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

The 44-mer suppresses hepatocyte apoptosis in CCl₄-treated mice.

<p>Apoptosis of hepatocytes was quantified 24 h after administration of a single dose of CCl₄ or CCl₄+44-mer. (A) Liver sections were double-stained with TUNEL, to identify apoptotic cells (<i>green</i>), and for albumin, to identify hepatocytes (<i>red</i>). (B) The number of TUNEL-positive cells was determined as the mean of 5 different areas at ×400 magnification of each section. *<i>P</i> <0.05 <i>versus</i> CCl₄+control peptide-treated group. (C) Western blot analysis of active caspase-3 protein in response to CCl₄ and CCl₄+ 44-mer and control peptides. Representative blots and densitometric analysis are shown from 3 independent experiments. *<i>P</i> <0.05 versus CCl₄+control peptide -treated group. The 44-mer partially sustains hepatic Bcl-xL protein levels in mice for 24 h, following a single injection of CCl₄. (D) Liver sections were immunostained with anti-Bcl-xL antibody and counterstained with hematoxylin. Secondary antibody only was used as negative control. (E) Liver protein extracts were harvested and subjected to western blot analysis with antibodies as indicated. Representative blots and densitometric analysis from 3 independent experiments are shown. The immunoblots were scanned and quantitated at individual sites and normalized to β-actin. *<i>P</i> <0.02 versus CCl₄+control peptide -treated group. (F) The Effect of CCl₄, 44-mer peptide, and their combination on the relative mRNA levels of PEDF in mouse liver. Results represent the mean ± SE from three independent experiments. *<i>P</i> <0.05 versus CCl₄+control peptide -treated group.</p

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

Knockdown of PEDF leads to a reduction in cell survival.

<p>Hepatocytes were transfected with control siRNA or PEDF specific siRNA as indicated. Two days later, the hepatocytes were serum starved for 24 h with or without 10 μM 44-mer. (A) Expression of the PEDF proteins was verified by Western blotting. PEDF levels were normalized to the β-actin. Graphs represent mean ± SE of 3 different experiments. *<i>P</i>< 0.002 versus SF/Cont siRNA-transfected cells. **<i>P</i>< 0.05 versus 44-mer/Cont siRNA-transfected cells. (B) Expression of the PEDF mRNA was verified by quantitative RT-PCR. The relative mRNA level was calculated from the mean value relative to the basal control. Results are means ± SE of triplicate transfections. *<i>P</i>< 0.001 versus SF/Cont siRNA-transfected cells. **<i>P</i>< 0.02 versus 44-mer/Cont siRNA-transfected cells. (C) TUNEL assay. The percentage of cell apoptosis was quantified by dividing the number of apoptotic nuclei to a population of 2000 counted cells per condition. Graphs represent means ± SE (n = 3). *<i>P</i>< 0.01 versus SF/Cont siRNA-transfected cells. **<i>P</i>< 0.001 relative to the Cont peptide/PEDF siRNA-transfected 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

Effect of the 44-mer on assessment of oxidative stress.

<p>Effect of the 44-mer on assessment of oxidative stress.</p

PEDF and the 44-mer induce endogenous PEDF expression in primary rat hepatocytes and human hepatoma cells.

<p>(A) Western blot analysis. Primary rat hepatocytes were treated with varying doses of PEDF, 44-mer or 34-mer peptide for 24 h. Protein expression were calculated as the fold increase of PEDF expression compared to the solvent (S) controls. Representative immunoblots and densitometric analysis are shown as the mean ± SE of 3 independent experiments. *<i>P</i><0.05 versus solvent-treated cells. (B) Relative mRNA levels of PEDF. Primary rat hepatocytes were treated with 10 nM PEDF, 10 μM 44-mer or 34-mer peptide for 24 h. PEDF mRNA was estimated by quantitative real-time RT-PCR. Results represent the mean ± SE from three independent experiments, each performed in quadruplicate. *<i>P</i> <0.05 versus solvent-treated cells. (C-E) Human hepatoma HepG2 and HuH7 cells were treated with 10 nM PEDF, 10 μM 44-mer or control peptide for 24 h. Cells were then harvested and assayed by western blot analysis and quantitative real-time PCR. Representative immunoblots and densitometric analysis are shown as the mean ± SE (n = 3). Real-time PCR data are presented as the mean ± SE (n = 4). *<i>P</i> <0.05 versus solvent-treated cells. **<i>P</i> <0.05 versus control peptide-treated cells.</p

Phosphorylation of HPV-16 E2 at Serine 243 Enables Binding to Brd4 and Mitotic Chromosomes

<div><p>The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding.</p></div