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The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light.sup.1,2. In mice, OPN5 is a known photoreceptor in the retina.sup.3 and skin.sup.4 but is also expressed in the hypothalamic preoptic area (POA).sup.5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis. Mice possess neurons in the preoptic area of the hypothalamus that are sensitive to violet light; these deep brain neurons sense light via OPN5 and regulate adaptive thermogenesis in brown fat., Author(s): Kevin X. Zhang [sup.1] [sup.2] [sup.3] [sup.4] , Shane D'Souza [sup.1] [sup.2] [sup.3] , Brian A. Upton [sup.1] [sup.2] [sup.3] [sup.4] , Stace Kernodle [sup.5] , Shruti Vemaraju [sup.1] [...]

The vitamin D receptor (VDR) is the single known regulatory mediator of hormonal 1,25-dihydroxyvitamin [D.sub.3] [1,25[(OH).sub.2][D.sub.3]] in higher vertebrates. It acts in the nucleus of vitamin D target cells to regulate the expression of genes whose products control diverse, cell type-specific biological functions that include mineral homeostasis. In this Review we describe progress that has been made in defining new cellular sites of action of this receptor, the mechanisms through which this mediator controls the expression of genes, the biology that ensues, and the translational impact of this receptor on human health and disease. We conclude with a brief discussion of what comes next in understanding vitamin D biology and the mechanisms that underlie its actions., Introduction In the early 1970s, 1,25-dihydroxyvitamin [D.sub.3] [1,25[(OH).sub.2][D.sub.3]] was identified as both the exclusive, metabolically active form of vitamin D and a key component of what proved to be an [...]

Mast cells (MCs) are present in various tissues and are responsible for initiating many of the early inflammatory responses to extrinsic challenges. Recent studies have demonstrated that MCs can tailor their responses, depending on the stimulus encountered and the tissue in which they are stimulated. In this issue of the JCI, Gaudenzio and colleagues examine the mechanistic differences between MC responses observed after engagement of Fcζ receptor I and those seen after MC stimulation via the recently identified G protein-coupled receptor MRGPRX2. By showing that discrete cellular activation patterns affect the phenotype of the MC response in vivo and in vitro, the authors provide important information about how MCs differentially process various stimuli into distinct degranulation programs., Mast cells: sentinel cells and first responders Increasing lines of evidence indicate that mast cells (MCs) are key coordinators of both homeostasis and inflammation in a variety of tissues. Conventionally, [...]

Angiopoietin-1/Tie2 (ANG1/Tie2) signaling is well documented as regulating angiogenesis and vessel maturation. This pathway is complicated by involvement of the orphan receptor Tie1, which has been implicated as both a positive and negative regulator of ANG1/Tie2 signaling, and ANG2, which can serve as both a Tie2 agonist and antagonist, depending on the context. Two papers in this issue of the JCI provide new insight into this complicated pathway. Korhonen et al. reveal that Tie1 acts to modulate the effects of ANG1 and ANG2 on Tie2 in vitro and in vivo. Kim et al. demonstrate that ANG2 acts as a Tie2 agonist in non-pathological conditions, whereas in the setting of inflammation, ANG2 functions as a Tie2 antagonist and promotes vascular dysfunction. Both studies indicate that inflammation promotes cleavage of the ectodomain of Tie1 and that this cleavage event corresponds with the switch of ANG2 from a Tie2 agonist to an antagonist. The results of these studies lay the groundwork for future strategies to therapeutically exploit this pathway in diseases characterized by adverse vascular remodeling and increased permeability., Angiopoietin/Tie receptor signaling: a complex relationship Tie receptors (tyrosine kinases with immunoglobulin and epidermal growth factor homology domains) are a family of receptor tyrosine kinases (RTKs) that are expressed primarily [...]

Tumor necrosis factor (TNF) superfamily member 11 (TNFSF11, also known as RANKL) regulates multiple physiological or pathological functions, including osteoclast differentiation and osteoporosis. TNFRSF11A (also called RANK) is considered to be the sole receptor for RANKL. Herein we report that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL. LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling during osteoclast differentiation. RANKL binding to LGR4 activates the Gaq and GSK3-p signaling pathway, an action that suppresses the expression and activity of nuclear factor of activated T cells, cytoplasmic, calcineurindependent 1 (NFATC1) during osteoclastogenesis. Both whole-body ([Lgr4.sup.-/-]) and monocyte conditional knockout mice of Lgr4 (Lgr4 CKO) exhibit osteoclast hyperactivation (including elevation of osteoclast number, surface area, and size) and increased bone erosion. The soluble LGR4 extracellular domain (ECD) binds RANKL and inhibits osteoclast differentiation in vivo. Moreover, LGR4-ECD therapeutically abrogated RANKL-induced bone loss in three mouse models of osteoporosis. Therefore, LGR4 acts as a second RANKL receptor that negatively regulates osteoclast differentiation and bone resorption., Bone-mass regulation depends on the dynamic balance between bone formation and bone resorption, which are driven by osteoblast activation and osteoclast activation, respectively. RANKL is a central positive regulator of [...]

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft- versushost disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an [HLA-A2.sup.-]specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by [HLA-A2.sup.+] T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases., Introduction The essential role of regulatory T cells (Tregs) in preventing autoimmunity and controlling responses to alloantigens is well established. Multiple Treg-based cell therapy approaches are now being tested in [...]

Tumor cells frequently escape from [CD8.sup.+] T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the 'self' TRH4 peptide/[D.sup.b] complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-[I.sup.lo]). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific [CD8.sup.+] T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies., Introduction Cytotoxic [CD8.sup.+] T cells are key players of the immune system that kill virus-infected and cancerous cells by sensing the state of the cellular proteome. A major focus of [...]

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding., Introduction Thrombosis-associated events are among the leading causes of death worldwide. Systemic infections caused by a plethora of bacterial genera can initiate thrombus development. While the mechanisms that underlie this [...]

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG- GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders., Introduction Mammalian platelets are small anucleated blood cells specialized to continuously monitor and preserve the integrity of the cardiovascular system (hemostasis) (1-3). Once released from megakaryocytes, they circulate for 10 [...]

Osteoclasts are bone-resorbing cells that are Important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis., Introduction Bone undergoes constant remodeling by balancing the antagonistic activities of bone-forming osteoblasts and bone lytic osteoclasts (1-3). Bone loss resulting from increased osteoclast differentiation and activity (1, 4) can [...]

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases., Fibrotic diseases impose a major socioeconomic burden on modern societies and account for up to 45% of deaths in the developed world (1). TGF-β is a master regulator of mesenchymal [...]

Learning and memory deficits are early clinical manifestations of Huntington's disease (HD). These cognitive impairments have been mainly associated with frontostriatal HD pathology; however, compelling evidence provided by several HD murine models suggests that the hippocampus may contribute to synaptic deficits and memory dysfunction in HD. The neurotrophin receptor [p75.sup.NTR] negatively regulates spine density, which is associated with learning and memory; therefore, we explored whether disturbed [p75.sup.NTR] function in the hippocampus could contribute to synaptic dysfunction and memory deficits in HD. Here, we determined that levels of [p75.sup.NTR] are markedly increased in the hippocampus of 2 distinct mouse models of HD and in HD patients. Normalization of [p75.sup.NTR] levels in HD mutant mice heterozygous for [p75.sup.NTR] prevented memory and synaptic plasticity deficits and ameliorated dendritic spine abnormalities, likely through normalization of the activity of the GTPase RhoA. Moreover, viral-mediated overexpression of [p75.sup.NTR] in the hippocampus of WT mice reproduced HD learning and memory deficits, while knockdown of [p75.sup.NTR] in the hippocampus of HD mice prevented cognitive decline. Together, these findings provide evidence of hippocampus-associated memory deficits in HD and demonstrate that [p75.sup.NTR] mediates synaptic, learning, and memory dysfunction in HD., Introduction Evidence of cognitive deficits including altered acquisition of new motor skills, paired attention, planning, and memory has been demonstrated in Huntington's disease (HD) patients before the onset of motor [...]

Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that [P2Y.sub.14] modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, [P2Y.sub.14] deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking [P2Y.sub.14]. Enhanced senescence coincided with increased ROS, elevated [p16.sup.INK4a] expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the [P2Y.sub.14] phenotype, suggesting that [P2Y.sub.14] mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking [P2Y.sub.14] were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that [P2Y.sub.14] on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that [P2Y.sub.14]-mediated responses prevent the premature decline of regenerative capacity after injury., Introduction Organisms inevitably encounter a variety of stresses during their lifetimes, including radiation, oxidation, and infection. The nature and efficiency of the response to stress is a fundamental determinant of [...]

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease., Introduction Sphingosine-1-phosphate receptor 1 (S1P1), originally named EDG-1, is the founding member of a family of 5 GPCRs with high affinity for the lipid ligand sphingosine-1-phosphate (S1P) (1-3). Activation ofS1P1 [...]

Background: Behavioural sensitization has been linked to drug craving in both clinical and preclinical studies of addiction. Increased motor activity is accompanied by enhanced dopamine (DA) release, particularly in the nucleus accumbens (NAcc). The neural bases of sensitization are linked to alterations in synaptic connections that also underlie learning and memory. The present study uses an 'interference' peptide, [Tat-GluA2.sub.3Y], that blocks long-term depression (LTD) at glutamatergic synapses by disrupting the endocytosis of aamino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs), to explore the role of this form of synaptic plasticity in the induction and maintenance of sensitization. Methods: Rats were given 5 injections of d-amphetamine (d-AMPH, 1.0 mg/kg, intraperitoneal) every second day. [Tat-GluA2.sub.3Y], was administered by 2 different routes (intravenously and intracerebrally to the ventral tegmental area [VTA] or to the NAcc) before each injection of d-AMPH. After a 14-day drug-free period, expression of behavioural sensitization was evoked by a challenge injection of d-AMPH (0.5 mg/kg, intraperitoneal). Dopamine efflux in the NAcc was measured by high-pressure liquid chromatography with electrochemical detection analyses of brain dialysates on days 1, 9 and 24 of the intravenous peptide experiment. Results: Systemic administration of [Tat-GluA2.sub.3Y] during the induction phase blocked maintenance of behavioural sensitization and attenuated the maintenance of neurochemical sensitization. Intra-VTA infusion of [Tat-GluA2.sub.3Y] before each administration of d-AMPH did not affect induction, but inhibited maintenance and subsequent expression of sensitization, whereas intra-NAcc infusion of the peptide did not affect induction or maintenance of sensitization. Limitations: The relevance of behavioural sensitization in rodents is related to the development of craving and does not provide direct measures of drug reinforcement. Conclusion: These findings confirm that drug-induced neuroplasticity is labile and may be subject to disruption at a time when long-lasting associations between drug reward and contextual stimuli are formed. Furthermore, the unique ability of [Tat-GluA2.sub.3Y] to block maintenance of behavioural sensitization implicates LTD in the consolidation of essential associative memories. [Tat-GluA2.sub.3Y] has the unique ability to disrupt functional neuroadaptations triggered by repeated psychostimulant exposure and therefore may protect against the development of craving and drug seeking behaviours., Introduction Persistent thoughts and actions related to the procurement and use of illicit drugs, along with vulnerability to relapse following exposure to contextual stimuli associated with drugs of abuse, are [...]

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection (1). However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response., Memory B lymphocytes contribute to the protective immune response to flu infection by producing immunoglobulins that bind and neutralize the virus (1). The lung of an exposed individual contains influenza-specific [...]

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific [...]

A systemic inflammatory response is observed in patients undergoing hemorrhagic shock and sepsis. Here we report increased levels of cold-inducible RNA-binding protein (CIRP) in the blood of individuals admitted to [...]

Jawed vertebrates (gnathostomes) and jawless vertebrates (cyclostomes) have different adaptive immune systems (1,2). Gnathostomes use T- and B-cell antigen receptors belonging to the immunoglobulin superfamily (3,4). Cyclostomes, the lampreys and hagfish, instead use leucine-rich repeat proteins to construct variable lymphocyte receptors (VLRs), two types of which, VLRA and VLRB, are reciprocally expressed by lymphocytes resembling gnathostome T and B cells (5-7). Here we define another lineage of T-cell-like lymphocytes that express the recently identified VLRC receptors (8,9). Both [VLRC.sup.+] and [VLRA.sup.+] lymphocytes express orthologues of genes that gnathostome γδ and αβ T cells use for their differentiation, undergo VLRC and VLRA assembly and repertoire diversification in the 'thymoid' gill region, and express their VLRs solely as cell-surface proteins. Our findings suggest that the genetic programmes for two primordial T-cell lineages and a prototypic B-cell lineage were already present in the last common vertebrate ancestor approximately 500 million years ago. We propose that functional specialization of distinct T-cell-like lineages was an ancient feature of a primordial immune system., The invariant stalk region of the sea lamprey VLRC shares approximately 20% sequence identity with the invariant VLRA and VLRB stalk regions, and distinguishing VLRC sequences are also present in [...]

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs [...]

Cholecystokinin (CCK) is a satiety hormone produced by discrete enteroendocrine cells scattered among absorptive cells of the small intestine. CCK is released into blood following a meal; however, the mechanisms [...]

The numbers and types of ionotropic glutamate receptors at most vertebrate central excitatory synapses are altered as a function of changes in input activity patterns that occur during postnatal development. Activity-dependent developmental alterations in glutamate receptors underlie lasting changes in synaptic efficacy (plasticity) and metaplasticity (the plasticity of synaptic plasticity), which are critical elements of normal brain maturation. Understanding the specific involvement of glutamate receptors in synaptic development and function is made multiplicatively complex by the existence of a large number of glutamate receptor subunits, numerous subunit-specific amino acid sequences that regulate receptor function, and subunit-specific synaptic insertion restrictions imposed by associated anchoring proteins. Many receptor properties are altered when subunits are switched, so it is unclear which individual receptor property or properties underlie changes in synaptic function and plasticity during postnatal development. As a result, a more detailed understanding of the factors that regulate synaptic and cognitive development will involve mutations in glutamate receptor subunits that separate individual receptor properties and permit synaptic insertion at both immature and mature synapses in genetically modified organisms. This position paper focuses on structural modifications in N-methyl-D-aspartate receptors (NMDARs) that occur during postnatal forebrain development and attempts to provide a method for pursuing a more complete understanding of the functional ramifications of developmental alterations in NMDAR subunit composition., Introduction N-methyl-D-aspartate receptors (NMDARs) are neurotransmitter receptors that are ubiquitous to glutamatergic synapses in the central nervous system. NMDARs are involved in basic synaptic transmission and serve as a triggering [...]

Cells of the immune system have evolved various molecular mechanisms to sense their environment and react to alterations of self. NK cells are lymphocytes with effector and regulatory functions, which are remarkably adaptable to changes in self. In a study published in this issue of the JCI, Tarek and colleagues report the clinical benefits of manipulating NK cell adaptation to self in an innovative mAb-based therapy against neuroblastoma (NB). This novel therapeutic strategy should stimulate further research on NK cell therapies., NK cells are involved in the elimination of tumor cells and infected cells (1); they can kill their cellular targets via cytotoxic granule exocytosis and also secrete cytokines such as [...]

PAX5, a B cell-specific transcription factor, is overexpressed through chromosomal translocations in a sub-set of B cell lymphomas. Previously, we had shown that activation of immunoreceptor tyrosine-based activa-tion motif (ITAM) proteins and B cell receptor (BCR) signaling by PAX5 contributes to B-lymphomagenesis. However, the effect of PAX5 on other oncogenic transcription factor-controlled pathways is unknown. Using a MYC-induced murine lymphoma model as well as MYC-transformed human B cell lines, we found that PAX5 controls c-MYC protein stability and steady-state levels. This promoter-independent, posttranslational mechanism of c-MYC regulation was independent of ITAM/BCR activity. Instead it was controlled by another PAX5 target, CD19, through the PI3K-AKT-GSK3 β axis. Consequently, MYC levels in B cells from CD19-defi-cient mice were sharply reduced. Conversely, reexpression of CD19 in murine lymphomas with spontaneous silencing of PAX5 boosted MYC levels, expression of its key target genes, cell proliferation in vitro, and over-all tumor growth in vivo. In human B-lymphomas, CD19 mRNA levels were found to correlate with those of MYC-activated genes. They also negatively correlated with the overall survival of patients with lymphoma in the same way that MYC levels do. Thus, CD19 is a major BCR-independent regulator of MYC-driven neoplastic growth in B cell neoplasms., Introduction The concept of oncogene addiction (1) is particularly well validated in hematological malignancies. 'Liquid' tumors are thought to depend on fewer genetic alterations and thus be more sensitive to [...]

In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8+ T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8+ T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination., Introduction Pressure to detect infection has shaped the evolution of the immune system. Several families of pathogen recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) and activate myeloid cells to [...]

DNGR-1 (CLEC9A) is a receptor for necrotic cells required by DCs to cross-prime CTLs against dead cell antigens in mice. It is currently unknown how DNGR-1 couples dead cell recognition to cross-priming. Here we found that DNGR-1 did not mediate DC activation by dead cells but rather diverted necrotic cell cargo into a recycling endosomal compartment, favoring cross-presentation to CD [8.sup.+] T cells. DNGR-1 regulated cross-priming in non-infectious settings such as immunization with antigen-bearing dead cells, as well as in highly immunogenic situations such as infection with herpes simplex virus type 1. Together, these results suggest that DNGR-1 is a dedicated receptor for cross-presentation of cell-associated antigens. Our work thus underscores the importance of cross-priming in immunity and indicates that antigenicity and adjuvanticity can be decoded by distinct innate immune receptors. The identification of specialized receptors that regulate antigenicity of virus-infected cells reveals determinants of antiviral immunity that might underlie the human response to infection and vaccination., Introduction DCs are central players in the establishment and maintenance of self-tolerance, as well as in the induction of immunity against invading microorganisms (1, 2). In mice and humans, distinct [...]

In the bacterial chemotaxis network, receptor clusters process input (1-3), and flagellar motors generate output (4). Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell (5). However, the motor is ultrasensitive, with a narrow operating range of CheY-P concentrations (6). How the match between receptor output and motor input might be optimized is unclear. Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase (7,8) and why motors show signal-dependent FliM turnover (9). Adaptive remodelling is likely to be a common feature in the operation of many molecular machines., The chemotaxis signalling pathway allows bacterial cells to sense and respond to changes in concentrations of chemical attractants or repellents (1,2). Binding of chemicals by receptors modulates the activity of [...]

Like all heterotrophic organisms, insects require a strict control of food intake and efficient digestion of food into nutrients to maintain homeostasis and to fulfill physiological tasks. Feeding and digestion are steered by both external and internal signals that are transduced by a multitude of regulatory factors, delivered either by neurons innervating the gut or mouthparts, or by midgut endocrine cells. The present review gives an overview of peptide regulators known to control feeding and digestion in insects. We describe the discovery and functional role in these processes for insect allatoregulatory peptides, diuretic hormones, FMRFamide-related peptides, (short) neuropeptide F, proctolin, saliva production stimulating peptides, kinins, and tachykinins. These peptides control either gut myoactivity, food intake, and (or) release of digestive enzymes. Some peptides exert their action at multiple levels, possibly having a biological function that depends on their site of delivery. Many regulatory peptides have been physically extracted from different insect species. However, multiple peptidomics, proteomics, transcriptomics, and genome sequencing projects have led to increased discovery and prediction of peptide (precursor) and receptor sequences. In combination with physiological experiments, these large-scale projects have already led to important steps forward in unraveling the physiology of feeding and digestion in insects. Key words: insect, midgut, digestion, neuropeptides, stomatogastric nervous system. Comme tous les organismes heterotrophes, les insectes requierent un controle strict de leur ingestion de nourriture et la digestion de leur nourriture en nutriments afin de maintenir leur homeostasie et de completer leurs taches physiologiques. L'alimentation et la digestion sont gouvernees par des signaux tant externes qu'internes qui sont transmis par un multitude de facteurs regulateurs, livres ou bien par les neurones qui innervent le tube digestif ou les pieces buccales ou alors par les cellules endocrines du tube digestif moyen. Notre revue fournit une synthese des regulateurs peptidiques connus pour controler l'alimentation et la digestion chez les insectes. Nous decrivons la decouverte et le role fonctionnel de ces processus dans le cas des peptides allatoregulateurs, des hormones diuretiques, des peptides apparentes aux amides FMRF, du neuropeptide F (court), de la proctoline, des peptides stimulateurs de la production de salive, des kinines et des tachykinines des insectes. Ces peptides controlent la myoactivite du tube digestif, l'ingestion de nourriture et(ou) la liberation des enzymes digestives. Certains peptides agissent a de multiples niveaux, possedant une fonction biologique qui depend de leur site d'action. Plusieurs peptides regulateurs ont ete extraits physiquement de differentes especes d'insectes. Cependant, plusieurs projets de peptidomique, de proteomique, de transcriptomique et de genomique ont mene a de nombreuses decouvertes supplementaires et des predictions de sequences de peptides (precurseurs) et de recepteurs. En combinaison avec les experiences physiologiques, ces projets a grande echelle ont deja mene a des progres importants dans l'elucidation de la physiologie de l'alimentation et de la digestion chez les insectes. Mots-cles: insecte, tube digestif moyen, digestion, neuropeptides, systeme nerveux stomogastrique. [Traduit par la Redaction], Introduction Eumetazoa are heterotrophs that acquire organic compounds through feeding. Therefore, strict control of food intake and efficient digestion of food into nutrients are indispensible for these organisms to maintain [...]

Diuresis, the removal of excess metabolic waste through production of primary urine while maintaining homeostasis, is an important biological process that is tightly regulated by endocrine factors. Several hormonal components that act as diuretic or antidiuretic factors in insects have been identified in the last few decades. Physiological mechanisms responsible for ion and water transport across biological membranes have been intensively studied. The large amount of data rapidly accumulating in the genomics era has led to an increased dependence on reverse genetic and physiological approaches, first identifying candidate genes and subsequently deriving functions. In many cases, the reverse approaches have been highly successful, especially in studies of the receptors for diuretic factors, which are mainly G-protein-coupled receptors. This review summarizes research on insect diuretic and antidiuretic endocrine factors, and their receptors. Emphases of the review are given to the genomics of ligands and their receptors, as well as to their implications for evolution and function. Key words: neuropeptide, hormone, G-protein-coupled receptor, guanylate cyclase, malpighian tubule. La diurese, le retrait des dechets metaboliques en exces par la production d'urine primaire, tout en maintenant l'homeostasie, est un processus biologique important qui est fortement regule par des facteurs endocrines. Plusieurs composantes hormonales qui agissent comme facteurs diuretiques ou antidiuretiques chez les insectes ont ete identifies au cours des quelques dernieres decennies. Les mecanismes physiologiques responsables du transport des ions et de l'eau a travers les membranes biologiques ont fait l'objet d'etudes approfondies. La grande quantite de donnees qui s'accumule rapidement dans notre ere de genomique a mene a une dependance accrue sur les methodologies de genetique inverse et de physiologie, identifiant d'abord les genes candidats et ensuite derivant leur fonction. Dans plusieurs cas, les methodologies inverses ont eu un franc succes, particulierement dans les etudes des recepteurs pour les facteurs diuretiques, qui sont principalement des recepteurs couples a des proteines G. Cette retrospective resume la recherche sur les facteurs endocrines diuretiques et antidiuretiques et leurs recepteurs. Une emphase particuliere est mise sur la genomique des ligands et de leurs recepteurs, de meme que sur les repercussions sur leur evolution et fonction. Mots-cles: neuropeptide, hormone, recepteur couple aux proteines G, guanylate cyclase, tubule de Malpighi. [Traduit par la Redaction], Introduction Diuresis is an essential physiological process for the removal of potentially toxic metabolic wastes and for maintaining water and ion balance in the medium surrounding vital cells and organs. [...]

Immunological and neural synapses share properties such as the synaptic cleft, adhesion molecules, stability, and polarity. However, the mismatch in scale has limited the utility of these comparisons. The discovery of phosphatase micro-exclusion from signaling elements in immunological synapses and innate phagocytic synapses define a common functional unit at a common sub-micron scale across synapse types. Bundling of information from multiple antigen receptor microclusters by an immunological synapse has parallels to bundling of multiple synaptic inputs into a single axonal output by neurons, allowing integration and coincidence detection. Bonafide neuroimmune synapses control the inflammatory reflex. A better understanding of the shared mechanisms between immunological and neural synapses could aid in the development of new therapeutic modalities for immunological, neurological, and neuroimmunological disorders alike., Introduction Cell-cell communication systems in the immune and nervous systems share several features, which has led to the adoption of the common term 'synapse' to describe the close cell-cell contacts [...]

Stroke is the third leading cause of death in the United States. Fewer than 5% of patients benefit from the only intervention approved to treat stroke. Thus, there is an [...]

BACKGROUND: The occurrence of halogenated analogs of the xenoestrogen bisphenol A (BPA) has been recently demonstrated both in environmental and human samples. These analogs include brominated [e.g., tetrabromobisphenol A (TBBPA)] [...]

The successful treatment of certain autoimmune conditions with the humanized anti-IL-6 receptor (IL-6R) antibody tocilizumab has emphasized the clinical importance of cytokines that signal through the β -receptor subunit glycoprotein 130 (gp130). In this Review, we explore how gp130 signaling controls disease progression and examine why IL-6 has a special role among these cytokines as an inflammatory regulator. Attention will be given to the role of the soluble IL-6R, and we will provide a perspective into the clinical blockade of IL-6 activity in autoimmunity, inflammation, and cancer., Introduction Inflammation is not a disease but a consequence of disease and is the body's defense against infection or injury. When effective, the inflammatory response ensures successful resolution of the [...]

The accumulation of AMPA receptors (AMPARs) at synapses is essential for excitatory synaptic transmission. However, the mechanisms underlying synaptic targeting of AMPARs remain elusive. We have now used a molecular replacement approach on an AMPAR-null background to investigate the targeting mechanisms necessary for regulating AMPAR trafficking in the hippocampus. Although there is an extensive literature on the role of the GluA1 C-tail in AMPAR trafficking, there is no effect of overexpressing the C-tail on basal transmission. Instead, we found that the first intracellular loop domain (Loop1) of GluA1, a previously overlooked region within AMPARs, is critical for receptor targeting to synapses, but not for delivery of receptors to the plasma membrane. We also identified a CaMKII phosphorylation site (S567) in the GluA1 Loop1, which is phosphorylated in vitro and in vivo. Furthermore, we show that S567 is a key residue that regulates Loop1-mediated AMPAR trafficking. Thus, our study reveals a unique mechanism for targeting AMPARs to synapses to mediate synaptic transmission. GluA2 | GluR1 | postsynaptic density | hippocampus | pyramidal neurons doi/ 10.1073/pnas.1016289107

Proteins of the major histocompatibility complex class I (MHCI) are known for their role in immunity and have recently been implicated in long-term plasticity of excitatory synaptic transmission. However, the mechanisms by which MHCI influences synaptic plasticity remain unknown. Here we show that endogenous MHCI regulates synaptic responses mediated by NMDA-type glutamate receptors (NMDARs) in the mammalian central nervous system (CNS). The AMPA/NMDA ratio is decreased at MHCI-deficient hippocampal synapses, reflecting an increase in NMDAR-mediated currents. This enhanced NMDAR response is not associated with changes in the levels, subunit composition, or gross subcellular distribution of NMDARs. Increased NMDAR-mediated currents in MHCI-deficient neurons are associated with characteristic changes in AMPA receptor trafficking in response to NMDAR activation. Thus, endogenous MHCI tonically inhibits NMDAR function and controls downstream NMDAR-induced AMPA receptor trafficking during the expression of plasticity. immune | GluR | hippocampus doi/ 10.1073/pnas.0914064107

The all-trans-retinoic acid (atRA) isomer, 9-cis-retinoic acid (9cRA), activates retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in vitro. RARs control multiple genes, whereas RXRs serve as partners for RARs and other nuclear receptors that regulate metabolism. Physiological function has not been determined for 9cRA, because it has not been detected in serum or multiple tissues with analytically validated assays. Here, we identify 9cRA in mouse pancreas by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and show that 9cRA decreases with feeding and after glucose dosing and varies inversely with serum insulin. 9cRA reduces glucose-stimulated insulin secretion (GSIS) in mouse islets and in the rat [beta]-cell line 832/13 within 15 min by reducing glucose transporter type 2 (Glut2) and glucokinase (GK) activities. 9cRA also reduces Pdx-1 and HNF4[alpha] mRNA expression, ~8- and 80-fold, respectively: defects in Pdx-1 or HNF4[alpha] cause maturity onset diabetes of the young (MODY4 and 1, respectively), as does a defective GK gene (MODY2). Pancreas [beta]-cells generate 9cRA, and mouse models of reduced [beta]-cell number, heterozygous Akita mice, and streptozotocin-treated mice have reduced 9cRA. 9cRA is abnormally high in glucose-intolerant mice, which have [beta]-cell hypertropy, including mice with diet-induced obesity (DIO) and ob/ob and db/db mice. These data establish 9cRA as a pancreas-specific autacoid with multiple mechanisms of action and provide unique insight into GSIS. retinol | vitamin A | rexinoids doi: 10.1073/pnas.1008859107

Acute intramedullary infusion of hyperosmotic NaCI, used to simulate a high-salt diet-induced increase of medullary osmolality, increases urine production and endothelin release from the kidney. To determine whether endothelin mediates this diuretic and natriuretic response, urine flow and [Na.sup.+] excretion rate were measured during acute intramedullary infusion of hyperosmotic NaC1 in anesthetized rats, with or without endothelin receptor antagonism. Isosmotic NaC1 was infused into the left renal medulla during an equilibration period and 30-min baseline period, followed by hyperosmotic NaC1 for two additional 30-min periods. Hyperosmotic NaC1 infusion significantly increased urine flow of vehicle-treated rats (from 5.9 [+ or -] 0.9 to 11.1 [+ or -] 1.8 [micro]l/min). Systemic [ET.sub.B] receptor blockade enhanced this effect (A-192621; from 7.7 [+ or -] 1.1 to 18.7 [+ or -] 2.9 [micro]l/min; P < 0.05), ETA receptor blockade (ABT-627) had no significant effect alone, but the diuresis was markedly attenuated by combined ABT-627 and A-192621 administration (from 4.4 [+ or -] 0.7 to 5.4 [+ or -] 0.9 [micro]l/min). Mean arterial pressures overall were not significantly different between groups. Surprisingly, the natriuretic response to hyperosmotic NaC1 infusion was not significantly altered by systemic endothelin receptor blockade, and furthermore, intramedullary [ET.sub.B] receptor blockade enhanced the diuretic and natriuretic response to hyperosmotic NaC1 infusion. ETA receptor blockade significantly attenuated both the diuretic and natriuretic responses to hyperosmotic NaC1 infusion in [ET.sub.B] receptor-deficient sl/sl rats. These results demonstrate an important role of endothelin in mediating diuretic responses to intramedullary infusion of hyperosmotic NaC1. Moreover, these data suggest ETA and ETa receptors are both required for the full diuretic and natriuretic actions of endothelin. salt and water excretion; blood pressure; kidney; endothelin doi: 10.1152/ajprenal.00015.2010

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody, cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover. lung; phospholipid; plasma membrane; lipid turnover doi: 10.1152/ajplung.00372.2009.

We have previously shown that mouse atherosclerosis regression involves monocyte-derived ([CD68.sup.+]) cell emigration from plaques and is dependent on the chemokine receptor CCR7. Concurrent with regression, mRNA levels of the gene encoding LXRα are increased in plaque [CD68.sup.+] cells, suggestive of a functional relationship between LXR and CCR7. To extend these results, atherosclerotic [Apoe.sup.-/-] mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque [CD68.sup.+] cells. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic [Apoe.sup.-/-] mice, or from [Apoe.sup.-/-] mice with BM deficiency of LXRα or LXRβ, into WT recipients. Plaques from both LXRα-and LXRβ-deficient [Apoe.sup.-/-] mice exhibited impaired regression. In addition, the [CD68.sup.+] cells displayed reduced emigration and CCR7 expression. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. This increase was blunted when LXRα and LXRβ levels were reduced by siRNAs. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. We conclude that LXR is required for maximal effects on plaque [CD68.sup.+] cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice., Introduction Atherosclerosis is the leading underlying cause of death worldwide (1). Absolute risk for cardiovascular disease rises with age because of the progression of coronary atherosclerosis (2-6). To date, the [...]

During the classic 'fight-or-flight' stress response, sympathetic nervous system activation leads to catechol-amine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium ([Ca.sup.2+]). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-[S2808A.sup.+/+] mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte [Ca.sup.2+] transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-[S2808A.sup.+/+] mice. The blunted cardiac response to catecholamines in RyR2-[S2808A.sup.+/+] mice resulted in impaired exercise capacity. RyR2-[S2808A.sup.+/+] mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation., Introduction During exercise, heart rate (chronotropy) and cardiac contractility (inotropy) increase to meet the metabolic demands of the organs. Stress-induced activation of the sympathetic nervous system (SNS) results in catecholamine [...]

Increased sarcoplasmic reticulum (SR) [Ca.sup.2+] leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-[S2808D.sup.+/+] mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-[S2808D.sup.+/+] mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR [Ca.sup.2+] store content, and increased diastolic SR [Ca.sup.2+] leak. After myocardial infarction, RyR2-[S2808D.sup.+/+] mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR [Ca.sup.2+] leak and reduced HF progression in WT and RyR2-[S2808D.sup.+/+] mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-[S2808D.sup.+/+] mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF., Introduction Heart failure (HF), a syndrome characterized by progressive deterioration of cardiac function, represents a major public health burden and is a leading cause of morbidity and mortality in the [...]

In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated [Ca.sup.2+] release from intracellular [Ca.sup.2+] stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and 'leaky,' leading to arrhythmias and other pathology. This 'leaky RyR2 hypothesis' is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory. Moreover, the experiments revealed an influence of redox modifications of RyR2 that may account for some discrepancies in the field., The heart has a remarkable capacity to react to altered demand by changing the rate at which it beats and the force with which it contracts, thereby changing its output. [...]

Many physiological events require transient increases in cytosolic [Ca.sup.2+] concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of [Ca.sup.2+] from the endoplasmic and sarcoplasmic reticulum (1). Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult (2,3). Here we show the 2.5 Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps (4,5) unambiguously places the domains in the cytoplasmic portion of the channel' forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains' disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods' and will be useful for developing new strategies to modulate RyR function in disease states., Ryanodine receptors are large (~2.2 MDa) tetrameric channels that control the release of [Ca.sup.2+] from the endoplasmic and sarcoplasmic reticulum. Three isoforms (RyR1, RyR2 and RyR3) have been isolated in [...]

In humans and mice, the early development of [alpha][beta] T cells is controlled by the pre--T-cell receptor [alpha] chain (pT[alpha]) that is covalently associated with the T-cell receptor [beta] (TCR[beta]) chain to form the pre--T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pT[alpha]. Using in silico and gene synteny-based approaches, we identified the pT[alpha] gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pT[alpha] functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pT[alpha] cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pT[alpha] was exclusively to enable expression of the TCR[beta] chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pT[alpha] signaling. doi/ 10.1073/pnas.1010166107

The pre--T-cell receptor (TCR) is crucial for the early T-cell development, but the ligand for pre-TCR remains unidentified. We recently proposed a model that pre-TCR complexes oligomerize spontaneously through interactions of the pre-TCR[alpha] chain. To investigate the mechanism underlying this ligand-independent signaling in vivo, we established knock-in mice that express a pre-TCR[alpha] mutant lacking charged amino acids ([D.sup.22][R.sup.24][R.sup.102][R.sup.117] to [A.sup.22][A.sup.24][A.sup.102][A.sup.117]; 4A). [CD4.sup.+][CD8.sup.+] thymocyte number was significantly reduced in invariant pre-TCR[alpha] ([pT[alpha].sup.4A/4A]) mice, whereas [CD4.sup.-][CD8.sup.-] thymocytes were unaffected. The percentages of double-negative 3 (DN3) cells and [gamma][delta] T cells were increased in the pT[alpha]4A/4A thymus, indicating that [beta]-selection is impaired in [pT[alpha].sup.4A/4A] mice. Pre-TCR-mediated tyrosine phosphorylation and clonal expansion into double-positive thymocytes were also defective in the knock-in mice. Pre-TCR was expressed at higher levels on [pT[alpha].sup.4A/4A] cell surfaces than on those of the wild type, suggesting that the charged residues in pT[alpha] are critical for autonomous engagement and subsequent internalization of pre-TCR. Pre-TCR--mediated allelic exclusion of the TCR[beta] gene was also inhibited in [pT[alpha].sup.4A/4A] mice, and thereby, dual TCR[beta]s were expressed on [pT[alpha].sup.4A/4A] T cells. Furthermore, the TCR[beta] chain variable region (V[beta]) repertoire of mature T cells was significantly altered in [pT[alpha].sup.4A/4A] mice. These results suggest that charged residues of pT[alpha] are critical for [beta]-selection, allelic exclusion, and TCR[beta] repertoire formation. thymic selection | signal transduction doi/ 10.1073/pnas.1011228107

Although an immunoregulatory role of aryl hydrocarbon receptor (Ahr) has been demonstrated in T cells and macrophages, little is known about its function in dendritic cells (DC). Here, we show that lipopolysaccharide (LPS) and CpG stimulate Ahr expression in bone marrow-derived dendritic cells (BMDC). Furthermore, we found that Ahr is required to induce indoleamine 2,3-dioxygenase (IDO) expression, an immunosuppressive enzyme that catabolizes tryptophan into kynurenine (Kyn) and other metabolites in DC. In the presence of LPS or CpG, Ahr-deficient ([Ahr.sup.-/-]) mature BMDC induced immune responses characterized by reduced Kyn and IL-10 production compared with results observed with tolerogenic mature WT BMDC. In a coculture system with LPS- or CpG-stimulated BMDC and naive T cells, [Ahr.sup.-/-] BMDC inhibited naive T-cell differentiation into regulatory T (Treg) cells, which likely facilitated Th17 cell development and promoted naive T-cell proliferation. Addition of synthetic L-Kyn to the coculture system skewed the differentiation of naive T cells to Treg cells rather than Th17 cells. Taken together, our results demonstrate a previously unknown negatively regulatory role for Ahr in DC-mediated immunogenesis in the presence of LPS or CpG, which, in turn, alters the Kyn-dependent generation of Treg cells and Th17 cells from naive T cells. dioxin receptor | tryptophan catabolism | immune regulation doi/ 10.1073/pnas.1014465107

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions. lateral diffusion | glutamate receptor | trafficking | biomimetic multivalent ligand | development doi/ 10.1073/pnas.1002690107