Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of V600EBRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of V600EBRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by V600EBRAF/MEK signalling in melanoma cells.

Oestrogen and progestin responses in human endometrium

Our understanding of the mechanisms of the actions of oestrogens and progestins have evolved from the simple concept of nuclear receptor-mediated regulation of transcription to a highly sophisticated, finely tuned interplay between various coregulators, other signaling cascades and transcription factors. The net result of these complex regulatory mechanisms is a steroid-, cell-, or tissue-specific action of oestrogens and progestins. their antagonists or selective modulators of their receptors. In this review, we have attempted to shed some light on the regulation of the actions of oestrogens and progestins on the human endometrium. (C) 2003 Elsevier Science Ltd. All rights reserved.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Quantification of gammaH2AX foci in response to ionising radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX(1). Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)(2). Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB(2,3). This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning approximately 2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete gammaH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy(2). The loss of gammaH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary(4-8). The disappearence of gammaH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C(5,6). Further, removal of gammaH2AX by redistribution involving histone exchange with H2A.Z has been implicated(7,8). Importantly, the quantitative analysis of gammaH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of gammaH2AX foci in gamma-irradiated adherent human keratinocytes(9)

Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.

Quantitation of gammaH2AX foci in tissue samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

Stress at the synapse : signal transduction mechanisms of adrenal steroids at neuronal membranes

As the key neuron-to-neuron interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. However, the signal transduction mechanisms by which stress mediates its lasting effects on synapse transmission and on memory are not fully understood. A key component of the stress response is the increased secretion of adrenal steroids. Adrenal steroids (e.g., cortisol) bind to genomic mineralocorticoid and glucocorticoid receptors (gMRs and gGRs) in the cytosol. In addition, they may act through membrane receptors (mMRs and mGRs), and signal transduction through these receptors may allow for rapid modulation of synaptic transmission as well as modulation of membrane ion currents. mMRs increase synaptic and neuronal excitability; mechanisms include the facilitation of glutamate release through extracellular signal-regulated kinase signal transduction. In contrast, mGRs decrease synaptic and neuronal excitability by reducing calcium currents through N-methyl-D-aspartate receptors and voltage-gated calcium channels by way of protein kinase A- and G protein-dependent mechanisms. This body of functional data complements anatomical evidence localizing GRs to the postsynaptic membrane. Finally, accumulating data also suggest the possibility that mMRs and mGRs may show an inverted U-shaped dose response, whereby glutamatergic synaptic transmission is increased by low doses of corticosterone acting at mMRs and decreased by higher doses acting at mGRs. Thus, synaptic transmission is regulated by mMRs and mGRs, and part of the stress signaling response is a direct and bidirectional modulation of the synapse itself by adrenal steroids.

A neuronal topography of visual and acoustic fear conditioning within the amygdala

The lateral amygdala (LA) receives information from auditory and visual sensory modalities, and uses this information to encode lasting memories that predict threat. One unresolved question about the amygdala is how multiple memories, derived from different sensory modalities, are organized at the level of neuronal ensembles. We previously showed that fear conditioning using an auditory conditioned stimulus (CS) was spatially allocated to a stable topography of neurons within the dorsolateral amygdala (LAd) (Bergstrom et al, 2011). Here, we asked how fear conditioning using a visual CS is topographically organized within the amygdala. To induce a lasting fear memory trace we paired either an auditory (2 khz, 55 dB, 20 s) or visual (1 Hz, 0.5 s on/0.5 s off, 35 lux, 20 s) CS with a mild foot shock unconditioned stimulus (0.6 mA, 0.5 s). To detect learning-induced plasticity in amygdala neurons, we used immunohistochemistry with an antibody for phosphorylated mitogen-activated protein kinase (pMAPK). Using a principal components analysis-based approach to extract and visualize spatial patterns, we uncovered two unique spatial patterns of activated neurons in the LA that were associated with auditory and visual fear conditioning. The first spatial pattern was specific to auditory cued fear conditioning and consisted of activated neurons topographically organized throughout the LAd and ventrolateral nuclei (LAvl) of the LA. The second spatial pattern overlapped for auditory and visual fear conditioning and was comprised of activated neurons located mainly within the LAvl. Overall, the density of pMAPK labeled cells throughout the LA was greatest in the auditory CS group, even though freezing in response to the visual and auditory CS was equivalent. There were no differences detected in the number of pMAPK activated neurons within the basal amygdala nuclei. Together, these results provide the first basic knowledge about the organizational structure of two different fear engrams within the amygdala and suggest they are dissociable at the level of neuronal ensembles within the LA

High and low fear phenotypes show differences in phosphorylated (p44/42 ERK) MAPK-expressing neurons in the lateral amygdala

Post traumatic stress disorder (PTSD) is a serious medical condition effecting both military and civilian populations. While its etiology remains poorly understood it is characterized by high and prolonged levels of fear responding. One biological unknown is whether individuals expressing high or low conditioned fear memory encode the memory differently and if that difference underlies fear response. In this study we examined cellular mechanisms that underlie high and low conditioned fear behavior by using an advanced intercrossed mouse line (B6D2F1) selected for high and low Pavlovian fear response. A known requirement for consolidation of fear memory, phosphorylated mitogen activated protein kinase (p44/42 (ERK) MAPK (pMAPK)) in the lateral amygdala (LA) is a reliable marker of fear learning-related plasticity. In this study, we asked whether high and low conditioned fear behavior is associated with differential pMAPK expression in the LA and if so, is it due to an increase in neurons expressing pMAPK or increased pMAPK per neuron. To examine this, we quantified pMAPK-expressing neurons in the LA at baseline and following Pavlovian fear conditioning. Results indicate that high fear phenotype mice have more pMAPK-expressing neurons in the LA. This finding suggests that increased endogenous plasticity in the LA may be a component of higher conditioned fear responses and begins to explain at the cellular level how different fear responders encode fear memories. Understanding how high and low fear responders encode fear memory will help identify novel ways in which fear-related illness risk can be better predicted and treated.

Neurobiological correlates of encoding strength of Pavlovian fear conditioned memory

Emotionally significant memories, especially those induced in conjunction with physical and mental trauma, are frequently retained for an individual’s lifetime. How these memories are organized and encoded within neural networks is a fundamental question. The lateral amygdala (LA) is a key nucleus for acquisition and maintenance of associative emotional memories. We used Pavlovian fear conditioning to study how ‘weaker’ and ‘stronger’ memories are encoded in neural networks of the LA. In Pavlovian fear conditioning a neutral stimulus, in this case a tone, is temporally paired with an aversive unconditioned stimulus (US), such as a foot shock. The previously neutral stimulus becomes a conditioned stimulus (CS) capable of eliciting defensive responses. We used time spent freezing when the CS is presented in a neutral context as a dependent variable measure of memory ‘strength’.

AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle

Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole- 4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P < 0.05) in blood glucose and an increase (P < 0.05) in insulin concentration without a change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P < 0.05) in skeletal muscle. While GLUT4 and GLUT1 protein expression remained unchanged, GLUT8 was increased (P < 0.05) following AICAR treatment. Up-regulation of GLUT8 protein expression by AICAR suggests that this novel GLUT isoform plays an important role in equine muscle glucose transport. In addition, the data suggest that AMPK activation enhances pancreatic insulin secretion. Collectively, the findings suggest that AICAR acutely promotes muscle glucose uptake in healthy horses and thus its therapeutic potential for managing IR requires investigation.

Novel mutations in ACVR1 result in atypical features in two fibrodysplasia ossificans progressiva patients

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, heritable condition typified by progression of extensive ossification within skeletal muscle, ligament and tendon together with defects in skeletal development. The condition is easily diagnosed by the presence of shortened great toes and there is severe advancement of disability with age. FOP has been shown to result from a point mutation (c.617G>A) in the ACVR1 gene in almost all patients reported. Very recently two other mutations have been described in three FOP patients. We present here evidence for two further unique mutations (c.605G>T and c.983G>A) in this gene in two FOP patients with some atypical digit abnormalities and other clinical features. The observation of disparate missense mutations mapped to the GS and kinase domains of the protein supports the disease model of mild kinase activation and provides a potential rationale for phenotypic variation. © 2009 Petrie et al.

Interleukin-23 mediates the intestinal response to microbial β-1,3-glucan and the development of spondyloarthritis pathology in SKG mice

Objective Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70W163C-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan). Methods Eight weeks after curdlan injection in wild-type or IL-17A-/- SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs. Results In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22. Conclusion In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance.