Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway

(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes

Deterioration of glucose homeostasis in type 2 diabetic patients one year after beginning of statins therapy

OBJECTIVE: We evaluated the long-term effects of rosuvastatin and simvastatin on insulin sensitivity and secretion in patients with well-controlled type 2 diabetes. METHODS: After a 3 weeks run-in, 27 eligible patients were randomly assigned to receive either rosuvastatin 20\ua0mg daily (Group 1) or simvastatin 20\ua0mg daily (Group 2) for 6 months; thereafter they were switched to the other treatment for additional 6 months. Patients were recruited among individuals attending the outpatient service of the Diabetology Unit of the "Policlinico Tor Vergata" University Hospital, Rome, Italy. Serum lipids, glucose and insulin, glycated hemoglobin, C-reactive protein, TNF-\u3b1, leptin, adiponectin, insulin sensitivity by euglycemic-hyperinsulinemic clamp, \u3b2-cells function by HOMA-\u3b2 were assessed at months 0, 6 and 12. Additionally, endothelial function was assessed by use of the brachial artery reactivity technique. RESULTS: Besides marked reduction in lipid levels, glycated hemoglobin significantly increased from baseline after 12 months in both Group 1 (+0.8\ua0\ub1\ua00.2%, p\ua0<\ua00.001) and Group 2 (+0.9\ua0\ub1\ua00.3%; p\ua0<\ua00.001). Similar trends were observed for fasting glucose in both groups. No changes in insulin sensitivity were detected throughout the study, whereas HOMA-\u3b2 significantly decreased from baseline after 12 months in both Group 1 (-21.9%, p\ua0<\ua00.01) and Group 2 (-38.9%; p\ua0<\ua00.001). In addition, both treatments similarly decreased C-reactive protein and leptin, as well as improved endothelial function. No changes in anthropometric measures were observed. CONCLUSIONS: In well-controlled type 2 diabetic patients both rosuvastatin and simvastatin significantly impaired glycemic control and insulin secretion, without affecting insulin sensitivity

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF

A Survey of Autoantibodies to Self Antigens in Graves' Disease Patients with Thyroid-Associated Ophthalmopathy

Thyroid Associated Ophthalmopathy (TAO) is a complex pathology to treat and a considerable threat for Graves' Disease (GD) patients. Thus, there is great interest to find tools able to predict the onset and prognosis of TAO. Chronic inflamed tissues are characterized by tissue damage and recruitment of cells from the bloodstream, events that can lead to self-antigen exposure and induce autoimmune phenomena. In this study, we determined whether the occurrence of antibody anti-extracellular matrix (ECM) molecules [Collagen CI, CIII, CIV, CV, laminin (LM) and fibronectin, (FN)], anti-smooth muscle (ASM), anti-nuclear antigen (ANA), anti-ribonucleoprotein (RNP) and anti-thyroid-stimulating hormone (TSH) receptor (TRAbs) were associated with TAO in GD patients. We analyzed serum of 50 patients affected by GD, 24 of whom were affected by TAO, and 40 healthy donors (HD). The occurrence of TRAbs or ANA, anti-SM and anti-RNP antibodies did not allow to discriminate TAO+ from TAO- GD patients. Among the 24 TAO+ and 26 TAO- patients, 15/24 and 17/26 displayed TRAbs, respectively. None of the GD patients displayed anti-RNP antibodies, while 20/24 TAO+ and 17/26 TAO- patients were found to be positive for ANA and 3/24 TAO+ and 4/26 TAO- patients showed anti-SM antibodies. Conversely, when compared with HD control sera, GD sera showed antibodies to all individual ECM molecules. Remarkably, anti-CHI autoantibodies of the IgG isotype were significantly associated with GD TAO+ patients (p=0.045). Indeed, 6 out of 24 GD TAO+ patients scored positive for anti-CIII IgGs as compared to only 1 out of 26 TAO-. Our results suggest the potential involvement of anti-ECM antibodies in GD and a contribution of anti-CIII IgGs in TAO pathogenesis of GD patients

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of Braf V600E in the intestinal tissue of an inducible mouse model. We show that Braf V600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, Braf V600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in Braf V600E-mutant mice. Overall, our work unveils the long-term impact of Braf V600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins

Notopterygium forbesii Boiss extract and its active constituents increase reactive species and heme oxygenase-1 in human fetal hepatocytes: Mechanisms of action

10.1021/tx800301fChemical Research in Toxicology21122414-2423CRTO