Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Calcium dependent CAMTA1 in adult stem cell commitment to a myocardial lineage.

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1.

Development of Stimulus-Responsive Ligands for the Modulation of Copper and Iron Coordination

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

Night-time neuronal activation of Cluster N in a day- and night-migrating songbird.

Magnetic compass orientation in a night-migratory songbird requires that Cluster N, a cluster of forebrain regions, is functional. Cluster N, which receives input from the eyes via the thalamofugal pathway, shows high neuronal activity in night-migrants performing magnetic compass-guided behaviour at night, whereas no activation is observed during the day, and covering up the birds' eyes strongly reduces neuronal activation. These findings suggest that Cluster N processes light-dependent magnetic compass information in night-migrating songbirds. The aim of this study was to test if Cluster N is active during daytime migration. We used behavioural molecular mapping based on ZENK activation to investigate if Cluster N is active in the meadow pipit (Anthus pratensis), a day- and night-migratory species. We found that Cluster N of meadow pipits shows high neuronal activity under dim-light at night, but not under full room-light conditions during the day. These data suggest that, in day- and night-migratory meadow pipits, the light-dependent magnetic compass, which requires an active Cluster N, may only be used during night-time, whereas another magnetosensory mechanism and/or other reference system(s), like the sun or polarized light, may be used as primary orientation cues during the day.

Evolution of networks and sequences in eukaryotic cell cycle control.

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.

Can Genetics Predict Response to Complex Behavioral Interventions? Evidence from a Genetic Analysis of the Fast Track Randomized Control Trial.

Early interventions are a preferred method for addressing behavioral problems in high-risk children, but often have only modest effects. Identifying sources of variation in intervention effects can suggest means to improve efficiency. One potential source of such variation is the genome. We conducted a genetic analysis of the Fast Track randomized control trial, a 10-year-long intervention to prevent high-risk kindergarteners from developing adult externalizing problems including substance abuse and antisocial behavior. We tested whether variants of the glucocorticoid receptor gene NR3C1 were associated with differences in response to the Fast Track intervention. We found that in European-American children, a variant of NR3C1 identified by the single-nucleotide polymorphism rs10482672 was associated with increased risk for externalizing psychopathology in control group children and decreased risk for externalizing psychopathology in intervention group children. Variation in NR3C1 measured in this study was not associated with differential intervention response in African-American children. We discuss implications for efforts to prevent externalizing problems in high-risk children and for public policy in the genomic era.

Co-activation of the amygdala, hippocampus and inferior frontal gyrus during autobiographical memory retrieval.

Functional MRI was used to investigate the role of medial temporal lobe and inferior frontal lobe regions in autobiographical recall. Prior to scanning, participants generated cue words for 50 autobiographical memories and rated their phenomenological properties using our autobiographical memory questionnaire (AMQ). During scanning, the cue words were presented and participants pressed a button when they retrieved the associated memory. The autobiographical retrieval task was interleaved in an event-related design with a semantic retrieval task (category generation). Region-of-interest analyses showed greater activation of the amygdala, hippocampus, and right inferior frontal gyrus during autobiographical retrieval relative to semantic retrieval. In addition, the left inferior frontal gyrus showed a more prolonged duration of activation in the semantic retrieval condition. A targeted correlational analysis revealed pronounced functional connectivity among the amygdala, hippocampus, and right inferior frontal gyrus during autobiographical retrieval but not during semantic retrieval. These results support theories of autobiographical memory that hypothesize co-activation of frontotemporal areas during recollection of episodes from the personal past.

Activation in mesolimbic and visuospatial neural circuits elicited by smoking cues: evidence from functional magnetic resonance imaging.

OBJECTIVE: The authors sought to increase understanding of the brain mechanisms involved in cigarette addiction by identifying neural substrates modulated by visual smoking cues in nicotine-deprived smokers. METHOD: Event-related functional magnetic resonance imaging (fMRI) was used to detect brain activation after exposure to smoking-related images in a group of nicotine-deprived smokers and a nonsmoking comparison group. Subjects viewed a pseudo-random sequence of smoking images, neutral nonsmoking images, and rare targets (photographs of animals). Subjects pressed a button whenever a rare target appeared. RESULTS: In smokers, the fMRI signal was greater after exposure to smoking-related images than after exposure to neutral images in mesolimbic dopamine reward circuits known to be activated by addictive drugs (right posterior amygdala, posterior hippocampus, ventral tegmental area, and medial thalamus) as well as in areas related to visuospatial attention (bilateral prefrontal and parietal cortex and right fusiform gyrus). In nonsmokers, no significant differences in fMRI signal following exposure to smoking-related and neutral images were detected. In most regions studied, both subject groups showed greater activation following presentation of rare target images than after exposure to neutral images. CONCLUSIONS: In nicotine-deprived smokers, both reward and attention circuits were activated by exposure to smoking-related images. Smoking cues are processed like rare targets in that they activate attentional regions. These cues are also processed like addictive drugs in that they activate mesolimbic reward regions.

Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

BACKGROUND: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Leptin metabolically licenses T cells for activation to link nutrition and immunity.

Immune responses are highly energy-dependent processes. Activated T cells increase glucose uptake and aerobic glycolysis to survive and function. Malnutrition and starvation limit nutrients and are associated with immune deficiency and increased susceptibility to infection. Although it is clear that immunity is suppressed in times of nutrient stress, mechanisms that link systemic nutrition to T cell function are poorly understood. We show in this study that fasting leads to persistent defects in T cell activation and metabolism, as T cells from fasted animals had low glucose uptake and decreased ability to produce inflammatory cytokines, even when stimulated in nutrient-rich media. To explore the mechanism of this long-lasting T cell metabolic defect, we examined leptin, an adipokine reduced in fasting that regulates systemic metabolism and promotes effector T cell function. We show that leptin is essential for activated T cells to upregulate glucose uptake and metabolism. This effect was cell intrinsic and specific to activated effector T cells, as naive T cells and regulatory T cells did not require leptin for metabolic regulation. Importantly, either leptin addition to cultured T cells from fasted animals or leptin injections to fasting animals was sufficient to rescue both T cell metabolic and functional defects. Leptin-mediated metabolic regulation was critical, as transgenic expression of the glucose transporter Glut1 rescued cytokine production of T cells from fasted mice. Together, these data demonstrate that induction of T cell metabolism upon activation is dependent on systemic nutritional status, and leptin links adipocytes to metabolically license activated T cells in states of nutritional sufficiency.

The liver kinase B1 is a central regulator of T cell development, activation, and metabolism.

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Application of a rank-based genetic association test to age-at-onset data from the Collaborative Study on the Genetics of Alcoholism study.

Association studies of quantitative traits have often relied on methods in which a normal distribution of the trait is assumed. However, quantitative phenotypes from complex human diseases are often censored, highly skewed, or contaminated with outlying values. We recently developed a rank-based association method that takes into account censoring and makes no distributional assumptions about the trait. In this study, we applied our new method to age-at-onset data on ALDX1 and ALDX2. Both traits are highly skewed (skewness > 1.9) and often censored. We performed a whole genome association study of age at onset of the ALDX1 trait using Illumina single-nucleotide polymorphisms. Only slightly more than 5% of markers were significant. However, we identified two regions on chromosomes 14 and 15, which each have at least four significant markers clustering together. These two regions may harbor genes that regulate age at onset of ALDX1 and ALDX2. Future fine mapping of these two regions with densely spaced markers is warranted.