Multiple E3 ligases control tankyrase stability and function.

Tankyrase 1 and 2 are ADP-ribosyltransferases that catalyze formation of polyADP-Ribose (PAR) onto themselves and their binding partners. Tankyrase protein levels are regulated by the PAR-binding E3 ligase RNF146, which promotes K48-linked polyubiquitylation and proteasomal degradation of tankyrase and its partners. We identified a novel interaction between tankyrase and a distinct class of E3 ligases: the RING-UIM (Ubiquitin-Interacting Motif) family.

Multiple E3 ligases control tankyrase stability and function.

Tankyrase 1 and 2 are ADP-ribosyltransferases that use NAD as a substrate to catalyze polyADP-Ribose (PAR) onto themselves and their protein binding partners. Tankyrases have diverse cellular functions, ranging from resolution of telomere cohesion to activation of the Wnt/β-catenin signaling pathway. Robust and specific small molecule tankyrase inhibitors have been developed and are being investigated for cancer therapies.

Deficiency of Stat1 in CD11c Cells Alters Adipose Tissue Inflammation and Improves Metabolic Dysfunctions in Mice Fed a High-Fat Diet.

CD11c macrophages/dendritic cells (MDCs) are increased and display the classically activated M1-like phenotype in obese adipose tissue (AT) and may contribute to AT inflammation and insulin resistance. Stat1 is a key transcription factor for MDC polarization into the M1-like phenotype. Here, we examined the role of Stat1 in obesity-induced AT MDC polarization and inflammation and insulin resistance using mice with specific knockout of Stat1 in MDCs (cKO).

DNA demethylation agent 5azadC downregulates HPV16 E6 expression in cervical cancer cell lines independently of TBX2 expression.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells.

Comparative RNA sequencing reveals that HPV16 E6 abrogates the effect of E6*I on ROS metabolism.

High-risk Human Papillomavirus infections are responsible for anogenital and oropharyngeal cancers. Alternative splicing is an important mechanism controlling HPV16 gene expression. Modulation in the splice pattern leads to polycistronic HPV16 early transcripts encoding a full length E6 oncoprotein or truncated E6 proteins, commonly named E6*.

EMT is associated with an epigenetic signature of ECM remodeling genes.

Type III epithelial-mesenchymal transition (EMT) has been previously associated with increased cell migration, invasion, metastasis, and therefore cancer aggressiveness. This reversible process is associated with an important gene expression reprogramming mainly due to epigenetic plasticity. Nevertheless, most of the studies describing the central role of epigenetic modifications during EMT were performed in a single-cell model and using only one mode of EMT induction.

PD-L1 expression is regulated by both DNA methylation and NF-kB during EMT signaling in non-small cell lung carcinoma.

Tumor cells, which undergo Epithelial-mesenchymal transition (EMT) acquire increased capacities of proliferation, invasion and have the ability to generate metastases by escaping the immune system during their systemic migration. To escape the immune system, cancer cells may induce tolerance or resist elimination by immune effectors multiple mechanisms and we hypothesized that EMT may control the expression of immune checkpoint inhibitors, then promoting immune evasion. PD-L1 (programmed cell death ligand 1) but not PD-L2 nor Galectin 9 or Death receptor (DR4, DR5 and Fas) and ligands (FasL and TRAIL) expression was up-regulated during cytokine-driven EMT in a reversible manner.

5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression.

High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter.

Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia.

Objective: To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis.

Approach And Results: In apoE(-/-) mice fed Western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr(-/-)Apobec1(-/-) (LDb) mice on WD.

Attenuated adipose tissue and skeletal muscle inflammation in obese mice with combined CD4+ and CD8+ T cell deficiency.

Objectives: High-fat diet (HFD) feeding in mice is characterized by accumulation of αβ T cells in adipose tissue. However, the contribution of αβ T cells to obesity-induced inflammation of skeletal muscle, a major organ of glucose uptake, is unknown. This study was undertaken to evaluate the effect of αβ T cells on insulin sensitivity and inflammatory state of skeletal muscle and adipose tissue in obesity.

Essential role of CD11a in CD8+ T-cell accumulation and activation in adipose tissue.

Objective: T cells, particularly CD8(+) T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. We examined the mechanisms of CD8(+) T-cell accumulation and activation in AT and the role of CD11a, a β2 integrin.

Approach And Results: CD8(+) T cells in AT of obese mice showed activated phenotypes with increased proliferation and interferon-γ expression.

ApoE and the role of very low density lipoproteins in adipose tissue inflammation.

Objective: To identify the role of triglyceride-rich lipoproteins (TGRLs) and apoE, a major apolipoprotein in TGRLs, in adipose tissue inflammation with high-fat diet (HFD)-induced obesity.

Methods: Male apoE(-/-) and C57BL/6J wild-type (WT) mice fed HFD for 12 weeks were assessed for metabolic and inflammatory parameters. ApoE(-/-) and WT mice were orally gavaged with [(3)H]palmitic acid to examine the role of apoE in fat delivery to adipose tissue.

Differential effect of weight loss with low-fat diet or high-fat diet restriction on inflammation in the liver and adipose tissue of mice with diet-induced obesity.

Objective: We studied the effects of weight loss induced by either a low-fat normal diet (ND) or restriction of high-fat diet (HFD) on hepatic steatosis, inflammation in the liver and adipose tissue (AT), and blood monocytes of obese mice.

Methods: In mice with HFD-induced obesity, weight loss was achieved by switching from HFD to ND and maintaining on ND ad libitum or by restricting HFD intake to match body weight of mice with ND-induced weight loss. After diet interventions for 4 weeks, hepatic steatosis, hepatic and AT inflammation, and blood CD11c(+) monocytes were examined.

Effect of the cannabinoid receptor-1 antagonist rimonabant on inflammation in mice with diet-induced obesity.

We studied whether cannabinoid receptor (CB1) blockade with rimonabant has an anti-inflammatory effect in obese mice, and whether this effect depends on weight loss and/or diet consumption. High-fat diet (HFD)-induced obese mice were treated orally with rimonabant (HFD-R) or vehicle (HFD-V) for 4 weeks. Paired-feeding was conducted in two additional groups of obese mice to achieve either the same body weight (HFD-BW) or the same HFD intake (HFD DI) as HFD-R.

An alternative splicing variant in Clcn7-/- mice prevents osteopetrosis but not neural and retinal degeneration.

The ubiquitously expressed chloride channel 7 (CLCN7) is present within the ruffled border of osteoclasts. Mutations in the CLCN7 gene in humans (homologous to murine Clcn7) are responsible for several types of osteopetrosis in humans, and deficiencies in CLCN7 can present with retinal degeneration and a neuronal storage disease. A previously reported Clcn7(-/-) mouse showed diffuse osteopetrosis accompanied by severe retinal and neuronal degeneration.

CD11c expression in adipose tissue and blood and its role in diet-induced obesity.

Objective: To examine CD11c, a beta(2)-integrin, on adipose tissue (AT) leukocytes and blood monocytes and its role in diet-induced obesity.

Methods And Results: High-fat diet-induced obese C57BL/6 mice, CD11c-deficient mice, and obese humans were studied. CD11c, leukocytes, and chemokines/cytokines were examined in AT and/or blood by flow cytometry, RNase protection assay, quantitative polymerase chain reaction, or enzyme-linked immunosorbent assay.

T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity.

Background: Obesity is associated with chronic inflammation, which includes increased macrophage accumulation in adipose tissue (AT) and upregulation of chemokines and cytokines. T cells also play important roles in chronic inflammatory diseases such as atherosclerosis but have not been well studied in obesity.

Methods And Results: Flow cytometric analysis showed higher numbers of T cells and macrophages in AT of diet-induced obese insulin-resistant male mice than in lean mice and obese females (P<0.

Deficiency of CD11b or CD11d results in reduced staphylococcal enterotoxin-induced T cell response and T cell phenotypic changes.

The beta(2) integrin CD11a is involved in T cell-APC interactions, but the roles of CD11b, CD11c, and CD11d in such interactions have not been examined. To evaluate the roles of each CD11/CD18 integrin in T cell-APC interactions, we tested the ability of splenocytes of CD11-knockout (KO) mice to respond to staphylococcal enterotoxins (SEs), a commonly used superantigen. The defect in T cell proliferation with SEA was more severe in splenocytes from mice deficient in CD18, CD11b, or CD11d than in CD11a-deficient splenocytes, with a normal response in CD11c-deficient splenocytes.

Biodistribution and safety studies of hDel-1 plasmid-based gene therapy in mouse and rabbit models.

A plasmid encoding the human developmentally regulated endothelial locus-1 (hDel-1) protein formulated with poloxamer 188 is a potential gene therapy for peripheral arterial disease in man. As a prelude to clinical trials, the biodistribution and safety of this therapy were evaluated after intramuscular and intravenous administration in mice and rabbits. In mice, plasmid DNA persisted at the intramuscular injection site for at least 28 days, but was barely detectable in distal tissues by 24 h and essentially cleared by 28 days.

Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation.

Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels.

Safety toxicity study of plasmid-based IL-12 therapy in Cynomolgus monkeys.

We have investigated the potential toxicity of hlL-12 DNA plasmid formulated with 5% polyvinylpyrrolidone (PVP) administered twice weekly via subcutaneous injections to Cynomolgus monkeys for four weeks, and have evaluated recovery from any effects of the test article over a four-week treatment-free period. Doses of the formulated hIL-12 plasmid were selected based on anti-tumour efficacy studies previously conducted in mice. The duration of the study and the frequency of dosing were designed to support clinical trials.

Ligand-dependent regulation of vascular endothelial growth factor and erythropoietin expression by a plasmid-based autoinducible GeneSwitch system.

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude.

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils.

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.

Background: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium.

Methods And Results: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction.

LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice.

To better define the specific function of Mac-1 (CD11b) versus LFA-1 (CD11a) and the other CD11 integrins in vivo, we have disrupted murine CD11b by targeted homologous recombination in embryonic stem cells and generated mice which are homozygous for a mutation in CD11b. A null mutation was confirmed by Southern blotting, RNase protection assay, immunohistochemistry, and flow cytometry. Neutrophils isolated from mice deficient in Mac-1 were defective in adherence to keyhole limpet hemocyanin-coated glass, iC3b-mediated phagocytosis, and homotypic aggregation.