Martina Buck

C/EBPβ Phosphorylation by RSK Creates a Functional XEXD Caspase Inhibitory Box Critical for Cell Survival

Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl(4) induced activation of RSK, phosphorylation of C/EBPbeta on Thr(217), and proliferation of stellate cells in normal mice, but caused apoptosis of these cells in C/EBPbeta-/- or C/EBPbeta-Ala(217) (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPbeta-PThr(217) and the phosphorylation mimic C/EBPbeta-Glu(217), but not C/EBPbeta-Ala(217), were associated with procaspases 1 and 8 in vivo and in vitro and inhibited their activation. Our data suggest that C/EBPbeta phosphorylation on Thr(217) creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T(217)VD) that is mimicked by C/EBPbeta-Glu(217) (KE(217)VD). C/EBPbeta-/- and C/EBPbeta-Ala(217) stellate cells were rescued from apoptosis by the cell permeant KE(217)VD tetrapeptide or C/EBPbeta-Glu(217).

Nuclear export of phosphorylated C/EBPβ mediates the inhibition of albumin expression by TNF-α

Decreased albumin expression is a frequent feature of cachexia patients afflicted with chronic diseases, including cancer, and a major contributor to their morbidity. Here we show that tumor necrosis‐α (TNF‐α) treatment of primary mouse hepatocytes or TNF‐α overexpression in a mouse model of cachexia induces oxidative stress, nitric oxide synthase (NOS) expression and phosphorylation of C/EBPβ on Ser239, within the nuclear localization signal, thus inducing its nuclear export, which inhibits transcription from the albumin gene. SIN‐1, a NO donor, duplicated the TNF‐α effects on hepatocytes. We found similar molecular abnormalities in the liver of patients with cancer‐cachexia. The cytoplasmic localization and association of C/EBPβ‐PSer239 with CRM1 (exportin‐1) in TNF‐α‐treated hepatocytes was inhibited by leptomycin B, a blocker of CRM1 activity. Hepatic cells expressing the non‐phosphorylatable C/EBPβ alanine mutant were refractory to the inhibitory effects of TNF‐α on albumin transcription since the mutant remained localized to the nucleus. Treatment of TNF‐α mice with antioxidants or NOS inhibitors prevented phosphorylation of C/EBPβ on Ser239 and its nuclear export, and rescued the abnormal albumin gene expression.

Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures

Background Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. Methodology/Principal Findings Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes. Conclusions/Significance This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.

A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

Background In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis. Methodology/Principal Findings Chronic treatment with the hepatotoxin CCl4 induced severe liver fibrosis in C/EBPβ+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ+/+ mice with CCl4-induced severe liver fibrosis, while continuing on CCl4, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis. Conclusions/Significance These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.

Decreased Jun-D and myogenin expression in muscle wasting of human cachexia.

Muscle wasting is a critical feature of patients afflicted by acquired immune deficiency syndrome (AIDS), cancer, or chronic inflammatory diseases. In a mouse model of muscle wasting, TNF-alpha induces oxidative stress and nitric oxide synthase-2 (NOS2) and decreases myogenin, Jun-D, and creatinine kinase muscle isoform (CKM) expression. Here, we studied 12 patients with muscle wasting due to cancer (N = 10) or AIDS (N = 2) and 4 control subjects. We show that in skeletal muscle of cachectic patients there is 1) increased expression and activity of the TNF-alpha signaling, including TNF-alpha mRNA, activation of TNFR1, and TNF-alpha-associated to TNFR1; 2) increased oxidative stress, as determined by the presence of malondialdehyde-lysine adducts; 3) increased NOS2 mRNA and protein; 4) decreased expression of Jun-D, myogenin, myosin, and CKM mRNA and protein; 5) impaired CKM-E box binding activities, associated with decreased Jun-D/myogenin activities; and 6) oxidative modification and ubiquitination of Jun-D. These studies show that these molecular pathways are modulated in association with muscle wasting in patients with cancer or AIDS, and whether or not they cause muscle wasting remains to be determined.

Novel inflammatory biomarkers of portal pressure in compensated cirrhosis patients

The rationale for screening inflammatory serum biomarkers of the hepatic vein pressure gradient (HVPG) is based on the fact that portal hypertension is pathogenically related to liver injury and fibrosis, and that in turn these are associated with the activation of inflammatory pathways. This was a nested cohort study in the setting of a randomized, clinical trial to assess the development of gastroesophageal varices (GEV) (N Engl J Med 2005;353:2254). Patients had cirrhosis and portal hypertension but did not have GEV. A total of 90 patients who had baseline day‐1 sera available were enrolled in the present study. The objective of this study was to determine whether inflammatory biomarkers in conjunction with clinical parameters could be used to develop a predictive paradigm for HVPG. The correlations between HVPG and interleukin (IL)‐1β (P = 0.0052); IL‐1R‐α (P = 0.0085); Fas‐R (P = 0.0354), and serum VCAM‐1 (P = 0.0007) were highly significant. By using multivariate logistic regression analysis and selected parameters (transforming growth factor beta [TGFβ]; heat shock protein [HSP]‐70; at‐risk alcohol use; and Child class B) we could exclude HVPG ≥12 mmHg with 86% accuracy (95% confidence interval [CI]: 67.78 to 96.16%) and the sensitivity was 87.01% (95% CI: 69.68 to 96.34%). Therefore, the composite test could identify 86% of compensated cirrhosis patients with HVPG below 12 mmHg and prevent unnecessary esophagogastroduodenoscopy with its associated morbidity and costs in these patients. Our diagnostic test was not efficient in predicting HVPG ≥12 mmHg. Conclusion: A blood test for HVPG could be performed in cirrhosis patients to prevent unnecessary esophagogastroduodenoscopy. (Hepatology 2014;59:1052–1059)

Pioglitazone decreases hepatitis C viral load in overweight, treatment naive, genotype 4 infected-patients: a pilot study.

Background Insulin resistance (IR) is induced by chronic hepatitis C virus (HCV) genotypes 1 and 4 infections. It is not known whether drugs that affect IR such as Pioglitazone and Prednisone also affect serum HCV RNA titers independently of PEG-Interferon-α2/ribavirin treatment. The primary aim was to assess whether Pioglitazone by improving IR and/or inflammation decreases HCV viral load independently of standard of care HCV treatment. A secondary aim was to assess whether Prednisone, a drug that induces insulin resistance and stimulates HCV viral entry and replication in replicon culture systems, increases HCV viral load in this population. Methodology/Principal Findings We designed a two-arm, parallel Pilot Study of overweight, treatment naïve genotype 4 HCV-infected patients at a public referral Liver Clinic in Giza, Egypt. The subjects received Pioglitazone (30 mg/day for 14 days) or Prednisone (40 mg/day for 4 days) in a randomized fashion, but the two arms can be considered independent pilot studies. Only changes from baseline within each arm were assessed and no contrasts of the interventions were made, as this was not an aim of the study. Among 105 consecutive HCV genotype 4 patients, 39 were enrolled based on the optimal sample size and power analysis according to the CONSORT statement; 20 to the Pioglitazone group and 19 to the Prednisone group. Pioglitazone was effective in decreasing serum HCV RNA at day-14 (n = 10; difference of means = 205,618 IU/ml; 95% CI 26,600 to 384,600; P<0.001). Although Prednisone did increase serum HCV RNA at day-4 (n = 10; change from baseline = −42,786 IU/ml; 95% CI −85,500 to −15,700; P = 0.049), the log10 HCV RNA titers were statistically not different from baseline day-0. Conclusion/Significance This is the first documentation that Pioglitazone decreases the serum HCV RNA titers independently of PEG-Interferon-α2/ribavirin treatment. The novel findings of our Study provide the foundation for basic and clinical investigations on the molecular mechanisms responsible for the Pioglitazone-induced decrease in HCV genotype 4 RNA titers. Trial Registration ClinicalTrials.gov NCT01157975

C/EBPβ Associates With Caspase 8 Complex Proteins and Modulates Apoptosis in Hepatic Stellate Cells

Goals To analyze the role of C/EBPβ phosphorylation on hepatic stellate cell survival/cell death. Background Activation and survival of stellate cells is critical for the development of liver fibrosis. C/EBPβ phosphorylation regulates stellate cell survival by affecting caspase 8 activation. The mechanisms responsible for these effects are unknown. Study We study the effects of caspase 8 activators signaling through death receptors. In addition, we assess the role of C/EBPβ phosphorylation on the susceptibility of stellate cells to apoptotic stimuli. Finally, we investigated whether C/EBPβ is associated with the caspase 8 complex protein FLIP, a critical inhibitor of caspase 8. Results Primary mouse stellate cells from C/EBPβ wild type and the phosphorylation mimic C/EBPβGlu217 transgenic mice were treated with lipopolysaccharide [an inducer of tumor necrosis factor-α (TNF-α)], FAS, or TNF-α. Stellate cell apoptosis was determined by assessing the binding of annexin-V to exposed phosphatidylserine of plasma membranes. TNF-α and FAS, but not lipopolysaccharide, induced annexin-V binding at 6 hours in C/EBPβ wild type stellate cell. However, the stimulation of apoptosis by TNF-α and FAS was markedly blocked in C/EBPβGlu217 stellate cells (P<0.001). Stellate cells activated on a collagen type 1 matrix expressed both C/EBPβ and FLIPL. Treatment of stellate cells with a MAP kinase kinase1 (MEK1) inhibitor blocked FLIPL cellular localization, suggesting that MEK1 signaling through C/EBPβ modulates FLIP activity. The colocalization of C/EBPβ and FLIPL was disrupted by activation of the FAS receptor, by blocking the association of C/EBPβ with the long form of FLIP, FLIPL. Conclusions The MAPK-RSK-C/EBPβ signaling may modulate stellate cell survival through caspase 8-associated protein FLIPL. This step is critical for liver fibrosis and if blocked with competitor peptides may prevent fibrogenesis.

C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

Background Although C/EBPβko mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-β on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice. Methodology/Principal Findings Mice expressing the non-phosphorylatable C/EBPβ-Ala217 transgene had a marked reduction in lung injury on day-13 after Bleomycin exposure, compared to C/EBPβwt mice, judging by the decrease of CD68+ activated monocytes/macrophages, bone marrow-derived CD45+ cells and lung cytokines as well as by the normal surfactant protein-C expression by lung pneumocytes. On day-21 after Bleomycin treatment, C/EBPβwt mice but not mice expressing the dominant negative C/EBPβ-Ala217 transgene developed severe lung fibrosis as determined by quantitative collagen assays. All mice were of identical genetic background and back-crossed to the parental wild-type inbreed FVB mice for at least ten generations. Treatment of C/EBPβwt mice with a cell permeant, C/EBPβ peptide that inhibits phosphorylation of C/EBPβ on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPβ on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis. Conclusions/Significance These data suggest that the RSK-C/EBPβ phosphorylation pathway may contribute to the development of lung injury and fibrosis.

A novel domain of BRCA1 interacts with p53 in breast cancer cells

The interactions between BRCA1 and p53 are relevant for understanding hereditary breast and ovarian cancer. Although in vitro studies reported that BRCA1 (amino acids 224-500) and the second BRCT domain of the BRCA1 C-terminus may interact with p53, quantitative biophysical measurements indicate that these regions of BRCA1 do not bind efficiently to p53. Here we show that BRCA1 interacts with p53 in vivo in breast cancer cells, through another BRCA1 domain (amino acids 772-1292). Expression of a truncated BRCA1 (amino acids 772-1292) stimulated p53 DNA-binding and transcription activities and apoptosis, recapitulating some effects of DNA damage. These results suggest that a novel domain of BRCA1 may interact with p53 in breast cancer cells.