS medium. **<i>P</i> < 0.02 relative to the cells cultured in SF medium. ***<i>P</i>< 0.001 versus cells cultured in SF medium. (C) Analysis of ROS generation induced by serum deprivation. Hepatocytes were either left cultured in SF medium or treated with PEDF peptides or with 1 mM NAC for 24 h. ROS production was expressed as DCF fluorescence intensity and quantified by spectrofluorimetry. *<i>P</i><0.001 versus cells cultured in 10% FBS medium. **<i>P</i> < 0.02 relative to the cells cultured in SF medium. ^#<i>P</i>< 0.001 versus cells cultured in SF medium. (D) Apoptosis was determined by TUNEL assay and the percentage of cell death was quantified. Graphs represent means ± SE (n = 4). *<i>P</i><0.001 versus cells cultured in SF medium. **<i>P</i> < 0.05 versus TGF-β1 treated cells. (E) Western blotting analysis of active caspase-3. Representative blots and densitometric analysis are shown. Graphs represent means ± SE (n = 3). *<i>P</i><0.01 versus cells cultured in SF medium. **<i>P</i> < 0.001 versus TGF-β1 treated cells.</p

Effects of the 44-mer on CCl₄-induced liver inflammation.

<p>(A) Mice were treated with CCl₄ for 12 h. Liver tissues were then harvested and assayed by quantitative real-time PCR. (B) Serum levels of cytokines. Values are expressed as mean ± SE in each group. *<i>P</i> <0.05 versus CCl₄+control peptide-treated group.</p

Phosphorylation at serine 243 of HPV-16 E2 mediates binding to Brd4.

<p>A) Phosphorylation analysis and HPV-16 E2 proteins. 293 cells were transfected with expression vectors for wild type or mutated hinge region of HPV-16 E2 fused to EGFP. Equivalent amounts of protein lysate were immunoprecipitated using anti-EGFP antibody and the samples were analyzed by western blotting with antibody against phosphor-serine. B) The graph presents the quantified intensities of the phosphorylated signal. The level of phosphorylated E2 hinge was normalized using total E2 immunoprecipitated. C) Mutational analysis of E2 proteins binding to Brd4. 293 cells were transfected with vectors expressing various E2 proteins containing amino acid substitutions in the hinge region. Cell lysates were collected and immunoprecipitated with anti-Brd4 antibody. The protein complexes were analyzed by SDS-PAGE and subjected to immunoblotting with anti-EGFP antibody. The expression level of E2 proteins was normalized using β-Actin, and the percentage of E2 at each time point relative to that of the control (without CHX treatment, set at 100) is shown (lower panel) Data shown are means of results from three independent experiments. Asterisk, non-specific band. D) Summary of the Brd4-binding activity of the HPV-16 E2 proteins and mutants.</p

Chromosome association phenotypes of Brd4 and E2 proteins with amino acid substitutions in the hinge.

<p>A, B) Phosphorylation at residue 243 is critical for the co-localization of HPV-16 E2 and Brd4 and association with mitotic chromosomes. COS-7 cells transfected with plasmids expressing HPV-16 wild type E2, S207-mutated E2 (S207A) or S243-mutated E2 (S243A, S243D, S243N, S243E, S243Q) were visualized by confocal microscopy to determine the location of E2 (green), Brd4 (red) and cellular DNA (blue) during mitosis. A) metaphase and B) anaphase.</p