Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.

BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.

A novel immunogenic mouse model of melanoma for the preclinical assessment of combination targeted and immune-based therapy.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1). We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.

Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters.

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters. Cells were "shotgun" transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a range of expression levels. We used flow cytometry to analyze the populations and generate high-resolution, nearly continuous Ras dose-response curves. These efforts revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression lead to even greater signal transduction, this increased expression had minimal or decreasing effects on the proliferation rate. In addition, this study introduces a general method to quantify genetic dose-response relationships and identify gene expression ranges that produce an optimized phenotypic response.

Assessment of the role of MAP kinase in mediating activity-dependent transcriptional activation of the immediate early gene Arc/Arg3.1 in the dentate gyrus in vivo.

Different physiological and behavioral events activate transcription of Arc/Arg3.1 in neurons in vivo, but the signal transduction pathways that mediate induction in particular situations remain to be defined. Here, we explore the relationships between induction of Arc/Arg3.1 transcription in dentate granule cells in vivo and activation of mitogen-activated protein (MAP) kinase as measured by extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. We show that ERK1/2 phosphorylation is strongly induced in dentate granule cells within minutes after induction of perforant path long-term potentiation (LTP). Phospho-ERK staining appears in nuclei within minutes after stimulation commences, and ERK phosphorylation returns to control levels within 60 min. Electroconvulsive seizures, which strongly induce prolonged Arc/Arg3.1 transcription in dentate granule cells, induced ERK1/2 phosphorylation in granule cells that returned to control levels within 30 min. Following 30, 60, and 120 min of exploration in a novel complex environment, Arc/Arg3.1 transcription was activated in many more granule cells than stained positively for p-ERK at all time points. Although Arc/Arg3.1 transcription was induced in most pyramidal neurons in CA1 following exploration, very few pyramidal neurons exhibited nuclear p-ERK1/2 staining. Local delivery of U0126 during the induction of perforant path LTP blocked transcriptional activation of Arc/Arg3.1 in a small region near the injection site and blocked Arc/Arg3.1 protein expression over a wider region. Our results indicate that activation of Arc/Arg3.1 transcription in dentate granule cells in vivo is mediated in part by MAP kinase activation, but other signaling pathways also contribute, especially in the case of Arc/Arg3.1 induction in response to experience.

Risk assessment of tetrabromobisphenol A on cyclooxygenase-2 expression via MAP kinase/NF-kappaB/AP-1 signaling pathways in murine macrophages.

Tetrabromobisphenol A [2,2-bis-(3,5-dibromo-4-hydroxyphenyl)propane; TBBPA] is used worldwide as a flame retardant in numerous products. In the present study, the effects of TBBPA were examined on the expression of cyclooxygenase-2 (COX-2), inflammation-related cytokines, transcription factors, and signaling pathways responsible for transcriptional activation of the COX-2 gene in murine RAW 264.7 macrophages. Exposure to TBBPA markedly enhanced the production of prostaglandin E(2), a major COX-2 metabolite, in macrophages. TBBPA concentration-dependently increased the levels of COX-2 protein and mRNA. In addition, TBBPA increased the secretion and mRNA levels of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Transfection of a human COX-2 promoter construct demonstrated that TBBPA induced COX-2 promoter activity. Furthermore, transfection with pNF-kappaB-Luc and pAP-1-Luc plasmid revealed that TBBPA activated the NF-kappaB and AP-1 sites. Phosphatidylinositol 3 (PI3) kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by TBBPA. Our data demonstrate TBBPA-induced COX-2 and proinflammatory cytokine expression occurs through the PI3-kinase/Akt/MAP kinase/NF-kappaB/AP-1 pathways.

Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma.

PURPOSE: To determine whether retinal glial cells exhibit an activated phenotype in glaucomatous human eyes and whether the mitogen-activated protein kinases (MAPKs) are associated with glial activation in glaucoma. METHODS: Activated phenotypes of retinal macroglia (astrocytes and Müller cells) and microglia were identified by morphologic assessment and immunostaining for the cell markers glial fibrillary acidic protein (GFAP) and HLA-DR, respectively, in 30 eyes obtained from glaucomatous donor eyes in comparison with normal control eyes from 20 age-matched donors. Cellular localization of the activated forms of MAPKs, including extracellular signal-regulated kinases (ERK), c-Jun amino(N)-terminal kinase (JNK), and p38, were studied in the retina of these eyes by immunoperoxidase staining and double immunofluorescence labeling with phosphorylation site-specific antibodies. RESULTS: Retinal astrocytes and Müller cells exhibited a hypertrophic morphology and increased immunostaining for GFAP in the glaucomatous retina. Although an increase was detectable in the number and size of cells positive for HLA-DR immunostaining in the glaucomatous retina compared with the control retina, microglial activation was not as prominent or widespread as the macroglial activation detected in the same eyes. The intensity of immunostaining and the number of immunostained cells for the activated MAPKs were greater in retina sections from glaucomatous eyes than in control eyes, being most prominent for phospho-ERK. Double immunofluorescence labeling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly, but not exclusively, localized to glial cells, whereas, the immunostaining for phospho-JNK or phospho-p38 was mainly associated with nonglial cells. CONCLUSIONS: These findings provide evidence that retinal glial cells undergo activation in the glaucomatous human retina. A prominent and persistent activation of ERK in activated glial cells suggests that this signaling pathway is probably associated with the induction and/or maintenance of the activated glial phenotype in glaucoma. Because MAPKs are involved in determination of ultimate cell fate, their differential activity in neuronal and activated glial cells in the glaucomatous retina may be associated, in part, with the differential susceptibility of these cell types to glaucomatous injury.