Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.</p> <p>Results</p> <p>Human <it>BRCA2 </it>gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G₂), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.</p> <p>Conclusions</p> <p><it>BRCA2 </it>gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter <it>in vivo </it>and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.</p

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH)

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF-beta significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH

GLP-1 Analogs Reduce Hepatocyte Steatosis and Improve Survival by Enhancing the Unfolded Protein Response and Promoting Macroautophagy

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress-induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD

Baseline urine metabolic phenotype in patients with severe alcoholic hepatitis and its association with outcome

International audienceSevere alcoholic hepatitis (SAH) has a high mortality rate, and corticosteroid therapy is effective in 60% patients. This study aimed to investigate a baseline metabolic phenotype that could help stratify patients not likely to respond to steroid therapy and to have an unfavorable outcome. Baseline urine metabolome was studied in patients with SAH using ultra‐high performance liquid chromatography and high‐resolution mass spectrometry. Patients were categorized as responders (Rs, n = 52) and nonresponders (NRs, n = 8) at day 7 according to the Lille score. Multivariate projection analysis identified metabolites in the discovery cohort (n = 60) and assessed these in a validation cohort of 80 patients (60 Rs, 20 NRs). A total of 212 features were annotated by using metabolomic/biochemical/spectral databases for metabolite identification. After a stringent selection procedure, a total of nine urinary metabolites linked to mitochondrial functions significantly discriminated nonresponders, most importantly by increased acetyl‐L‐carnitine (12‐fold), octanoylcarnitine (4‐fold), decanoylcarnitine (4‐fold), and alpha‐ketoglutaric acid (2‐fold) levels. Additionally, urinary acetyl‐L‐carnitine and 3‐hydroxysebasic acid discriminated nonsurvivors ( P 0.3; P 3.0; P 2,500 ng/mL reliably segregated survivors from nonsurvivors ( P < 0.01, log‐rank test) in our study cohort. Conclusion: Urinary metabolome signatures related to mitochondrial functions can predict pretherapy steroid response and disease outcome in patients with SAH. ( Hepatology Communications 2018;2:628‐643

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis

We point out that there is a high-frequency tail of the stochastic inflationary gravitational wave background that scales as $f^{-1/2}$ with frequency $f$. This contribution comes from the graviton vacuum fluctuation amplified by the inflaton coherent oscillation during the reheating stage. It contains information on inflaton properties such as the inflaton mass as well as the thermal history of the early Universe

Suboptimal Level of Bone‐Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy

Bone loss is common in advanced cirrhosis, although the precise mechanisms underlying bone loss in cirrhosis are unknown. We studied the profile and functionality of bone‐forming cells and bone‐building proteins in bone marrow (BM) of individuals with cirrhosis (n = 61) and individuals without cirrhosis as normal controls (n = 50). We also performed dual energy X‐ray absorptiometry for clinical correlation. BM mesenchymal cells (MSCs) were analyzed for colony‐forming units‐fibroblasts and their osteogenic (fibronectin‐1 [FN1], insulin‐like growth factor binding protein 3 [IGFBP3], collagen type 1 alpha 1 chain [COL1A1], runt‐related transcription factor 2 [RUNX2], and alkaline phosphatase, liver [ALPL]) and adipogenic ( adiponectin, C1Q, and collagen domain containing [ADIPOQ], peroxisome proliferator‐activated receptor gamma [PPARγ], and fatty acid binding protein 4 [FABP4]) potentials. Colony‐forming units‐fibroblasts were lower in patients with cirrhosis (P = 0.002) than in controls. Cirrhotic BM‐MSCs showed >2‐fold decrease in osteogenic markers. Compared to controls, patients with cirrhosis showed fewer osteocytes (P = 0.05), osteoblasts, chondroblasts, osteocalcin‐positive (osteocalcin+) area, clusters of differentiation (CD)169+ macrophages (P 2‐fold decreased anti‐osteoclastic and increased pro‐osteoclastic factors were noted in patients with CTP C compared to CTP A. Bone‐building proteins (osteocalcin [P = 0.008], osteonectin [P < 0.001], and bone morphogenic protein 2 [P = 0.001]) were decreased while anti‐bone repair factors (fibroblast growth factor 23 [P = 0.015] and dipeptidyl peptidase 4 [P < 0.001]) were increased in BM and peripheral blood; this was more apparent in advanced cirrhosis. The dual energy X‐ray absorptiometry scan T score significantly correlated with the population of osteoblasts, osteocytes, MSCs, and CD169+ macrophages. Conclusion: Osteoprogenitor cells are substantially reduced in patients with cirrhosis and more so in advanced disease. Additionally, increased anti‐bone repair proteins enhance the ineffective bone repair and development of osteoporosis in cirrhosis. Hepatology Communications 2018;0:0‐0