Science.gov

Sample records for activated receptor-1 par-1

  1. Galectin-3 Facilitates Cell Motility in Gastric Cancer by Up-Regulating Protease-Activated Receptor-1(PAR-1) and Matrix Metalloproteinase-1(MMP-1)

    PubMed Central

    Kim, Seok-Jun; Shin, Ji-Young; Lee, Kang-Duck; Bae, Young-Ki; Choi, Il-Ju; Park, Seok Hee; Chun, Kyung-Hee

    2011-01-01

    Background Galectin-3 is known to regulate cancer metastasis. However, the underlying mechanism has not been defined. Through the DNA microarray studies after galectin-3 silencing, we demonstrated here that galectin-3 plays a key role in up-regulating the expressions of protease-activated receptor-1(PAR-1) and matrix metalloproteinase-1(MMP-1) PAR-1 thereby promoting gastric cancer metastasis. Methodology/Principal Findings We examined the expression levels of Galectin-3, PAR-1, and MMP-1 in gastric cancer patient tissues and also the effects of silencing these proteins with specific siRNAs and of over-expressing them using specific lenti-viral constructs. We also employed zebrafish embryo model for analysis of in vivo gastric cancer cell invasion. These studies demonstrated that: a) galectin-3 silencing decreases the expression of PAR-1. b) galectin-3 over-expression increases cell migration and invasion and this increase can be reversed by PAR-1 silencing, indicating that galectin-3 increases cell migration and invasion via PAR-1 up-regulation. c) galectin-3 directly interacts with AP-1 transcriptional factor, and this complex binds to PAR-1 promoter and drives PAR-1 transcription. d) galectin-3 also amplifies phospho-paxillin, a PAR-1 downstream target, by increasing MMP-1 expression. MMP-1 silencing blocks phospho-paxillin amplification and cell invasion caused by galectin-3 over-expression. e) Silencing of either galectin-3, PAR-1 or MMP-1 significantly reduced cell migration into the vessels in zebrafish embryo model. f) Galectin-3, PAR-1, and MMP-1 are highly expressed and co-localized in malignant tissues from gastric cancer patients. Conclusions/Significance Galectin-3 plays the key role of activating cell surface receptor through production of protease and boosts gastric cancer metastasis. Galectin-3 has the potential to serve as a useful pharmacological target for prevention of gastric cancer metastasis. PMID:21966428

  2. Protease activated receptor 1 (PAR1) enhances Src-mediated tyrosine phosphorylation of NMDA receptor in intracerebral hemorrhage (ICH)

    PubMed Central

    Duan, Zhen-Zhen; Zhang, Feng; Li, Feng-Ying; Luan, Yi-Fei; Guo, Peng; Li, Yi-Hang; Liu, Yong; Qi, Su-Hua

    2016-01-01

    It has been demonstrated that Src could modulate NMDA receptor, and PAR1 could also affect NMDAR signaling. However, whether PAR1 could regulate NMDAR through Src under ICH has not yet been investigated. In this study, we demonstrated the role of Src-PSD95-GluN2A signaling cascades in rat ICH model and in vitro thrombin challenged model. Using the PAR1 agonist SFLLR, antagonist RLLFS and Src inhibitor PP2, electrophysiological analysis showed that PAR1 regulated NMDA-induced whole-cell currents (INMDA) though Src in primary cultured neurons. Both in vivo and in vitro results showed the elevated phosphorylation of tyrosine in Src and GluN2A and enhanced interaction of the Src-PSD95-GluN2A under model conditions. Treatment with the PAR1 antagonist RLLFS, AS-PSD95 (Antisense oligonucleotide against PSD95) and Src inhibitor PP2 inhibited the interaction among Src-PSD95-GluN2A, and p-Src, p-GluN2A. Co-application of SFLLR and AS-PSD95, PP2, or MK801 (NMDAR inhibitor) abolished the effect of SF. In conclusion, our results demonstrated that activated thrombin receptor PAR1 induced Src activation, enhanced the interaction among Src-PSD95-GluN2A signaling modules, and up-regulated GluN2A phosphorylation after ICH injury. Elucidation of such signaling cascades would possibly provide novel targets for ICH treatment. PMID:27385592

  3. PAR1 activation induces rapid changes in glutamate uptake and astrocyte morphology

    PubMed Central

    Sweeney, Amanda M.; Fleming, Kelsey E.; McCauley, John P.; Rodriguez, Marvin F.; Martin, Elliot T.; Sousa, Alioscka A.; Leapman, Richard D.; Scimemi, Annalisa

    2017-01-01

    The G-protein coupled, protease-activated receptor 1 (PAR1) is a membrane protein expressed in astrocytes. Fine astrocytic processes are in tight contact with neurons and blood vessels and shape excitatory synaptic transmission due to their abundant expression of glutamate transporters. PAR1 is proteolytically-activated by bloodstream serine proteases also involved in the formation of blood clots. PAR1 activation has been suggested to play a key role in pathological states like thrombosis, hemostasis and inflammation. What remains unclear is whether PAR1 activation also regulates glutamate uptake in astrocytes and how this shapes excitatory synaptic transmission among neurons. Here we show that, in the mouse hippocampus, PAR1 activation induces a rapid structural re-organization of the neuropil surrounding glutamatergic synapses, which is associated with faster clearance of synaptically-released glutamate from the extracellular space. This effect can be recapitulated using realistic 3D Monte Carlo reaction-diffusion simulations, based on axial scanning transmission electron microscopy (STEM) tomography reconstructions of excitatory synapses. The faster glutamate clearance induced by PAR1 activation leads to short- and long-term changes in excitatory synaptic transmission. Together, these findings identify PAR1 as an important regulator of glutamatergic signaling in the hippocampus and a possible target molecule to limit brain damage during hemorrhagic stroke. PMID:28256580

  4. Protease-activated receptor-1 deficiency protects against streptozotocin-induced diabetic nephropathy in mice

    PubMed Central

    Waasdorp, Maaike; Duitman, JanWillem; Florquin, Sandrine; Spek, C. Arnold

    2016-01-01

    Endogenously administered activated protein C ameliorates diabetic nephropathy (DN) in a protease-activated receptor-1 (PAR-1)-dependent manner, suggesting that PAR-1 activation limits the progression of DN. Activation of PAR-1 in fibroblast-like cells, however, induces proliferation and extracellular matrix production, thereby driving fibrotic disease. Considering the key role of mesangial proliferation and extracellular matrix production during DN, PAR-1 may in fact potentiate diabetes-induced kidney injury. To determine the net effect of PAR-1 in DN, streptozotocin-induced DN was studied in wild type and PAR-1 deficient mice. Subsequent mechanistic insight was obtained by assessing profibrotic responses of mesangial and tubular epithelial cells in vitro, following PAR-1 stimulation and inhibition. Despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes, as evidenced by diminished proteinuria, plasma cystatin C levels, expansion of the mesangial area, and tubular atrophy. In vitro, PAR-1 signaling in mesangial cells led to increased proliferation and expression of matrix proteins fibronectin and collagen IV. Conversely, a reduction in both proliferation and fibronectin deposition was observed in diabetic PAR-1 deficient mice. Overall, we show that PAR-1 plays an important role in the development of DN and PAR-1 might therefore be an attractive therapeutic target to pursue in DN. PMID:27618774

  5. Activation of protease activated receptor 1 increases the excitability of the dentate granule neurons of hippocampus

    PubMed Central

    2011-01-01

    Protease activated receptor-1 (PAR1) is expressed in multiple cell types in the CNS, with the most prominent expression in glial cells. PAR1 activation enhances excitatory synaptic transmission secondary to the release of glutamate from astrocytes following activation of astrocytically-expressed PAR1. In addition, PAR1 activation exacerbates neuronal damage in multiple in vivo models of brain injury in a manner that is dependent on NMDA receptors. In the hippocampal formation, PAR1 mRNA appears to be expressed by a subset of neurons, including granule cells in the dentate gyrus. In this study we investigate the role of PAR activation in controlling neuronal excitability of dentate granule cells. We confirm that PAR1 protein is expressed in neurons of the dentate cell body layer as well as in astrocytes throughout the dentate. Activation of PAR1 receptors by the selective peptide agonist TFLLR increased the intracellular Ca2+ concentration in a subset of acutely dissociated dentate neurons as well as non-neuronal cells. Bath application of TFLLR in acute hippocampal slices depolarized the dentate gyrus, including the hilar region in wild type but not in the PAR1-/- mice. PAR1 activation increased the frequency of action potential generation in a subset of dentate granule neurons; cells in which PAR1 activation triggered action potentials showed a significant depolarization. The activation of PAR1 by thrombin increased the amplitude of NMDA receptor-mediated component of EPSPs. These data suggest that activation of PAR1 during normal function or pathological conditions, such as during ischemia or hemorrhage, can increase the excitability of dentate granule cells. PMID:21827709

  6. Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

    SciTech Connect

    Beaulieu, Lea M.; Church, Frank C. . E-mail: fchurch@email.unc.edu

    2007-02-15

    Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1-50 {mu}g/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified 'checkerboard' analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1.

  7. Synthesis of Arylpiperazine Derivatives As Protease Activated Receptor 1 Antagonists And Their Evaluation As Antiproliferative Agents.

    PubMed

    Zotti, Andrea Ilaria; Di Gennaro, Elena; Corvino, Angela; Frecentese, Francesco; Magli, Elisa; Perissutti, Elisa; Cirino, Giuseppe; Roviezzo, Fiorentina; Terranova-Barberio, Manuela; Iannelli, Federica; Caliendo, Giuseppe; Santagada, Vincenzo; Fiorino, Ferdinando; Budillon, Alfredo; Severino, Beatrice

    2016-09-26

    Protease activated receptor-1 (PAR1) is a G-coupled receptor activated by α-thrombin and other proteases. Several reports demonstrate PAR1 involvement in tumorigenesis and tumor progression. In order to investigate on potential use of PAR1 antagonists as antiproliferative agents, we have identified a series of arylpiperazine derivatives acting as PAR1 antagonists; the selected molecules have been evaluated for their antiproliferative properties. All the compounds inhibited the growth of a panel of cell lines expressing PAR1; two of them, compounds 13 and 15, were able to inhibit, in a dose dependent manner, the growth of the selected cell lines with the lowest IC50 values, and were further characterized to define the mechanism responsible for the observed antiproliferative effect. This study directed us to the identification of two interesting leads that may help to further validate PAR1 as an important therapeutic target for cancer treatment.

  8. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

    PubMed

    Almonte, Antoine G; Qadri, Laura H; Sultan, Faraz A; Watson, Jennifer A; Mount, Daniel J; Rumbaugh, Gavin; Sweatt, J David

    2013-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

  9. Protease-Activated Receptor-1 Supports Locomotor Recovery by Biased Agonist Activated Protein C after Contusive Spinal Cord Injury

    PubMed Central

    Whetstone, William D.; Walker, Breset; Trivedi, Alpa; Lee, Sangmi; Noble-Haeusslein, Linda J.; Hsu, Jung-Yu C.

    2017-01-01

    Thrombin-induced secondary injury is mediated through its receptor, protease activated receptor-1 (PAR-1), by "biased agonism." Activated protein C (APC) acts through the same PAR-1 receptor but functions as an anti-coagulant and anti-inflammatory protein, which counteracts many of the effects of thrombin. Although the working mechanism of PAR-1 is becoming clear, the functional role of PAR-1 and its correlation with APC in the injured spinal cord remains to be elucidated. Here we investigated if PAR-1 and APC are determinants of long-term functional recovery after a spinal cord contusive injury using PAR-1 null and wild-type mice. We found that neutrophil infiltration and disruption of the blood-spinal cord barrier were significantly reduced in spinal cord injured PAR-1 null mice relative to the wild-type group. Both locomotor recovery and ability to descend an inclined grid were significantly improved in the PAR-1 null group 42 days after injury and this improvement was associated with greater long-term sparing of white matter and a reduction in glial scarring. Wild-type mice treated with APC acutely after injury showed a similar level of improved locomotor recovery to that of PAR-1 null mice. However, improvement of APC-treated PAR-1 null mice was indistinguishable from that of vehicle-treated PAR-1 null mice, suggesting that APC acts through PAR-1. Collectively, our findings define a detrimental role of thrombin-activated PAR-1 in wound healing and further validate APC, also acting through the PAR-1 by biased agonism, as a promising therapeutic target for spinal cord injury. PMID:28122028

  10. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  11. N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop

    PubMed Central

    Soto, Antonio G.; Trejo, JoAnn

    2010-01-01

    Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling. PMID:20368337

  12. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  13. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase

    PubMed Central

    Barber, Kara R.; Sherman, Michael

    2017-01-01

    Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs) and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated. PMID:28222093

  14. The anticoagulant activated protein C (aPC) promotes metaplasticity in the hippocampus through an EPCR-PAR1-S1P1 receptors dependent mechanism.

    PubMed

    Maggio, Nicola; Itsekson, Zeev; Ikenberg, Benno; Strehl, Andreas; Vlachos, Andreas; Blatt, Ilan; Tanne, David; Chapman, Joab

    2014-08-01

    Thrombin and other clotting factors regulate long-term potentiation (LTP) in the hippocampus through the activation of the protease activated receptor 1 (PAR1) and consequent potentiation of N-methyl-d-aspartate receptor (NMDAR) functions. We have recently shown that the activation of PAR1 either by thrombin or the anticoagulant factor activated protein C (aPC) has differential effects on LTP. While thrombin activation of PAR1 induces an NMDAR-mediated slow onset LTP, which saturates the ability to induce further LTP in the exposed network, aPC stimulation of PAR1 enhances tetanus induced LTP through a voltage-gated calcium channels mediated mechanism. In this study, we addressed the mechanisms by which aPC enhances LTP in hippocampal slices. Using extracellular recordings, we show that a short tetanic stimulation, which does not induce LTP, is able to enhance plasticity in the presence of aPC through a mechanism that requires the activation of sphingosine-1 phosphate receptor 1 and intracellular Ca(2+) stores. These data identify aPC as a "metaplastic molecule", capable of shifting the threshold of LTP towards further potentiation. Our findings propose novel strategies to enhance plasticity in neurological diseases associated with the breakdown of the blood brain barrier and alterations in synaptic plasticity.

  15. Learning and memory deficits in mice lacking protease activated receptor-1.

    PubMed

    Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F

    2007-10-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.

  16. CONTRIBUTION OF PROTEASE-ACTIVATED RECEPTOR 1 IN STATUS EPILEPTICUS-INDUCED EPILEPTOGENESIS

    PubMed Central

    Isaev, D.; Lushnikova, I.; Lunko, O.; Zapukhliak, O.; Maximyuk, O.; Romanov, A.; Skibo, G.G.; Tian, C.; Holmes, G.L.; Isaeva, E.

    2015-01-01

    Clinical observations and studies on different animal models of acquired epilepsy consistently demonstrate that blood-brain barrier (BBB) leakage can be an important risk factor for developing recurrent seizures. However, the involved signaling pathways remain largely unclear. Given the important role of thrombin and its major receptor in the brain, protease-activated receptor 1 (PAR1), in the pathophysiology of neurological injury, we hypothesized that PAR1 may contribute to status epilepticus (SE)-induced epileptogenesis and that its inhibition shortly after SE will have neuroprotective and antiepileptogenic effects. Adult rats subjected to lithium-pilocarpine SE were administrated SCH79797 (a PAR1 selective antagonist) after SE termination. Thrombin and PAR1 levels and neuronal cell survival were evaluated 48 hr following SE. The effect of PAR1 inhibition on animal survival, interictal spikes (IIS) and electrographic seizures during the first two weeks after SE and behavioral seizures during the chronic period were evaluated. SE resulted in a high mortality rate and incidence of IIS and seizures in the surviving animals. There was a marked increase in thrombin, decrease in PAR1 immunoreactivity and hippocampal cell loss in the SE-treated rats. Inhibition of PAR1 following SE resulted in a decrease in mortality and morbidity, increase in neuronal cell survival in the hippocampus and suppression of IIS, electrographic and behavioral seizures following SE. These data suggest that the PAR1 signaling pathway contributes to epileptogenesis following SE. Because breakdown of the BBB occurs frequently in brain injuries, PAR1 inhibition may have beneficial effects in a variety of acquired injuries leading to epilepsy. PMID:25843668

  17. Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins*

    PubMed Central

    Grimsey, Neil J.; Coronel, Luisa J.; Cordova, Isabel Canto; Trejo, JoAnn

    2016-01-01

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms. PMID:26635365

  18. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    SciTech Connect

    Wang, Junru; Kulkarni, Ashwini; Chintala, Madhu; Fink, Louis M.; Hauer-Jensen, Martin

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  19. Specific activation, signalling and secretion profiles of human platelets following PAR-1 and PAR-4 stimulation.

    PubMed

    Nguyen, Kim Anh; Hamzeh-Cognasse, Hind; Laradi, Sandrine; Pozzetto, Bruno; Garraud, Olivier; Cognasse, Fabrice

    2015-01-01

    Blood platelets play a central haemostatic function; however, they also play a role in inflammation and are capable of secreting various cytokines, chemokines and related products. The purpose of this study was to identify subtle variations in platelet physiology using proteomics. We compared the levels of membrane proteins (n = 3), α and δ granule proteins (n = 18), and signalling proteins (n = 30) from unstimulated platelets with those of protease-activated receptor (PAR)-1- and PAR-4-stimulated platelets (n = 10). The vast majority of these proteins responded similarly to PAR-1 or PAR-4 engagement. However, differences were observed within membrane CD40L expressed, and α granule GRO-α and MDC secreted proteins.

  20. Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death.

    PubMed

    Burns, Kelly E; Thévenin, Damien

    2015-12-15

    Even though abnormal expression of G protein-coupled receptors (GPCRs) and of their ligands is observed in many cancer cells of various origins, only a few anti-cancer compounds directly act on their signalling. One promising approach to modulate their activity consists of targeting the receptor cytoplasmic surfaces interacting with the associated G-proteins using peptides mimicking the intracellular loops of the receptor. Thus, to be fully effective, the peptide mimics must be selectively targeted to the tumour while sparing healthy tissues, translocated across the cell membrane and stay anchored to the cytoplasmic leaflet of the plasma membrane. In the present study, we introduce a novel way to selectively target and inhibit the activity of a GPCR in cancer cells under acidic conditions, such as those found in solid tumours. We find that the conjugation of a peptide fragment derived from the third intracellular loop (i3) of the protease-activated receptor 1 (PAR1) to a peptide that can selectively target tumours solely based on their acidity [pH(Low) Insertion Peptide (pHLIP)], produces a construct capable of effectively down-regulating PAR1 activity in a concentration- and pH-dependent manner and of inducing a potent cytotoxic effect in a panel of cancer cells that is proportional to the relative level of receptor expression at the cell surface. This strategy not only allows for a more selective targeting and specific intracellular delivery than current approaches, but also offers new possibilities for developing novel anti-cancer drugs targeting GPCRs.

  1. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

    PubMed

    Soto, Antonio G; Smith, Thomas H; Chen, Buxin; Bhattacharya, Supriyo; Cordova, Isabel Canto; Kenakin, Terry; Vaidehi, Nagarajan; Trejo, JoAnn

    2015-07-07

    Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

  2. Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.

    PubMed

    Duvernay, Matthew; Young, Summer; Gailani, David; Schoenecker, Jonathan; Hamm, Heidi E; Hamm, Heidi

    2013-04-01

    With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

  3. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

  4. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  5. Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets

    PubMed Central

    French, Shauna L.; Paramitha, Antonia C.; Moon, Mitchell J.; Dickins, Ross A.; Hamilton, Justin R.

    2016-01-01

    Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets–PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs. PMID

  6. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

    PubMed Central

    Allen, Megan; Ghosh, Suhasini; Ahern, Gerard P.; Villapol, Sonia; Maguire-Zeiss, Kathleen A.; Conant, Katherine

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism. PMID:27762280

  7. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder.

    PubMed

    Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A; Power, Christopher; Hollenberg, Morley D; Seidah, Nabil G

    2015-11-01

    The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.

  8. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

    PubMed Central

    Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A.; Power, Christopher; Hollenberg, Morley D.

    2015-01-01

    The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND. PMID:26283733

  9. [Peptide-agonist of protease-activated receptor (PAR 1), similar to activated protein C, promotes proliferation in keratinocytes and wound healing of epithelial layer].

    PubMed

    Kiseleva, E V; Sidorova, M V; Gorbacheva, L R; Strukova, S M

    2014-01-01

    Activated protein C (APC) is serine protease hemostasis, independent of its anticoagulant activity, exhibits anti-inflammatory and anti-apoptotic properties that determine the possibility of the protective effects of APC in different diseases, including sepsis and chronic wound healing. APC, binding of endothelial protein C receptor (EPCR) and specifically cleaving PAR1 receptor and releasing peptide agonist PAR1 stabilizes not only endothelial cells, but also many others, including epidermal keratinocytes of the skin. We develop the hypothesis that the cytoprotective effect of APC on the cells, involved in wound healing, seem to imitate peptide - analogous of PAR1 "tethered ligand" that activate PAR1. In our work, we synthesized a peptide (AP9) - analogue of PAR1 tethered ligand, released by APC, and firstly showed that peptide AP9 (0.1-10 мM), like to APC (0.01-100 nM), stimulates the proliferative activity of human primary keratinocytes. Using a model of the formation of epithelial wounds in vitro we found that peptide AP9, as well as protease APC, accelerates wound healing. Using specific antibodies to the receptor PAR1 and EPCR was studied the receptor mechanism of AP9 action in wound healing compared with the action of APС. The necessity of both receptors - PAR1 and EPСR, for proliferative activity of agonists was revealed. Identified in our work imitation by peptide AP9 - PAR1 ligand, APC acts on keratinocytes suggests the possibility of using a peptide AP9 to stimulate tissue repair.

  10. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    PubMed

    Fortunato, Tiago M; Vara, Dina S; Wheeler-Jones, Caroline P; Pula, Giordano

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  11. Heat stress-induced disruption of endothelial barrier function is via PAR1 signaling and suppressed by Xuebijing injection.

    PubMed

    Xu, Qiulin; Liu, Jingxian; Wang, Zhenglian; Guo, Xiaohua; Zhou, Gengbiao; Liu, Yanan; Huang, Qiaobing; Su, Lei

    2015-01-01

    Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential

  12. Elastase and metalloproteinase activities regulate soluble complement receptor 1 release.

    PubMed

    Sadallah, S; Hess, C; Miot, S; Spertini, O; Lutz, H; Schifferli, J A

    1999-11-01

    Complement receptor 1 (CR1) is cleaved from the surface of polymorphonuclear cells (PMN) in the membrane-proximal region to yield a soluble fragment (sCR1) that contains the functional domains. The enzymes involved in this cleavage are produced by the PMN itself, since in vitro stimulation of purified PMN is followed by sCR1 release. Purified human neutrophil elastase (HNE) cleaved CR1 from erythrocytes and urinary vesicles originating from podocytes and enhanced tenfold the cleavage of CR1 from activated PMN. The largest fragment released from PMN by HNE was identical in size to CR1 shed spontaneously. The CR1 fragments cleaved from erythrocytes were functional. The shedding of sCR1 by activated PMN was inhibited by phenylmethylsulfonyl fluoride (80 +/- 10%), alpha1-antiprotease (50 +/- 5%) and elafin (60 +/- 5%). Furthermore the cleavage was blocked by the metalloprotease inhibitor 1,10-phenanthroline (70 +/- 6 %) as well as by a monoclonal antibody against human neutrophil collagenase MMP8 (40 +/- 10%). Maximal inhibition of sCR1 shedding was obtained by a combination of 1,10-phenanthroline with elafin (86 +/- 6%). These inhibitors had no effect on L-selectin shedding, indicating that the cleavage of CR1 was specific. In conclusion, elastase or elastase-like activity may be responsible for the shedding of functional sCR1 in vivo, and this activity is controlled by the local release of PMN metalloproteases and alpha1antiprotease.

  13. Inhibition of proinflammatory cytokines by SCH79797, a selective protease-activated receptor 1 antagonist, protects rat kidney against ischemia-reperfusion injury.

    PubMed

    El Eter, Eman Abdelazeem; Aldrees, Abdulmajeed

    2012-06-01

    Renal ischemia-reperfusion injury (I/R) is the most common cause of acute renal failure. It is partially mediated by thrombin as it is attenuated by thrombin inhibition or deletion of its receptor protease-activated receptor 1 (PAR1). However, the role of PAR1 in renal I/R injury needs to be further elucidated. The present study investigated the effect of PAR1 antagonist, SCH79797 (SCH), on renal protection and downstream effectors involved. Male Wistar rats were pretreated with SCH (25 μg/kg i.p.) or vehicle, 15 min before 45 min of clamping of left renal pedicle after right nephrectomy. To investigate the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt, a group of rats was subjected to pretreatment with an inhibitor of PI3K/Akt (LY 29004, 3 mg/kg i.p.) before renal ischemia and SCH treatment. A sham-operated group served as control and received saline. All rats were killed 24 h after reperfusion or sham operation, and blood samples collected and kidney tissues processed either for immunostaining and histological assessment or for biochemical analysis. SCH79797 markedly attenuated kidney damage histologically and by improving serum creatinine. Both plasma and protein expression of P selectin were markedly reduced as well as neutrophil infiltration, cytokine-induced neutrophil chemoattractant 1, and tumor necrosis factor α. These protective effects of blocking PAR1 receptor were abolished by preadministration of LY29004. These results suggest that PAR1 mediates renal I/R injury and that blocking PAR1 using SCH limits renal injury by an anti-inflammatory effect possibly signaling via PI3K/Akt.

  14. Kallikrein 6 Signals through PAR1 and PAR2 to Promote Neuron Injury and Exacerbate Glutamate Neurotoxicity

    PubMed Central

    Yoon, Hyesook; Radulovic, Maja; Wu, Jianmin; Blaber, Sachiko I.; Blaber, Michael; Fehlings, Michael G.; Scarisbrick, Isobel A.

    2014-01-01

    CNS trauma generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine protease implicated in neurodegeneration and here we investigate the role of protease activated receptors 1 (PAR1) and PAR2 in mediating these effects. First we demonstrate Klk6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and PAR2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant Klk6 triggered ERK1/2 signaling in cerebellar granule neurons and in the NSC34 spinal cord motoneuron cell line, in a PI3K and MEK-dependent fashion. Importantly, lipopeptide inhibitors of PAR1 or PAR2, and PAR1 genetic deletion, each reduced Klk6-ERK1/2 activation. In addition, Klk6 and thrombin promoted degeneration of cerebellar neurons and exacerbated glutamate neurotoxicity. Moreover, genetic deletion of PAR1 blocked thrombin-mediated cerebellar neurotoxicity and reduced the neurotoxic effects of Klk6. Klk6 also increased glutamate-mediated Bim signaling, PARP cleavage and lactate dehydrogenase (LDH) release in NSC34 motoneurons and these effects were blocked by PAR1 and PAR2 lipopeptide inhibitors. Taken together these data point to a novel Klk6-signaling axis in CNS neurons that is mediated by PAR1 and PAR2 and is positioned to contribute to neurodegeneration. PMID:23647384

  15. NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro

    PubMed Central

    Nguyen, Chi Huu; Senfter, Daniel; Basilio, Jose; Holzner, Silvio; Stadler, Serena; Krieger, Sigurd; Huttary, Nicole; Milovanovic, Daniela; Viola, Katharina; Simonitsch-Klupp, Ingrid; Jäger, Walter; de Martin, Rainer; Krupitza, Georg

    2015-01-01

    RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited “circular chemo-repellent induced defects” (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20–30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10–15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca2+ release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 – MMP1 (MDA-MB231) – PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression. PMID:26513020

  16. NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro.

    PubMed

    Nguyen, Chi Huu; Senfter, Daniel; Basilio, Jose; Holzner, Silvio; Stadler, Serena; Krieger, Sigurd; Huttary, Nicole; Milovanovic, Daniela; Viola, Katharina; Simonitsch-Klupp, Ingrid; Jäger, Walter; de Martin, Rainer; Krupitza, Georg

    2015-11-17

    RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited "circular chemo-repellent induced defects" (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20-30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10-15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca(2+) release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 - MMP1 (MDA-MB231) - PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.

  17. AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

    PubMed Central

    Dores, Michael R.; Paing, May M.; Lin, Huilan; Montagne, William A.; Marchese, Adriano; Trejo, JoAnn

    2012-01-01

    The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting. PMID:22833563

  18. Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells

    PubMed Central

    Schuldt, Gerlind; Galanis, Christos; Strehl, Andreas; Hick, Meike; Schiener, Sabine; Lenz, Maximilian; Deller, Thomas; Maggio, Nicola; Vlachos, Andreas

    2016-01-01

    Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions. PMID:27378862

  19. A protease-activated receptor 1 antagonist protects against global cerebral ischemia/reperfusion injury after asphyxial cardiac arrest in rabbits

    PubMed Central

    Yang, Jing-ning; Chen, Jun; Xiao, Min

    2017-01-01

    Cerebral ischemia/reperfusion injury is partially mediated by thrombin, which causes brain damage through protease-activated receptor 1 (PAR1). However, the role and mechanisms underlying the effects of PAR1 activation require further elucidation. Therefore, the present study investigated the effects of the PAR1 antagonist SCH79797 in a rabbit model of global cerebral ischemia induced by cardiac arrest. SCH79797 was intravenously administered 10 minutes after the model was established. Forty-eight hours later, compared with those administered saline, rabbits receiving SCH79797 showed markedly decreased neuronal damage as assessed by serum neuron specific enolase levels and less neurological dysfunction as determined using cerebral performance category scores. Additionally, in the hippocampus, cell apoptosis, polymorphonuclear cell infiltration, and c-Jun levels were decreased, whereas extracellular signal-regulated kinase phosphorylation levels were increased. All of these changes were inhibited by the intravenous administration of the phosphoinositide 3-kinase/Akt pathway inhibitor LY29004 (3 mg/kg) 10 minutes before the SCH79797 intervention. These findings suggest that SCH79797 mitigates brain injury via anti-inflammatory and anti-apoptotic effects, possibly by modulating the extracellular signal-regulated kinase, c-Jun N-terminal kinase/c-Jun and phosphoinositide 3-kinase/Akt pathways.

  20. Protease-Activated Receptor-1 is Upregulated in Reactive Stroma of Primary Prostate Cancer and Bone Metastasis

    PubMed Central

    Zhang, Xiaotun; Wang, Wenbin; True, Lawrence D.; Vessella, Robert L.; Takayama, Thomas K.

    2009-01-01

    BACKGROUND Prostate cancer progression is partly facilitated by tumor-stroma interactions. We recently reported that protease-activated receptors (PAR-1 and PAR-2) are overexpressed in prostate cancer, and PAR-1 expression in peritumoral stroma is associated with biochemical recurrence. However, the nature of PAR expression in prostate tumor microenvironment is not fully understood. We therefore evaluated PAR-1 and PAR-2 expression in primary prostate cancer and bone metastasis. METHODS PAR-1 and PAR-2 expression in normal, primary prostate cancer and the corresponding bone metastatic tissues were examined by immunohistochemistry, and double-label immunohistochemistry with the use of additional markers. RESULTS PAR-1 was expressed in peritumoral stroma in the majority of primary cancer tissues (83%). Serial sections and double-label immunohistochemistry determined that these PAR-1 expressing stromal cells were predominantly myofibroblasts, the primary cell type in reactive stroma. Analysis of cancer glands revealed that PAR-1 expression was significantly increased in the reactive stroma around higher Gleason grade cancers. PAR-2 was predominantly expressed in the primary cancer cells as well as smooth muscle cells but not in reactive stroma. In bone metastasis, PAR-1 expression in cancer cells was elevated compared to the primary site from the same patient. In the bone reactive stroma, PAR-1 was present in vascular endothelial cells and fibroblasts, while both PAR-1 and PAR-2 were expressed in osteoblasts and osteoclasts. CONCLUSIONS In primary prostate cancer and bone metastasis, PAR-1 is upregulated in reactive stroma and PAR-2 is uniformly overexpressed in carcinoma cells, suggesting these receptors may play potentially different roles in prostate cancer development and metastasis. PMID:19170048

  1. PAR-1 mediated apoptosis of breast cancer cells by V. cholerae hemagglutinin protease.

    PubMed

    Ray, Tanusree; Pal, Amit

    2016-05-01

    Bacterial toxins have emerged as promising agents in cancer treatment strategy. Hemagglutinin (HAP) protease secreted by Vibrio cholerae induced apoptosis in breast cancer cells and regresses tumor growth in mice model. The success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity for normal tissues. Increased expression of Protease Activated Receptor-1 (PAR-1) has been reported in different malignant cells. In this study we report that HAP induced activation and over expression of PAR-1 in breast cancer cells (EAC). Immunoprecipitation studies have shown that HAP specifically binds with PAR-1. HAP mediated activation of PAR-1 caused nuclear translocation of p50-p65 and the phosphorylation of p38 which triggered the activation of NFκB and MAP kinase signaling pathways. These signaling pathways enhanced the cellular ROS level in malignant cells that induced the intrinsic pathway of cell apoptosis. PAR-1 mediated apoptosis by HAP of malignant breast cells without effecting normal healthy cells in the same environment makes it a good therapeutic agent for treatment of cancer.

  2. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

    PubMed

    Citron, Bruce A; Ameenuddin, Syed; Uchida, K; Suo, William Z; SantaCruz, Karen; Festoff, Barry W

    2016-07-15

    Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

  3. Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis

    PubMed Central

    2012-01-01

    Background Glutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear. Findings We report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes. Conclusions These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release. PMID:23062602

  4. Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

    PubMed

    Diaz, Jorge; Aranda, Evelyn; Henriquez, Soledad; Quezada, Marisol; Espinoza, Estefanía; Bravo, Maria Loreto; Oliva, Bárbara; Lange, Soledad; Villalon, Manuel; Jones, Marius; Brosens, Jan J; Kato, Sumie; Cuello, Mauricio A; Knutson, Todd P; Lange, Carol A; Leyton, Lisette; Owen, Gareth I

    2012-08-01

    Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

  5. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

    PubMed

    Perez, Michel; Lamothe, Marie; Maraval, Catherine; Mirabel, Etienne; Loubat, Chantal; Planty, Bruno; Horn, Clemens; Michaux, Julien; Marrot, Sebastien; Letienne, Robert; Pignier, Christophe; Bocquet, Arnaud; Nadal-Wollbold, Florence; Cussac, Didier; de Vries, Luc; Le Grand, Bruno

    2009-10-08

    Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

  6. F16357, a novel protease‐activated receptor 1 antagonist, improves urodynamic parameters in a rat model of interstitial cystitis

    PubMed Central

    Gillespie, J; Farrié, M; Le Grand, B; Junquero, D; Vergnolle, N

    2016-01-01

    Background and Purpose The aims of the present study were to characterize the role of PAR1 in rat bladder under inflammatory conditions and determine whether a selective PAR1 antagonist, F16357, can prevent the pathophysiological symptoms of cyclophosphamide‐induced interstitial cystitis (IC). Experimental Approach Immunohistochemistry, contractile activity in isolated bladder and urodynamics were determined before and after cyclophosphamide treatment. F16357 was administered intravesically during the acute phase of inflammation, and effects on PAR1 and PAR1‐related bladder contraction evaluated 24 h after cyclophosphamide injection. Urodynamics and associated voided volumes were recorded 7 and 24 h after cyclophosphamide. Key Results In control conditions, PAR1 was present only in some umbrella cells. Cyclophosphamide disrupted the urothelium and expression of PAR1 by all remaining urothelial cells. After F16357 treatment, urothelial damage was absent and PAR1 immunoreactivity similar to control tissues. Thrombin and TFLLR‐NH2 induced bladder contractions. These were increased in inflammatory conditions and antagonized by F16357 in a concentration‐dependent manner. In telemetric experiments, furosemide increased urine production and voiding frequency for 60 min, 7 h after cyclophosphamide injection. Intravesical administration of F16357 blocked these changes with a return to a physiological profile; 24 h after cyclophosphamide, the volume of micturition was still lower with no increase in number of micturitions. F16357 30 μM reduced the number of micturitions and improved bladder capacity, but did not affect diuresis. Under similar experimental conditions, lidocaine 2% induced comparable effects. Conclusions and Implications PAR1 is expressed in rat bladder, overactivated in inflammatory conditions and involved in bladder function and sensation. F16357 could represent an interesting candidate for IC treatment. PMID:27111354

  7. Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1

    PubMed Central

    Ba, Qian; Zhou, Naiyuan; Duan, Juan; Chen, Tao; Hao, Miao; Yang, Xinying; Li, Junyang; Yin, Jun; Chu, Ruiai; Wang, Hui

    2012-01-01

    Artemisinin and its main active metabolite dihydroartemisinin, clinically used antimalarial agents with low host toxicity, have recently shown potent anticancer activities in a variety of human cancer models. Although iron mediated oxidative damage is involved, the mechanisms underlying these activities remain unclear. In the current study, we found that dihydroartemisinin caused cellular iron depletion in time- and concentration-dependent manners. It decreased iron uptake and disturbed iron homeostasis in cancer cells, which were independent of oxidative damage. Moreover, dihydroartemisinin reduced the level of transferrin receptor-1 associated with cell membrane. The regulation of dihydroartemisinin to transferrin receptor-1 could be reversed by nystatin, a cholesterol-sequestering agent but not the inhibitor of clathrin-dependent endocytosis. Dihydroartemisinin also induced transferrin receptor-1 palmitoylation and colocalization with caveolin-1, suggesting a lipid rafts mediated internalization pathway was involved in the process. Also, nystatin reversed the influences of dihydroartemisinin on cell cycle and apoptosis related genes and the siRNA induced downregulation of transferrin receptor-1 decreased the sensitivity to dihydroartemisinin efficiently in the cells. These results indicate that dihydroartemisinin can counteract cancer through regulating cell-surface transferrin receptor-1 in a non-classical endocytic pathway, which may be a new action mechanism of DHA independently of oxidative damage. PMID:22900042

  8. TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells

    PubMed Central

    Smoktunowicz, Natalia; Platé, Manuela; Stern, Alejandro Ortiz; D'Antongiovanni, Vanessa; Robinson, Eifion; Chudasama, Vijay; Caddick, Stephen; Scotton, Chris J.; Jarai, Gabor; Chambers, Rachel C.

    2016-01-01

    The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer. PMID:27566553

  9. Critical Role for PAR1 in Kallikrein 6-Mediated Oligodendrogliopathy

    PubMed Central

    Burda, Joshua E.; Radulovic, Maja; Yoon, Hyesook; Scarisbrick, Isobel A.

    2014-01-01

    Kallikrein 6 (Klk6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of Klk6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke and glioblastoma. Taken with recent evidence establishing Klk6 as a CNS-endogenous activator of protease-activated receptors (PARs), we hypothesized that Klk6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1-deficient mice and the murine oligodendrocyte cell line, Oli-neu, were used to demonstrate that Klk6 mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1-dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1-activating peptides (PAR1-APs). Klk6 also exacerbated ATP-mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1-mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1-APs, into the dorsal column white matter of PAR+/+ but not PAR−/− mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC-1+ oligodendrocytes. These results demonstrate a functional role for Klk6-PAR1 signaling in oligodendroglial pathophysiology and suggest that PAR1 or PAR1-agonists may represent new targets to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease. PMID:23832758

  10. Inhibition of diacylglycerol lipase (DAGL) in the lateral hypothalamus of rats prevents the increase in REMS and food ingestion induced by PAR1 stimulation.

    PubMed

    Pérez-Morales, Marcel; López-Colomé, Ana María; Méndez-Díaz, Mónica; Ruiz-Contreras, Alejandra E; Prospéro-García, Oscar

    2014-08-22

    Stimulation of the protease-activated receptor 1 (PAR1) in vitro, was shown to induce synaptic retrograde signaling through the endocannabinoid 2-arachidonoylglycerol (2-AG) synthesis and activation of the cannabinoid receptor type 1 (CB1R). The activation of PAR1 by the agonist S1820 in the lateral hypothalamus (LH) increases rapid eye movement sleep (REMS) and food intake in rats, and both effects are prevented by the CB1R inverse agonist AM251. In the present study, we implanted rats with electrodes and with cannulae aimed bilaterally to the LH. We administered tetrahydrolipstatin (THL), an inhibitor of the diacylglycerol lipase (DAGL), the enzyme responsible for 2-AG synthesis, to evaluate the sleep-wake cycle and food ingestion. THL in the LH readily prevented the increase in REMS and food intake induced by PAR1 stimulation, further supporting 2-AG as an upstream activator of PAR1. Our results demonstrate that the effect of PAR1 on REMS and food intake is blocked by the inhibition of DAGL, further suggesting that PAR1 stimulation in the lateral hypothalamus of rats induces an increase in sleep and food intake through 2-AG.

  11. PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells

    PubMed Central

    Gur-Cohen, Shiri; Itkin, Tomer; Chakrabarty, Sagarika; Graf, Claudine; Kollet, Orit; Ludin, Aya; Golan, Karin; Kalinkovich, Alexander; Ledergor, Guy; Wong, Eitan; Niemeyer, Elisabeth; Porat, Ziv; Erez, Ayelet; Sagi, Irit; Esmon, Charles T; Ruf, Wolfram; Lapidot, Tsvee

    2016-01-01

    Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation-related also control retention of EPCR+ LT-HSCs in the bone marrow and their recruitment to the blood via two different protease activated receptor 1 (PAR1)-mediated pathways. Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to TACE-mediated EPCR shedding, enhanced CXCL12-CXCR4-induced motility, and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with protein C that retain EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing VLA4 affinity and adhesion. Inhibition of NO production by activated protein C (aPC)-EPCR-PAR1 signaling reduces progenitor cell egress, increases NOlow bone marrow EPCR+ LT-HSCs retention and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR that control NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs with clinical relevance. PMID:26457757

  12. NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets

    PubMed Central

    Damien, Pauline; Cognasse, Fabrice; Payrastre, Bernard; Spinelli, Sherry L.; Blumberg, Neil; Arthaud, Charles-Antoine; Eyraud, Marie-Ange; Phipps, Richard P.; McNicol, Archibald; Pozzetto, Bruno; Garraud, Olivier; Hamzeh-Cognasse, Hind

    2017-01-01

    The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions. PMID:28220122

  13. Blockade of PAR1 signaling with cell-penetrating pepducins inhibits Akt-survival pathways in breast cancer cells and suppresses tumor survival and metastasis

    PubMed Central

    Yang, Eric; Boire, Adrienne; Agarwal, Anika; Nguyen, Nga; O'Callaghan, Katie; Tu, Powen; Kuliopulos, Athan; Covic, Lidija

    2009-01-01

    Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor that is not expressed in normal breast epithelia, but is up-regulated in invasive breast carcinomas. In the present study, we found that matrix metalloprotease-1 (MMP-1) robustly activates the PAR1-Akt survival pathway in breast carcinoma cells. This process is blocked by a cell-penetrating lipopeptide ‘pepducin’, P1pal-7, which is a potent inhibitor of cell viability in breast carcinoma cells expressing PAR1. Both a MMP-1 inhibitor and P1pal-7 significantly promote apoptosis in breast tumor xenografts and inhibit metastasis to the lungs by up to 88%. Dual therapy with P1pal-7 and taxotere inhibits the growth of MDA-MB-231 xenografts by 95%. Consistently, biochemical analysis of xenograft tumors treated with P1pal-7 or MMP-1 inhibitor demonstrated attenuated Akt activity. Ectopic expression of constitutively active Akt rescues breast cancer cells from the synergistic cytotoxicity of P1pal-7 and taxotere, suggesting that Akt is a critical component of PAR1-dependent cancer cell viability. Together, these findings indicate that blockade of MMP1-PAR1 signaling may provide a benefit beyond treatment with taxotere alone in advanced, metastatic breast cancer. PMID:19622769

  14. The MMP-1/PAR-1 Axis Enhances Proliferation and Neuronal Differentiation of Adult Hippocampal Neural Progenitor Cells

    PubMed Central

    Valente, Maria Maddalena; Allen, Megan; Bortolotto, Valeria; Lim, Seung T.; Conant, Katherine; Grilli, Mariagrazia

    2015-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a role in varied forms of developmental and postnatal neuroplasticity. MMP substrates include protease-activated receptor-1 (PAR-1), a G-protein coupled receptor expressed in hippocampus. We examined proliferation and differentiation of adult neural progenitor cells (aNPCs) from hippocampi of mice that overexpress the potent PAR-1 agonist MMP-1. We found that, as compared to aNPCs from littermate controls, MMP-1 tg aNPCs display enhanced proliferation. Under differentiating conditions, these cells give rise to a higher percentage of MAP-2+ neurons and a reduced number of oligodendrocyte precursors, and no change in the number of astrocytes. The fact that these results are MMP and PAR-1 dependent is supported by studies with distinct antagonists. Moreover, JSH-23, an inhibitor of NF-κB p65 nuclear translocation, counteracted both the proliferation and differentiation changes seen in MMP-1 tg-derived NPCs. In complementary studies, we found that the percentage of Sox2+ undifferentiated progenitor cells is increased in hippocampi of MMP-1 tg animals, compared to wt mice. Together, these results add to a growing body of data suggesting that MMPs are effectors of hippocampal neuroplasticity in the adult CNS and that the MMP-1/PAR-1 axis may play a role in neurogenesis following physiological and/or pathological stimuli. PMID:26783471

  15. Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    PubMed Central

    Florence, Jon M.; Booshehri, Laela M.; Allen, Timothy C.; Kurdowska, Anna K.

    2017-01-01

    The atherosclerotic process begins when vascular endothelial cells undergo pro-inflammatory changes such as aberrant activation to dysfunctional phenotypes and apoptosis, leading to loss of vascular integrity. Our laboratory has demonstrated that exposure of mice to second hand smoke triggers an increase in expression of metalloproteinase-9. Further, metalloproteinase-9 released by second hand smoke—activated leukocytes may propagate pro-atherogenic alterations in endothelial cells. We have shown that levels of metalloproteinase-9 were increased in the plasma from apolipoprotein E deficient (ApoE-/-) mice exposed to second hand smoke relative to non-exposed controls. Moreover, we have collected data from two different, but complementary, treatments of second hand smoke exposed atherosclerotic mice. Animals received either cell specific metalloproteinase-9 directed siRNA to minimize metalloproteinase-9 expression in neutrophils and endothelial cells, or a pharmacological inhibitor of Bruton’s tyrosine kinase which indirectly limits metalloproteinase-9 production in neutrophils. These treatments reduced atherosclerotic changes in mice and improved overall vascular health. We also demonstrated that metalloproteinase-9 could activate endothelial cells and induce their apoptosis via cleavage of protease activated receptor-1. In summary, better understanding of metalloproteinase-9’s pathogenic capabilities as well as novel signaling pathways involved may lead to development of treatments which may provide additional benefits to atherosclerosis patients with a history of second hand smoke exposure. PMID:28166283

  16. Vasodilator-Stimulated Phosphoprotein Deficiency Potentiates PAR-1-induced Increase in Endothelial Permeability in Mouse Lungs

    PubMed Central

    Profirovic, Jasmina; Han, Jingyan; Andreeva, Alexandra V.; Neamu, Radu F.; Pavlovic, Sasha; Vogel, Stephen M.; Walter, Ulrich; Voyno-Yasenetskaya, Tatyana A.

    2010-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. VASP function in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP−/− mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP−/− mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function. PMID:20945373

  17. Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis

    PubMed Central

    Gao, Yang; Vincent, David F.; Davis, Anna Jane; Sansom, Owen J.; Bartholin, Laurent; Li, Qinglei

    2016-01-01

    Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies. PMID:27344183

  18. Niacin ameliorates ulcerative colitis via prostaglandin D2-mediated D prostanoid receptor 1 activation.

    PubMed

    Li, Juanjuan; Kong, Deping; Wang, Qi; Wu, Wei; Tang, Yanping; Bai, Tingting; Guo, Liang; Wei, Lumin; Zhang, Qianqian; Yu, Yu; Qian, Yuting; Zuo, Shengkai; Liu, Guizhu; Liu, Qian; Wu, Sheng; Zang, Yi; Zhu, Qian; Jia, Daile; Wang, Yuanyang; Yao, Weiyan; Ji, Yong; Yin, Huiyong; Nakamura, Masataka; Lazarus, Michael; Breyer, Richard M; Wang, Lifu; Yu, Ying

    2017-03-24

    Niacin, as an antidyslipidemic drug, elicits a strong flushing response by release of prostaglandin (PG) D2 However, whether niacin is beneficial for inflammatory bowel disease (IBD) remains unclear. Here, we observed niacin administration-enhanced PGD2 production in colon tissues in dextran sulfate sodium (DSS)-challenged mice, and protected mice against DSS or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in D prostanoid receptor 1 (DP1)-dependent manner. Specific ablation of DP1 receptor in vascular endothelial cells, colonic epithelium, and myeloid cells augmented DSS/TNBS-induced colitis in mice through increasing vascular permeability, promoting apoptosis of epithelial cells, and stimulating pro-inflammatory cytokine secretion of macrophages, respectively. Niacin treatment improved vascular permeability, reduced apoptotic epithelial cells, promoted epithelial cell update, and suppressed pro-inflammatory gene expression of macrophages. Moreover, treatment with niacin-containing retention enema effectively promoted UC clinical remission and mucosal healing in patients with moderately active disease. Therefore, niacin displayed multiple beneficial effects on DSS/TNBS-induced colitis in mice by activation of PGD2/DP1 axis. The potential efficacy of niacin in management of IBD warrants further investigation.

  19. AP-2{alpha} suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    SciTech Connect

    Mitchell, Darrion L.; DiMario, Joseph X.

    2010-01-15

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2{alpha} is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2{alpha} binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2{alpha}-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2{alpha} interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2{alpha} is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  20. Soluble complement receptor 1 inhibits both complement and granulocyte activation during ex vivo hemodialysis.

    PubMed

    Himmelfarb, J; McMonagle, E; Holbrook, D; Toth, C

    1995-10-01

    Hemodialysis with cellulosic membranes results in both complement and granulocyte activation. We investigated the effects of soluble complement receptor 1 (sCR1), a potent complement inhibitor, on both complement and granulocyte activation in an ex vivo model of dialysis. Measurements were made of complement activation (radioimmunoassay for C3a desArg) as well as granulocyte activation (flow cytometric measurements of reactive oxygen species production, granulocyte CD11b/CD18 (MAC-1) expression and CD62L (L-selectin) expression). sCR1 completely abolished the generation of plasma C3a desArg during ex vivo hemodialysis. Without sCR1, C3a desArg levels rose from 968 +/- 373 ng/ml to 4961 +/- 40 ng/ml by the end of the ex vivo procedure (p < 0.001). sCR1 also completely inhibited MAC-1 upregulation and L-selectin shedding from granulocytes during ex vivo hemodialysis. With sCR1 there was still a statistically significant increase in granulocyte reactive oxygen species production (from 2.42 +/- 0.1 fluorescence channels to 6.47 +/- 0.7 fluorescence channels, p < 0.01) but a 50% inhibition when compared with experiments without sCR1 (3.15 +/- 0.5 to 11.2 +/- 1.9, p < 0.01). We conclude that sCR1 completely abolishes complement activation and changes in granulocyte cell adhesion molecules during ex vivo hemodialysis with cellulosic membranes. sCR1 partially inhibits granulocyte reactive oxygen species formation.

  1. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    PubMed

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

  2. Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

    PubMed

    Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

    2011-03-17

    The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions.

  3. PAR1 is selectively over expressed in high grade breast cancer patients: a cohort study

    PubMed Central

    Hernández, Norma A; Correa, Elma; Avila, Esther P; Vela, Teresa A; Pérez, Víctor M

    2009-01-01

    Background The protease-activated receptor (PAR1) expression is correlated with the degree of invasiveness in cell lines. Nevertheless it has never been directed involved in breast cancer patients progression. The aim of this study was to determine whether PAR1 expression could be used as predictor of metastases and mortality. Methods In a cohort of patients with infiltrating ductal carcinoma studied longitudinally since 1996 and until 2007, PAR1 over-expression was assessed by immunoblotting, immunohistochemistry, and flow citometry. Chi-square and log rank tests were used to determine whether there was a statistical association between PAR1 overexpression and metastases, mortality, and survival. Multivariate analysis was performed including HER1, stage, ER and nodes status to evaluate PAR1 as an independent prognostic factor. Results Follow up was 95 months (range: 2–130 months). We assayed PAR1 in a cohort of patients composed of 136 patients; we found PAR1 expression assayed by immunoblotting was selectively associated with high grade patients (50 cases of the study cohort; P = 0.001). Twenty-nine of 50 (58%) patients overexpressed PAR1, and 23 of these (46%) developed metastases. HER1, stage, ER and PAR1 overexpression were robustly correlated (Cox regression, P = 0.002, P = 0.024 and P = 0.002 respectively). Twenty-one of the 50 patients (42%) expressed both receptors (PAR1 and HER1 P = 0.0004). We also found a statistically significant correlation between PAR1 overexpression and increased mortality (P = 0.0001) and development of metastases (P = 0.0009). Conclusion Our data suggest PAR1 overexpression may be involved in the development of metastases in breast cancer patient and is associated with undifferentiated cellular progression of the tumor. Further studies are needed to understand PAR1 mechanism of action and in a near future assay its potential use as risk factor for metastasis development in high grade breast cancer patients. PMID:19538737

  4. Interference with Protease-activated Receptor 1 Alleviates Neuronal Cell Death Induced by Lipopolysaccharide-Stimulated Microglial Cells through the PI3K/Akt Pathway

    PubMed Central

    Li, Yuxin; Yang, Wuyang; Quinones-Hinojosa, Alfredo; Wang, Baocheng; Xu, Shujun; Zhu, Weijie; Yu, Feng; Yuan, Shaoji; Lu, Peigang

    2016-01-01

    Excessive microglial cells activation in response to inflammatory stimuli leads to synaptic loss, dysfunction, and neuronal cell death. Activated microglia are involved in the pathogenesis of neurological conditions and frequently contribute to several complications. Accumulating evidence suggests that signaling through PAR-1 is involved in inflammation, however, its function has yet to be fully elucidated. Here, we have demonstrated that the suppression of PAR-1 leads to down-regulation of inflammatory factors including IL-1β, IL-6, TNF-α, NO, as well as the prevention of activation of NF-κB in BV2 cells. In addition, we found that a PAR-1 antagonist, SCH, prevented LPS-induced excessive microglial activation in a dose-dependent manner. As a result of SCH treatment, neuronal cell death via up-regulation of Akt-mediated pathways was reduced. Our results demonstrate that the beneficial effects of SCH are linked to its ability to block an inflammatory response. Further, we found that SCH inhibited the death of PC12 neurons from the cytotoxicity of activated BV2 cells via activation of the PI3K/Akt pathway. These neuro-protective effects appear to be related to inhibition of PAR-1, and represents a novel neuroprotective strategy that could has potential for use in therapeutic interventions of neuroinflammatory disease. PMID:27910893

  5. PAR-1 contributes to the innate immune response during viral infection

    PubMed Central

    Antoniak, Silvio; Owens, A. Phillip; Baunacke, Martin; Williams, Julie C.; Lee, Rebecca D.; Weithäuser, Alice; Sheridan, Patricia A.; Malz, Ronny; Luyendyk, James P.; Esserman, Denise A.; Trejo, JoAnn; Kirchhofer, Daniel; Blaxall, Burns C.; Pawlinski, Rafal; Beck, Melinda A.; Rauch, Ursula; Mackman, Nigel

    2013-01-01

    Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3–induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1–/– mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1+/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1–/– mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1+/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection. PMID:23391721

  6. Exploration of a new series of PAR1 antagonists.

    PubMed

    Planty, Bruno; Pujol, Chantal; Lamothe, Marie; Maraval, Catherine; Horn, Clemens; Le Grand, Bruno; Perez, Michel

    2010-03-01

    Two series of new PAR1 antagonists have been identified. The first incorporates a cinnamoylpiperidine motif and the second a cinnamoylpyridine pattern. The synthesis, biological activity and structure-activity relationship of these compounds are presented. In each series, one analog showed potent in vivo antithrombotic activity in a rat AV shunt model, with up to 53% inhibition at 1.25mpk iv for compound 30.

  7. Recombination in the Human Pseudoautosomal Region PAR1

    PubMed Central

    Hinch, Anjali G.; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R.

    2014-01-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  8. Recombination in the human Pseudoautosomal region PAR1.

    PubMed

    Hinch, Anjali G; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R

    2014-07-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

  9. Artificial neural networks from MATLAB in medicinal chemistry. Bayesian-regularized genetic neural networks (BRGNN): application to the prediction of the antagonistic activity against human platelet thrombin receptor (PAR-1).

    PubMed

    Caballero, Julio; Fernández, Michael

    2008-01-01

    Artificial neural networks (ANNs) have been widely used for medicinal chemistry modeling. In the last two decades, too many reports used MATLAB environment as an adequate platform for programming ANNs. Some of these reports comprise a variety of applications intended to quantitatively or qualitatively describe structure-activity relationships. A powerful tool is obtained when there are combined Bayesian-regularized neural networks (BRANNs) and genetic algorithm (GA): Bayesian-regularized genetic neural networks (BRGNNs). BRGNNs can model complicated relationships between explanatory variables and dependent variables. Thus, this methodology is regarded as useful tool for QSAR analysis. In order to demonstrate the use of BRGNNs, we developed a reliable method for predicting the antagonistic activity of 5-amino-3-arylisoxazole derivatives against Human Platelet Thrombin Receptor (PAR-1), using classical 3D-QSAR methodologies: Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). In addition, 3D vectors generated from the molecular structures were correlated with antagonistic activities by multivariate linear regression (MLR) and Bayesian-regularized neural networks (BRGNNs). All models were trained with 34 compounds, after which they were evaluated for predictive ability with additional 6 compounds. CoMFA and CoMSIA were unable to describe this structure-activity relationship, while BRGNN methodology brings the best results according to validation statistics.

  10. Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling

    PubMed Central

    Gur-Cohen, Shiri; Kollet, Orit; Graf, Claudine; Esmon, Charles T.; Ruf, Wolfram; Lapidot, Tsvee

    2016-01-01

    The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of several cell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs), endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR), which is used as a marker to isolate them. EPCR/PAR1 signaling in endothelial cells has anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflammation. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR+ LT-HSC BM retention and egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production, maintaining NOlow LT-HSC BM retention with increased VLA4 expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generation and activate different PAR1 signaling which overcomes BM EPCR+ LT-HSC retention, inducing their recruitment to the bloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulation of CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses new roles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1 signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production. PMID:26928241

  11. Downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated by the prostaglandin E receptor EP2 through cAMP elevation and protein kinase A.

    PubMed

    Sokolova, Elena; Hartig, Roland; Reiser, Georg

    2008-07-01

    Many cellular functions of lung fibroblasts are controlled by protease-activated receptors (PARs). In fibrotic diseases, PAR-1 plays a major role in controlling fibroproliferative and inflammatory responses. Therefore, in these diseases, regulation of PAR-1 expression plays an important role. Using the selective prostaglandin EP2 receptor agonist butaprost and cAMP-elevating agents, we show here that prostaglandin (PG)E(2), via the prostanoid receptor EP2 and subsequent cAMP elevation, downregulates mRNA and protein levels of PAR-1 in human lung fibroblasts. Under these conditions, the functional response of PAR-1 in fibroblasts is reduced. These effects are specific for PGE(2). Activation of other receptors coupled to cAMP elevation, such as beta-adrenergic and adenosine receptors, does not reproduce the effects of PGE(2). PGE(2)-mediated downregulation of PAR-1 depends mainly on protein kinase A activity, but does not depend on another cAMP effector, the exchange protein activated by cAMP. PGE(2)-induced reduction of PAR-1 level is not due to a decrease of PAR-1 mRNA stability, but rather to transcriptional regulation. The present results provide further insights into the therapeutic potential of PGE(2) to specifically control fibroblast function in fibrotic diseases.

  12. PAR1-dependent and independent increases in COX-2 and PGE2 in human colonic myofibroblasts stimulated by thrombin.

    PubMed

    Seymour, Michelle L; Zaidi, Nosheen F; Hollenberg, Morley D; MacNaughton, Wallace K

    2003-05-01

    Subepithelial myofibroblast-derived prostaglandin E(2) (PGE(2)) regulates epithelial chloride secretion in the intestine. Thrombin is elevated in inflammatory conditions of the bowel. Therefore, we sought to determine a role for thrombin in regulating PGE(2) synthesis by colonic myofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblasts with thrombin, the proteinase-activated receptor 1 (PAR(1))-activating peptide (Cit-NH(2)), and peptides corresponding to 2 noncatalytic regions of thrombin (TP367 and TP508) for 18 h increased both cyclooxygenase (COX)-2 expression (immunocytochemistry) and PGE(2) synthesis (enzyme immunoassay). Inhibition of thrombin by D-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reduce PGE(2) synthesis, which remained elevated compared with control. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE(2) elevations. Recombinant human bFGF concentration dependently increased PGE(2) synthesis, and a bFGF neutralizing antibody inhibited PGE(2) synthesis induced by TP367 and TP508 (approximately 40%) and by thrombin (approximately 20%) (but not Cit-NH(2)). Thrombin, therefore, upregulates COX-2-derived PGE(2) synthesis by both catalytic cleavage of PAR(1) and bFGF-dependent noncatalytic activity. This presents a novel mechanism by which intestinal myofibroblasts might regulate epithelial chloride secretion.

  13. Plasmin induces in vivo monocyte recruitment through protease-activated receptor-1-, MEK/ERK-, and CCR2-mediated signaling.

    PubMed

    Carmo, Aline A F; Costa, Bruno R C; Vago, Juliana P; de Oliveira, Leonardo C; Tavares, Luciana P; Nogueira, Camila R C; Ribeiro, Ana Luíza C; Garcia, Cristiana C; Barbosa, Alan S; Brasil, Bruno S A F; Dusse, Luci M; Barcelos, Lucíola S; Bonjardim, Cláudio A; Teixeira, Mauro M; Sousa, Lirlândia P

    2014-10-01

    The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.

  14. ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.

    PubMed

    Dores, Michael R; Chen, Buxin; Lin, Huilan; Soh, Unice J K; Paing, May M; Montagne, William A; Meerloo, Timo; Trejo, JoAnn

    2012-04-30

    The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.

  15. Identification of California Condor Estrogen Receptors 1 and 2 and Their Activation by Endocrine Disrupting Chemicals.

    PubMed

    Felton, Rachel G; Steiner, Cynthia C; Durrant, Barbara S; Keisler, Duane H; Milnes, Matthew R; Tubbs, Christopher W

    2015-12-01

    Recently, California condors (Gymnogyps californianus) have been reintroduced to coastal regions of California where they feed on marine mammal carcasses. There is evidence that coastal-dwelling condors experience reproductive issues, such as eggshell thinning, likely resulting from exposure to endocrine-disrupting chemicals (EDCs). To address this problem, we have identified and cloned condor estrogen receptors (ESRs) 1 and 2 and characterized their activation by EDCs present in the coastal habitats where condors reside. Dichlorodiphenyltrichloroethane (DDT) and its metabolites all activated ESR1 and ESR2, although their relative potency differed between the receptors. Bisphenol A, dieldrin, trans-nonachlor, and polychlorinated biphenyl 52 (PCB52) moderately activated both ESRs, whereas PCB138 and PCB153 stimulated little to no activation. Overall, EDC activation of condor ESR2, which is the first ESR2 cloned from a raptor species, was greater than that of ESR1. Significant activation of both condor ESRs by EDCs occurred at high concentrations (≥1μM), which are within the range of plasma levels of certain EDCs (eg, dichlorodiphenyldichloroethylene [p'p-DDE]) in coastal-dwelling condors. Finally, phylogenetic analyses of ESRs of 41 avian species identified a single amino acid position in ESR2 under positive selection. Mutation of this amino acid affected receptor activation by EDCs, suggesting the identity of this amino acid may influence EDC sensitivity of avian species. Together, these findings broaden our understanding of EDC interactions with ESRs in avian species. For condors specifically, these data could be used to evaluate EDC exposure risk at future release sites to identify those least likely to compromise the continued recovery of this species.

  16. Molecular mechanisms in the selective basal activation of pyrabactin receptor 1: Comparative analysis of mutants.

    PubMed

    Dorosh, Lyudmyla; Rajagopalan, Nandhakishore; Loewen, Michele C; Stepanova, Maria

    2014-01-01

    Pyrabactin receptors (PYR) play a central role in abscisic acid (ABA) signal transduction; they are ABA receptors that inhibit type 2C protein phosphatases (PP2C). Molecular aspects contributing to increased basal activity of PYR against PP2C are studied by molecular dynamics (MD) simulations. An extensive series of MD simulations of the apo-form of mutagenized PYR1 as a homodimer and in complex with homology to ABA-insensitive 1 (HAB1) phosphatase are reported. In order to investigate the detailed molecular mechanisms mediating PYR1 activity, the MD data was analyzed by essential collective dynamics (ECD), a novel approach that allows the identification, with atomic resolution, of persistent dynamic correlations based on relatively short MD trajectories. Employing the ECD method, the effects of select mutations on the structure and dynamics of the PYR1 complexes were investigated and considered in the context of experimentally determined constitutive activities against HAB1. Approaches to rationally design constitutively active PYR1 constructs to increase PP2C inhibition are discussed.

  17. Dopamine receptor 1 neurons in the dorsal striatum regulate food anticipatory circadian activity rhythms in mice

    PubMed Central

    Gallardo, Christian M; Darvas, Martin; Oviatt, Mia; Chang, Chris H; Michalik, Mateusz; Huddy, Timothy F; Meyer, Emily E; Shuster, Scott A; Aguayo, Antonio; Hill, Elizabeth M; Kiani, Karun; Ikpeazu, Jonathan; Martinez, Johan S; Purpura, Mari; Smit, Andrea N; Patton, Danica F; Mistlberger, Ralph E; Palmiter, Richard D; Steele, Andrew D

    2014-01-01

    Daily rhythms of food anticipatory activity (FAA) are regulated independently of the suprachiasmatic nucleus, which mediates entrainment of rhythms to light, but the neural circuits that establish FAA remain elusive. In this study, we show that mice lacking the dopamine D1 receptor (D1R KO mice) manifest greatly reduced FAA, whereas mice lacking the dopamine D2 receptor have normal FAA. To determine where dopamine exerts its effect, we limited expression of dopamine signaling to the dorsal striatum of dopamine-deficient mice; these mice developed FAA. Within the dorsal striatum, the daily rhythm of clock gene period2 expression was markedly suppressed in D1R KO mice. Pharmacological activation of D1R at the same time daily was sufficient to establish anticipatory activity in wild-type mice. These results demonstrate that dopamine signaling to D1R-expressing neurons in the dorsal striatum plays an important role in manifestation of FAA, possibly by synchronizing circadian oscillators that modulate motivational processes and behavioral output. DOI: http://dx.doi.org/10.7554/eLife.03781.001 PMID:25217530

  18. Mammary tumorigenesis induced by fibroblast growth factor receptor 1 requires activation of the epidermal growth factor receptor.

    PubMed

    Bade, Lindsey K; Goldberg, Jodi E; Dehut, Hazel A; Hall, Majken K; Schwertfeger, Kathryn L

    2011-09-15

    Fibroblast growth factor receptor 1 (FGFR1) is an oncoprotein with known involvement in mammary tumorigenesis. To understand how FGFR1 signaling promotes mammary tumorigenesis, an inducible FGFR1 (iFGFR1) system was created previously. Previous studies have demonstrated that upon iFGFR1 activation in vivo, the epidermal growth factor (EGF) ligands amphiregulin (AREG) and epiregulin (EREG) are upregulated. Both AREG and EREG interact with the EGF receptor (EGFR). Here, we investigated whether the FGFR1-induced increase in AREG and EREG expression might coordinately increase EGFR signaling to promote mammary tumorigenesis. Treatment of mouse mammary epithelial cells with either AREG or EREG conferred a greater migratory potential, increased cellular proliferation and increased extracellular regulated kinase 1/2 (ERK1/2) activation. These effects could be blocked with the EGFR-specific inhibitor erlotinib, suggesting that they are EGFR-dependent. In transgenic mice with iFGFR1 under the control of the mouse mammary tumor virus (MMTV) promoter, iFGFR1 activation also led to increased mammary epithelial cell proliferation that was inhibited with erlotinib. Taken together, these data suggest that AREG and EREG mediate tumorigenic phenotypes by activating EGFR signaling, and that the oncogenic potential of FGFR1 requires EGFR activation to promote mammary tumorigenesis.

  19. Constitutive activation of transforming growth factor Beta receptor 1 in the mouse uterus impairs uterine morphology and function.

    PubMed

    Gao, Yang; Duran, Samantha; Lydon, John P; DeMayo, Francesco J; Burghardt, Robert C; Bayless, Kayla J; Bartholin, Laurent; Li, Qinglei

    2015-02-01

    Despite increasing evidence pointing to the essential involvement of the transforming growth factor beta (TGFB) superfamily in reproduction, a definitive role of TGFB signaling in the uterus remains to be unveiled. In this study, we generated a gain-of-function mouse model harboring a constitutively active (CA) TGFB receptor 1 (TGFBR1), the expression of which was conditionally induced by the progesterone receptor (Pgr)-Cre recombinase. Overactivation of TGFB signaling was verified by enhanced phosphorylation of SMAD2 and increased expression of TGFB target genes in the uterus. TGFBR1 Pgr-Cre CA mice were sterile. Histological, cellular, and molecular analyses demonstrated that constitutive activation of TGFBR1 in the mouse uterus promoted formation of hypermuscled uteri. Accompanying this phenotype was the upregulation of a battery of smooth muscle genes in the uterus. Furthermore, TGFB ligands activated SMAD2/3 and stimulated the expression of a smooth muscle maker gene, alpha smooth muscle actin (ACTA2), in human uterine smooth muscle cells. Immunofluorescence microscopy identified a marked reduction of uterine glands in TGFBR1 Pgr-Cre CA mice within the endometrial compartment that contained myofibroblast-like cells. Thus, constitutive activation of TGFBR1 in the mouse uterus caused defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced uterine glands, and impaired uterine decidualization. These results underscore the importance of a precisely controlled TGFB signaling system in establishing a uterine microenvironment conducive to normal development and function.

  20. Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

    PubMed Central

    Nam, Gaewon; Jeong, Se Kyoo; Park, Bu Man; Lee, Sin Hee; Kim, Hyun Jong; Hong, Seung-Phil; Kim, Beomjoon

    2016-01-01

    Background Many inflammatory mediators, including various cytokines (e.g. interleukins and tumor necrosis factor [TNF]), inflammatory proteases, and histamine are released following mast cell activation. However, the endogenous modulators for mast cell activation and the underlying mechanism have yet to be elucidated. Endogenous cannabinoids such as palmitoylethanolamide (PEA) and N-arachidonoylethanolamine (anandamide or AEA), were found in peripheral tissues and have been proposed to possess autacoid activity, implying that cannabinoids may downregulate mast cell activation and local inflammation. Objective In order to investigate the effect of cannabinoid receptor-1 (CB1R) agonists on mast cell activation, AEA-derived compounds were newly synthesized and evaluated for their effect on mast cell activation. Methods The effects of selected compounds on FcεRI-induced histamine and β-hexosaminidase release were evaluated in a rat basophilic leukemia cell line (RBL-2H3). To further investigate the inhibitory effects of CB1R agonist in vivo, an oxazolone-induced atopic dermatitis mouse model was exploited. Results We found that CB1R inhibited the release of inflammatory mediators without causing cytotoxicity in RBL-2H3 cells and that CB1R agonists markedly and dose-dependently suppressed mast cell proliferation indicating that CB1R plays an important role in modulating antigen-dependent immunoglobulin E (IgE)-mediated mast cell activation. We also found that topical application of CB1R agonists suppressed the recruitment of mast cells into the skin and reduced the level of blood histamine. Conclusion Our results indicate that CB1R agonists down-regulate mast cell activation and may be used for relieving inflammatory symptoms mediated by mast cell activation, such as atopic dermatitis, psoriasis, and contact dermatitis. PMID:26848215

  1. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    PubMed

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  2. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  3. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  4. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    SciTech Connect

    Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro; Matsuda, Kouhei; Asaoka, Yoichi; Nishina, Hiroshi; Nakakura, Takashi; Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu; Tomura, Hideaki

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  5. Discoidin domain receptor 1 promotes Th17 cell migration by activating the RhoA/ROCK/MAPK/ERK signaling pathway

    PubMed Central

    Azreq, Mohammed-Amine El; Kadiri, Maleck; Boisvert, Marc; Pagé, Nathalie; Tessier, Philippe A.; Aoudjit, Fawzi

    2016-01-01

    Effector T cell migration through the tissue extracellular matrix (ECM) is an important step of the adaptive immune response and in the development of inflammatory diseases. However, the mechanisms involved in this process are still poorly understood. In this study, we addressed the role of a collagen receptor, the discoidin domain receptor 1 (DDR1), in the migration of Th17 cells. We showed that the vast majority of human Th17 cells express DDR1 and that silencing DDR1 or using the blocking recombinant receptor DDR1:Fc significantly reduced their motility and invasion in three-dimensional (3D) collagen. DDR1 promoted Th17 migration by activating RhoA/ROCK and MAPK/ERK signaling pathways. Interestingly, the RhoA/ROCK signaling module was required for MAPK/ERK activation. Finally, we showed that DDR1 is important for the recruitment of Th17 cells into the mouse dorsal air pouch containing the chemoattractant CCL20. Collectively, our results indicate that DDR1, via the activation of RhoA/ROCK/MAPK/ERK signaling axis, is a key pathway of effector T cell migration through collagen of perivascular tissues. As such, DDR1 can contribute to the development of Th17-dependent inflammatory diseases. PMID:27391444

  6. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish.

    PubMed

    Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro; Matsuda, Kouhei; Asaoka, Yoichi; Nishina, Hiroshi; Nakakura, Takashi; Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu; Tomura, Hideaki

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors.

  7. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    PubMed

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds.

  8. Screening for cardiovascular safety: a structure-activity approach for guiding lead selection of melanin concentrating hormone receptor 1 antagonists.

    PubMed

    Kym, Philip R; Souers, Andrew J; Campbell, Thomas J; Lynch, John K; Judd, Andrew S; Iyengar, Rajesh; Vasudevan, Anil; Gao, Ju; Freeman, Jennifer C; Wodka, Dariusz; Mulhern, Mathew; Zhao, Gang; Wagaw, Seble H; Napier, James J; Brodjian, Sevan; Dayton, Brian D; Reilly, Regina M; Segreti, Jason A; Fryer, Ryan M; Preusser, Lee C; Reinhart, Glenn A; Hernandez, Lisa; Marsh, Kennan C; Sham, Hing L; Collins, Christine A; Polakowski, James S

    2006-04-06

    An inactin-anesthetized rat cardiovascular (CV) assay was employed in a screening mode to triage multiple classes of melanin-concentrating hormone receptor 1 (MCHr1) antagonists. Lead identification was based on a compound profile producing high drug concentration in both plasma (>40 microM) and brain (>20 microg/g) with <15% change in cardiovascular endpoints. As a result of these stringent requirements, lead optimization activities on multiple classes of MCHr1 antagonists were terminated. After providing evidence that the cardiovascular liabilities were not a function of MCHr1 antagonism, continued screening identified the chromone-substituted aminopiperidine amides as a class of MCHr1 antagonists that demonstrated a safe cardiovascular profile at high drug concentrations in both plasma and brain. The high incidence of adverse cardiovascular effects associated with an array of MCHr1 antagonists of significant chemical diversity, combined with the stringent safety requirements for antiobesity drugs, highlight the importance of incorporating cardiovascular safety assessment early in the lead selection process.

  9. Design, synthesis and structure-activity relationship studies of novel free fatty acid receptor 1 agonists bearing amide linker.

    PubMed

    Yang, Jianyong; Li, Zheng; Li, Huilan; Liu, Chunxia; Wang, Nasi; Shi, Wei; Liao, Chen; Cai, Xingguang; Huang, Wenlong; Qian, Hai

    2017-04-15

    The free fatty acid receptor 1 (FFA1/GPR40) has attracted extensive attention as a novel antidiabetic target. Aiming to explore the chemical space of FFA1 agonists, a new series of lead compounds with amide linker were designed and synthesized by combining the scaffolds of NIH screened lead compound 1 and GW9508. Among them, the optimal lead compound 17 exhibited a considerable agonistic activity (45.78%) compared to the NIH screened compound 1 (15.32%). During OGTT in normal mice, the compound 17 revealed a significant glucose-lowering effect (-23.7%) at the dose of 50mg/kg, proximity to the hypoglycemic effect (-27.8%) of Metformin (200mg/kg). In addition, the compound 17 (100mg/kg) also exhibited a significant improvement in glucose tolerance with a 29.1% reduction of glucose AUC0-2h in type 2 diabetic mice. All of these results indicated that compound 17 was considered to be a promising lead structure suitable for further optimization.

  10. Predicted PAR1 inhibitors from multiple computational methods

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Liu, Jinfeng; Zhu, Tong; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

    2016-08-01

    Multiple computational approaches are employed in order to find potentially strong binders of PAR1 from the two molecular databases: the Specs database containing more than 200,000 commercially available molecules and the traditional Chinese medicine (TCM) database. By combining the use of popular docking scoring functions together with detailed molecular dynamics simulation and protein-ligand free energy calculations, a total of fourteen molecules are found to be potentially strong binders of PAR1. The atomic details in protein-ligand interactions of these molecules with PAR1 are analyzed to help understand the binding mechanism which should be very useful in design of new drugs.

  11. Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

    SciTech Connect

    Gandhi, Prafull S.; Chen, Zhiwei; Di Cera, Enrico

    2010-05-11

    Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 {angstrom} resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp{sup 39}, does not hinder binding to the active site and actually makes favorable interactions with Gly{sup 219} of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.

  12. Discoidin Domain Receptor 1

    PubMed Central

    Song, Sunmi; Shackel, Nicholas A.; Wang, Xin M.; Ajami, Katerina; McCaughan, Geoffrey W.; Gorrell, Mark D.

    2011-01-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell–collagen interactions in chronic liver injury. PMID:21356365

  13. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    SciTech Connect

    Gao, Lin; Chao, Lee; Chao, Julie

    2010-02-01

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  14. Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCɛ pathway

    PubMed Central

    Mousseaux, Delphine; Le Gallic, Lionel; Ryan, Joanne; Oiry, Catherine; Gagne, Didier; Fehrentz, Jean-Alain; Galleyrand, Jean-Claude; Martinez, Jean

    2006-01-01

    The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dep endent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Gö6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Gö6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCɛ-dependent pathway. PMID:16582936

  15. Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway.

    PubMed

    Mousseaux, Delphine; Le Gallic, Lionel; Ryan, Joanne; Oiry, Catherine; Gagne, Didier; Fehrentz, Jean-Alain; Galleyrand, Jean-Claude; Martinez, Jean

    2006-06-01

    1. The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2. To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3. Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4. We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Gö6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Gö6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (epsilon, delta), but not conventional or atypical PKCs. Further analyses suggest that PKCepsilon is required for the activation of ERK1/2. 5. Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCepsilon-dependent pathway.

  16. Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

    PubMed Central

    Huang, Zaohua; Myhr, Courtney; Bathgate, Ross A. D.; Ho, Brian A.; Bueno, Amaya; Hu, Xin; Xiao, Jingbo; Southall, Noel; Barnaeva, Elena; Agoulnik, Irina U.; Marugan, Juan J.; Ferrer, Marc; Agoulnik, Alexander I.

    2015-01-01

    Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing. PMID:26347712

  17. Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1

    PubMed Central

    Winther, Malene; Holdfeldt, André; Gabl, Michael; Wang, Ji Ming; Forsman, Huamei; Dahlgren, Claes

    2016-01-01

    Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits. PMID:27907124

  18. Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis.

    PubMed

    Gaudreault, Eric; Stankova, Jana; Rola-Pleszczynski, Marek

    2005-01-01

    Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.

  19. CRF receptor 1 antagonism and brain distribution of active components contribute to the ameliorative effect of rikkunshito on stress-induced anorexia.

    PubMed

    Mogami, Sachiko; Sadakane, Chiharu; Nahata, Miwa; Mizuhara, Yasuharu; Yamada, Chihiro; Hattori, Tomohisa; Takeda, Hiroshi

    2016-06-08

    Rikkunshito (RKT), a Kampo medicine, has been reported to show an ameliorative effect on sustained hypophagia after novelty stress exposure in aged mice through serotonin 2C receptor (5-HT2CR) antagonism. We aimed to determine (1) whether the activation of anorexigenic neurons, corticotropin-releasing factor (CRF), and pro-opiomelanocortin (POMC) neurons, is involved in the initiation of hypophagia induced by novelty stress in aged mice; (2) whether the ameliorative effect of RKT is associated with CRF and POMC neurons and downstream signal transduction; and (3) the plasma and brain distribution of the active components of RKT. The administration of RKT or 5-HT2CR, CRF receptor 1 (CRFR1), and melanocortin-4 receptor antagonists significantly restored the decreased food intake observed in aged male C57BL/6 mice in the early stage after novelty stress exposure. Seven components of RKT exhibited antagonistic activity against CRFR1. Hesperetin and isoliquiritigenin, which showed antagonistic effects against both CRFR1 and 5-HT2CR, were distributed in the plasma and brain of male Sprague-Dawley rats after a single oral administration of RKT. In conclusion, the ameliorative effect of RKT in this model is assumed to be at least partly due to brain-distributed active components possessing 5-HT2CR and CRFR1 antagonistic activities.

  20. Sortilin-related receptor 1 interacts with amyloid precursor protein and is activated by 6-shogaol, leading to inhibition of the amyloidogenic pathway.

    PubMed

    Na, Ji-Young; Song, Kibbeum; Lee, Ju-Woon; Kim, Sokho; Kwon, Jungkee

    2017-03-18

    Sortilin-related receptor 1 (SORL1) is a neuronal sorting protein that reduces amyloid precursor protein (APP) trafficking to secretases that generate amyloid beta (Aβ). Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, research regarding the activation of SORL1 has not yet been reported. Here, we aimed to investigate whether 6-shogaol contributes to the increases in SORL1 that are related to Alzheimer's disease (AD). To clarify the effect of 6-shogaol as a possible activator of SORL1, we used SORL1 siRNA as a blockade of SORL1 in hippocampal neuronal cells (HT22). We found that SORL1 siRNA treatment naturally inhibited SORL1 and led to increases in β-secretase APP cleaving enzyme (BACE), secreted APP-β (sAPPβ) and Aβ. In contrast, 6-shogaol-mediated activation of SORL1 significantly downregulated BACE, sAPPβ, and Aβ in both in vitro HT22 cells and in vivo APPSw/PS1-dE9 Tg mice. Therefore, SORL1 activation by 6-shogaol provides neuronal cell survival through the inhibition of Aβ production. These results indicate that 6-shogaol should be regarded as an SORL1 activator and a potential preventive agent for the treatment of AD.

  1. CRF receptor 1 antagonism and brain distribution of active components contribute to the ameliorative effect of rikkunshito on stress-induced anorexia

    PubMed Central

    Mogami, Sachiko; Sadakane, Chiharu; Nahata, Miwa; Mizuhara, Yasuharu; Yamada, Chihiro; Hattori, Tomohisa; Takeda, Hiroshi

    2016-01-01

    Rikkunshito (RKT), a Kampo medicine, has been reported to show an ameliorative effect on sustained hypophagia after novelty stress exposure in aged mice through serotonin 2C receptor (5-HT2CR) antagonism. We aimed to determine (1) whether the activation of anorexigenic neurons, corticotropin-releasing factor (CRF), and pro-opiomelanocortin (POMC) neurons, is involved in the initiation of hypophagia induced by novelty stress in aged mice; (2) whether the ameliorative effect of RKT is associated with CRF and POMC neurons and downstream signal transduction; and (3) the plasma and brain distribution of the active components of RKT. The administration of RKT or 5-HT2CR, CRF receptor 1 (CRFR1), and melanocortin-4 receptor antagonists significantly restored the decreased food intake observed in aged male C57BL/6 mice in the early stage after novelty stress exposure. Seven components of RKT exhibited antagonistic activity against CRFR1. Hesperetin and isoliquiritigenin, which showed antagonistic effects against both CRFR1 and 5-HT2CR, were distributed in the plasma and brain of male Sprague-Dawley rats after a single oral administration of RKT. In conclusion, the ameliorative effect of RKT in this model is assumed to be at least partly due to brain-distributed active components possessing 5-HT2CR and CRFR1 antagonistic activities. PMID:27273195

  2. Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

    PubMed Central

    McElroy, Steven J.; Frey, Mark R.; Yan, Fang; Edelblum, Karen L.; Goettel, Jeremy A.; John, Sutha; Polk, D. Brent

    2008-01-01

    Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2−/−, but not TNFR1−/−, mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1−/− MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases. PMID:18467504

  3. 1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea

    SciTech Connect

    Kikuno, Shota; Taguchi, Keiko; Iwamoto, Noriko; Yamano, Shigeru; Cho, Arthur K.; Froines, John R.; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-01-15

    1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM{sub 2.5}. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC{sub 5} value of 18.7 {mu}M. The contraction required extracellular calcium and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated phospholipase A2 (PLA2)/lipoxygenase (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation of PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells.

  4. Structure-activity relationships of 1,4-dihydropyridines that act as enhancers of the vanilloid receptor 1 (TRPV1).

    PubMed

    Roh, Eun Joo; Keller, Jason M; Olah, Zoltan; Iadarola, Michael J; Jacobson, Kenneth A

    2008-10-15

    Vanilloid agonists such as capsaicin activate ion flux through the TRPV1 channel, a heat- and ligand-gated cation channel that transduces painful chemical or thermal stimuli applied to peripheral nerve endings in skin or deep tissues. We have probed the SAR of a variety of 1,4-dihydropyridine (DHP) derivatives as novel 'enhancers' of TRPV1 activity by examining changes in capsaicin-induced elevations in (45)Ca(2+)-uptake in either cells ectopically expressing TRPV1 or in cultured dorsal root ganglion (DRG) neurons. The enhancers increased the maximal capsaicin effect on (45)Ca(2+)-uptake by typically 2- to 3-fold without producing an action when used alone. The DHP enhancers contained 6-aryl substitution and small alkyl groups at the 1 and 4 positions, and a 3-phenylalkylthioester was tolerated. Levels of free intracellular Ca(2+), as measured by calcium imaging, were also increased in DRG neurons when exposed to the combination of capsaicin and the most efficacious enhancer 23 compared to capsaicin alone. Thus, DHPs can modulate TRPV1 channels in a positive fashion.

  5. HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver

    PubMed Central

    Sun, Yue; Chen, Yutian; Swendeman, Steven L.; Jung, Bongnam; Chavez, Deebly; Cao, Zhongwei; Christoffersen, Christina; Nielsen, Lars Bo; Schwab, Susan R.; Rafii, Shahin; Hla, Timothy

    2016-01-01

    Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this “maladaptive repair” phenotype was recapitulated in mice that lack S1P1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis. PMID:28018969

  6. AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin

    PubMed Central

    Cai, Junting; Suber, Tomeka

    2017-01-01

    Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin. PMID:28348461

  7. C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity.

    PubMed

    Chillakuri, Chandramouli Reddy; Reinhart, Christoph; Michel, Hartmut

    2007-12-01

    We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.

  8. Raloxifene activates G protein-coupled estrogen receptor 1/Akt signaling to protect dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice.

    PubMed

    Bourque, Mélanie; Morissette, Marc; Di Paolo, Thérèse

    2014-10-01

    Raloxifene, used in the clinic, is reported to protect brain dopaminergic neurons in mice. Raloxifene was shown to mediate an effect through the G protein-coupled estrogen receptor 1 (GPER1). We investigated if raloxifene neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated male mice is mediated through GPER1 by using its antagonist G15. Striatal concentrations of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid to dopamine ratio as well as dopamine transporter and vesicular monoamine transporter 2 showed that raloxifene neuroprotection of dopaminergic neurons was blocked by G15. Protection by raloxifene was accompanied by activation of striatal Akt signaling (but not ERK1/2 signaling) and increased Bcl-2 and brain-derived neurotrophic factor levels; these effects were abolished by coadministration with G15. The effect of raloxifene was not mediated through increased levels of 17β-estradiol. MPTP mice had decreased plasma testosterone, dihydrotestosterone, and 3β-diol levels; this was prevented in raloxifene-treated MPTP mice. Our results suggest that raloxifene acted through GPER1 to mediate Akt activation, increase Bcl-2 and brain-derived neurotrophic factor levels, and protection of dopaminergic neurons and plasma androgens.

  9. Allele-specific differences in activity of a novel cannabinoid receptor 1 (CNR1) gene intronic enhancer in hypothalamus, dorsal root ganglia, and hippocampus.

    PubMed

    Nicoll, Gemma; Davidson, Scott; Shanley, Lynne; Hing, Ben; Lear, Marissa; McGuffin, Peter; Ross, Ruth; MacKenzie, Alasdair

    2012-04-13

    Polymorphisms within intron 2 of the CNR1 gene, which encodes cannabinoid receptor 1 (CB(1)), have been associated with addiction, obesity, and brain volume deficits. We used comparative genomics to identify a polymorphic (rs9444584-C/T) sequence (ECR1) in intron 2 of the CNR1 gene that had been conserved for 310 million years. The C-allele of ECR1 (ECR1(C)) acted as an enhancer in hypothalamic and dorsal root ganglia cells and responded to MAPK activation through the MEKK pathway but not in hippocampal cells. However, ECR1(T) was significantly more active in hypothalamic and dorsal root ganglia cells but, significantly, and in contrast to ECR1(C), was highly active in hippocampal cells where it also responded strongly to activation of MAPK. Intriguingly, rs9444584 is in strong linkage disequilibrium with two other SNPs (rs9450898 (r(2) = 0.841) and rs2023239 (r(2) = 0.920)) that have been associated with addiction, obesity (rs2023239), and reduced fronto-temporal white matter volumes in schizophrenia patients as a result of cannabis misuse (rs9450898). Considering their high linkage disequilibrium and the increased response of ECR1(T) to MAPK signaling when compared with ECR1(C), it is possible that the functional effects of the different alleles of rs9444584 may play a role in the conditions associated with rs9450898 and rs2023239. Further analysis of the different alleles of ECR1 may lead to a greater understanding of the role of CNR1 gene misregulation in these conditions as well as chronic inflammatory pain.

  10. REPEATED ANABOLIC/ANDROGENIC STEROID EXPOSURE DURING ADOLESCENCE ALTERS PHOSPHATE-ACTIVATED GLUTAMINASE AND GLUTAMATE RECEPTOR 1 SUBUNIT IMMUNOREACTIVITY IN HAMSTER BRAIN: CORRELATION WITH OFFENSIVE AGGRESSION

    PubMed Central

    Fischer, Shannon G.; Ricci, Lesley A.; Melloni, Richard H.

    2007-01-01

    Male Syrian hamsters (Mesocricetus auratus) treated with moderately high doses (5.0mg/kg/day) of anabolic/androgenic steroids (AAS) during adolescence (P27–P56) display highly escalated offensive aggression. The current study examined whether adolescent AAS-exposure influenced the immunohistochemical localization of phosphate-activated glutaminase (PAG), the rate-limiting enzyme in the synthesis of glutamate, a fast-acting neurotransmitter implicated in the modulation of aggression in various species and models of aggression, as well as glutamate receptor 1 subunit (GluR1). Hamsters were administered AAS during adolescence, scored for offensive aggression using the resident-intruder paradigm, and then examined for changes in PAG and GluR1 immunoreactivity in areas of the brain implicated in aggression control. When compared with sesame oil-treated control animals, aggressive AAS-treated hamsters displayed a significant increase in the number of PAG- and area density of GluR1- containing neurons in several notable aggression regions, although the differential pattern of expression did not appear to overlap across brain regions. Together, these results suggest that altered glutamate synthesis and GluR1 receptor expression in specific aggression areas may be involved in adolescent AAS-induced offensive aggression. PMID:17418431

  11. Par1b links lumen polarity with LGN–NuMA positioning for distinct epithelial cell division phenotypes

    PubMed Central

    Lázaro-Diéguez, Francisco; Cohen, David; Fernandez, Dawn; Hodgson, Louis; van IJzendoorn, Sven C.D.

    2013-01-01

    Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal domains and their cleavage furrow rarely bifurcates the luminal domains. We determine that the serine/threonine kinase Par1b defines lumen position in concert with the position of the astral microtubule anchoring complex LGN–NuMA to yield the distinct epithelial division phenotypes. Par1b signaling via the extracellular matrix (ECM) in polarizing cells determined RhoA/Rho-kinase activity at cell–cell contact sites. Columnar MDCK and Par1b-depleted hepatocytic HepG2 cells featured high RhoA activity that correlated with robust LGN–NuMA recruitment to the metaphase cortex, spindle alignment with the substratum, and columnar organization. Reduced RhoA activity at the metaphase cortex in HepG2 cells and Par1b-overexpressing MDCK cells correlated with a single or no LGN–NuMA crescent, tilted spindles, and the development of lateral lumen polarity. PMID:24165937

  12. Selective activation of the trace amine-associated receptor 1 decreases cocaine's reinforcing efficacy and prevents cocaine-induced changes in brain reward thresholds.

    PubMed

    Pei, Yue; Mortas, Patrick; Hoener, Marius C; Canales, Juan J

    2015-12-03

    The newly discovered trace amine-associated receptor 1 (TAAR1) has emerged as a promising target for medication development in stimulant addiction due to its ability to regulate dopamine (DA) function and modulate stimulants' effects. Recent findings indicate that TAAR1 activation blocks some of the abuse-related physiological and behavioral effects of cocaine. However, findings from existing self-administration studies are inconclusive due to the very limited range of cocaine unit doses tested. Here, in order to shed light on the influence of TAAR1 on cocaine's reward and reinforcement, we studied the effects of partial and full activation of TAAR1on (1) the dose-response curve for cocaine self-administration and (2) cocaine-induced changes in intracranial self-stimulation (ICSS). In the first experiment, we examined the effects of the selective full and partial TAAR1 agonists, RO5256390 and RO5203648, on self-administration of five unit-injection doses of cocaine (0.03, 0.1, 0.2, 0.45, and 1mg/kg/infusion). Both agonists induced dose-dependent downward shifts in the cocaine dose-response curve, indicating that both partial and full TAAR1 activation decrease cocaine, reinforcing efficacy. In the second experiment, RO5256390 and the partial agonist, RO5263397, dose-dependently prevented cocaine-induced lowering of ICSS thresholds. Taken together, these data demonstrated that TAAR1 stimulation effectively suppresses the rewarding and reinforcing effects of cocaine in self-administration and ICSS models, supporting the candidacy of TAAR1 as a drug discovery target for cocaine addiction.

  13. Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

    PubMed Central

    Tentori, Lucio; Scimeca, Manuel; Dorio, Annalisa S.; Atzori, Maria Grazia; Failla, Cristina M.; Morea, Veronica; Bonanno, Elena; D'Atri, Stefania; Lacal, Pedro M.

    2016-01-01

    Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. PMID:27655684

  14. Decreased dopamine receptor 1 activity and impaired motor-skill transfer in Dyt1 ΔGAG heterozygous knock-in mice

    PubMed Central

    Yokoi, Fumiaki; Dang, Mai T.; Liu, Jun; Gandre, Jason R.; Kwon, Kelly; Yuen, Robert; Li, Yuqing

    2014-01-01

    DYT1 dystonia is a movement disorder caused by a trinucleotide deletion (ΔGAG) in DYT1 (TOR1A), corresponding to a glutamic acid loss in the C-terminal region of torsinA. Functional alterations in the basal ganglia circuits have been reported in both DYT1 dystonia patients and rodent models. Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits and decreased striatal dopamine receptor 2 (D2R) binding activity, suggesting a malfunction of the indirect pathway. However, the role of the direct pathway in pathogenesis of dystonia is not yet clear. Here, we report that Dyt1 KI mice exhibit significantly decreased striatal dopamine receptor 1 (D1R) binding activity and D1R protein levels, suggesting the alteration of the direct pathway. The decreased D1R may be caused by translational or post-translational processes since Dyt1 KI mice had normal levels of striatal D1R mRNA and a normal number of striatal neurons expressing D1R. Levels of striatal ionotropic glutamate receptor subunits, dopamine transporter, acetylcholine muscarinic M4 receptor and adenosine A2A receptor were not altered suggesting a specificity of affected polytopic membrane-associated proteins. Contribution of the direct pathway to motor-skill learning has been suggested in another pharmacological rat model injected with a D1R antagonist. In the present study, we developed a novel motor skill transfer test for mice and found deficits in Dyt1 KI mice. Further characterization of both the direct and the indirect pathways in Dyt1 KI mice will aid the development of novel therapeutic drugs. PMID:25451552

  15. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    SciTech Connect

    Takeshita, Harunori; Kitano, Masayasu; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto; Miyazawa, Keiji; Hla, Timothy; Sano, Hajime

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  16. Both PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2 Promote Seedling Photomorphogenesis in Multiple Light Signaling Pathways1[C][W][OPEN

    PubMed Central

    Zhou, Peng; Song, Meifang; Yang, Qinghua; Su, Liang; Hou, Pei; Guo, Lin; Zheng, Xu; Xi, Yulin; Meng, Fanhua; Xiao, Yang; Yang, Li; Yang, Jianping

    2014-01-01

    Arabidopsis (Arabidopsis thaliana) seedlings undergo photomorphogenesis in the light and etiolation in the dark. Light-activated photoreceptors transduce the light signals through a series of photomorphogenesis promoting or repressing factors to modulate many developmental processes in plants, such as photomorphogenesis and shade avoidance. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a conserved RING finger E3 ubiquitin ligase, which mediates degradation of several photomorphogenesis promoting factors, including ELONGATED HYPOCOTYL5 (HY5) and LONG HYPOCOTYL IN FAR-RED1 (HFR1), through a 26S proteasome-dependent pathway. PHYTOCHROME RAPIDLY REGULATED1 (PAR1) was first detected as an early repressed gene in both phytochrome A (phyA)-mediated far-red and phyB-mediated red signaling pathways, and subsequent studies showed that both PAR1 and PAR2 are negative factors of shade avoidance in Arabidopsis. However, the role of PAR1 and PAR2 in seedling deetiolation, and their relationships with other photomorphogenesis promoting and repressing factors are largely unknown. Here, we confirmed that both PAR1 and PAR2 redundantly enhance seedling deetiolation in multiple photoreceptor signaling pathways. Their transcript abundances are repressed by phyA, phyB, and cryptochrome1 under far-red, red, and blue light conditions, respectively. Both PAR1 and PAR2 act downstream of COP1, and COP1 mediates the degradation of PAR1 and PAR2 through the 26S proteasome pathway. Both PAR1 and PAR2 act in a separate pathway from HY5 and HFR1 under different light conditions, except for sharing in the same pathway with HFR1 under far-red light. Together, our results substantiate that PAR1 and PAR2 are positive factors functioning in multiple photoreceptor signaling pathways during seedling deetiolation. PMID:24335334

  17. Augmented motor activity and reduced striatal preprodynorphin mRNA induction in response to acute amphetamine administration in metabotropic glutamate receptor 1 knockout mice.

    PubMed

    Mao, L; Conquet, F; Wang, J Q

    2001-01-01

    Metabotropic glutamate receptor 1 (mGluR1) is a G-protein-coupled receptor and is expressed in the medium spiny projection neurons of mouse striatum. To define the role of mGluR1 in actions of psychostimulant, we compared both motor behavior and striatal neuropeptide mRNA expression between mGluR1 mutant and wild-type control mice after a single injection of amphetamine. We found that acute amphetamine injection increased motor activity in both mutant and control mice in a dose-dependent manner (1, 4, and 12 mg/kg, i.p.). However, the overall motor responses of mGluR1 -/- mice to all three doses of amphetamine were significantly greater than those of wild-type +/+ mice. Amphetamine also induced a dose-dependent elevation of preprodynorphin mRNA in the dorsal and ventral striatum of mutant and wild-type mice as revealed by quantitative in situ hybridization. In contrast to behavioral responses, the induction of dynorphin mRNA in both the dorsal and ventral striatum of mutant mice was significantly less than that of wild-type mice in response to the two higher doses of amphetamine. In addition, amphetamine elevated basal levels of substance P mRNA in the dorsal and ventral striatum of mGluR1 mutant mice to a similar level as that of wild-type mice. There were no differences in basal levels and distribution patterns of the two mRNAs between the two genotypes of mice treated with saline. These results demonstrate a clear augmented behavioral response of mGluR1 knockout mice to acute amphetamine exposure that is closely correlated with reduced dynorphin mRNA induction in the same mice. It appears that an intact mGluR1 is specifically critical for full dynorphin induction, and impaired mobilization of inhibitory dynorphin system as a result of lacking mGluR1 may contribute to an augmentation of motor stimulation in response to acute administration of psychostimulant.

  18. Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

    PubMed

    Quach, Hue Tu; Hirano, Seiya; Fukuhara, Sayuri; Watanabe, Tsubasa; Kanoh, Naoki; Iwabuchi, Yoshiharu; Usui, Takeo; Kataoka, Takao

    2015-01-01

    Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

  19. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

    PubMed Central

    Dowal, Louisa; Sim, Derek S.; Dilks, James R.; Blair, Price; Beaudry, Sarah; Denker, Bradley M.; Koukos, Georgios; Kuliopulos, Athan; Flaumenhaft, Robert

    2011-01-01

    G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation. PMID:21282664

  20. Thrombin-induced regulation of CD95(Fas) expression in the N9 microglial cell line: evidence for involvement of proteinase-activated receptor(1) and extracellular signal-regulated kinase 1/2.

    PubMed

    Weinstein, Jonathan R; Zhang, Matthew; Kutlubaev, Mansur; Lee, Richard; Bishop, Caroline; Andersen, Henrik; Hanisch, Uwe-Karsten; Möller, Thomas

    2009-03-01

    Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase alpha-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human alpha-thrombin (rh-thr) induces a restricted set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose-response and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR(1) agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2). Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate the neuroinflammatory response in a variety of neurological disorders.

  1. Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation.

    PubMed

    Benton, Richard; Palacios, Isabel M; St Johnston, Daniel

    2002-11-01

    PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. We identified 14-3-3 proteins as Drosophila PAR-1 interactors and show that PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display identical phenotypes to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries.

  2. Vorapaxar: emerging evidence and clinical questions in a new era of PAR-1 inhibition.

    PubMed

    Ungar, Leo; Rodriguez, Fatima; Mahaffey, Kenneth W

    2016-11-01

    Despite the use of therapies recommended in practice guidelines for secondary prevention in patients with atherosclerotic coronary artery disease, the residual risk for cardiovascular events remains high. Some of the residual risk is believed to result from incomplete platelet inhibition with current therapy. Vorapaxar is a first-in-class, novel antiplatelet agent that acts by antagonizing the PAR-1 receptor, inhibiting thrombin-mediated platelet activation. Vorapaxar was recently approved by the Food and Drug Administration for secondary prevention of cardiovascular events in patients with a history of myocardial infarction or peripheral artery disease who do not have a history of transient ischemic attack or stroke. We review the data from two key phase III cardiovascular outcome trials with vorapaxar: TRACER and TRA 2P-TIMI 50. We will focus on identifying the key patient populations that should be identified for treatment, highlight practical clinical issues when prescribing vorapaxar, and review unanswered questions. Vorapaxar should be considered in patients at high risk for recurrent ischemic events and low risk of bleeding.

  3. TNF-α has tropic rather than apoptotic activity in human hematopoietic progenitors: involvement of TNF receptor-1 and caspase-8.

    PubMed

    Mizrahi, Keren; Stein, Jerry; Yaniv, Isaac; Kaplan, Offer; Askenasy, Nadir

    2013-01-01

    Tumor necrosis factor-α (TNF-α) has been suggested to exert detrimental effects on hematopoietic progenitor function that might limit the success of transplants. In this study, we assessed the influences of TNF-α and its two cognate receptors on the function of fresh umbilical cord blood (UCB) and cryopreserved mobilized peripheral blood (mPB). CD34(+) progenitors from both sources are less susceptible to spontaneous apoptosis than lineage-committed cells and are not induced into apoptosis by TNF-α. Consequently, the activity of UCB-derived severe combined immune deficiency (SCID) reconstituting cells and long-term culture-initiating cells is unaffected by this cytokine. On the contrary, transient exposure of cells from both sources to TNF-α stimulates the activity of myeloid progenitors, which persists in vivo in UCB cell transplants. Progenitor stimulation is selectively mediated by TNF-R1 and involves activation of caspase-8, without redundant activity of TNF-R2. Despite significant differences between fresh UCB cells and cryopreserved mPB cells in susceptibility to apoptosis and time to activation, TNF-α is primarily involved in tropic signaling in hematopoietic progenitors from both sources. Cytokine-mediated tropism cautions against TNF-α neutralization under conditions of stress hematopoiesis and may be particularly beneficial in overcoming the limitations of UCB cell transplants.

  4. Peptide length and folding state govern the capacity of staphylococcal β-type phenol-soluble modulins to activate human formyl-peptide receptors 1 or 2.

    PubMed

    Kretschmer, Dorothee; Rautenberg, Maren; Linke, Dirk; Peschel, Andreas

    2015-04-01

    Most staphylococci produce short α-type PSMs and about twice as long β-type PSMs that are potent leukocyte attractants and toxins. PSMs are usually secreted with the N-terminal formyl group but are only weak agonists for the leukocyte FPR1. Instead, the FPR1-related FPR2 senses PSMs efficiently and is crucial for leukocyte recruitment in infection. Which structural features distinguish FPR1 from FPR2 ligands has remained elusive. To analyze which peptide properties may govern the capacities of β-type PSMs to activate FPRs, full-length and truncated variants of such peptides from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis were synthesized. FPR2 activation was observed even for short N- or C-terminal β-type PSM variants once they were longer than 18 aa, and this activity increased with length. In contrast, the shortest tested peptides were potent FPR1 agonists, and this property declined with increasing peptide length. Whereas full-length β-type PSMs formed α-helices and exhibited no FPR1-specific activity, the truncated peptides had less-stable secondary structures, were weak agonists for FPR1, and required N-terminal formyl-methionine residues to be FPR2 agonists. Together, these data suggest that FPR1 and FPR2 have opposed ligand preferences. Short, flexible PSM structures may favor FPR1 but not FPR2 activation, whereas longer peptides with α-helical, amphipathic properties are strong FPR2 but only weak FPR1 agonists. These findings should help to unravel the ligand specificities of 2 critical human PRRs, and they may be important for new, anti-infective and anti-inflammatory strategies.

  5. Hypothesizing that, A Pro-Dopamine Regulator (KB220Z) Should Optimize, but Not Hyper-Activate the Activity of Trace Amine-Associated Receptor 1 (TAAR-1) and Induce Anti-Craving of Psychostimulants in the Long-Term.

    PubMed

    Blum, Kenneth; Badgaiyan, Rajendra D; Braverman, Eric R; Dushaj, Kristina; Li, Mona; Thanos, Peter K; Demetrovics, Zsolt; Febo, Marcelo

    2016-01-01

    Unlike other drugs of abuse such as alcohol, nicotine, opiates/opioids, the FDA has not approved any agent to treat psychostimulant dependence. Certainly, it is widely acceptable that dopaminergic signaling is a key factor in both the initiation and continued motivation to abuse this class of stimulant substances. It is also well accepted that psychostimulants such as cocaine affect not only the release of neuronal dopamine at the nucleus accumbens (NAc), but also has powerful inhibitory actions on the dopamine transporter system. Understandably, certain individuals are at high risk and very vulnerable to abuse this class of substances. Trace-amine-associated receptor 1 (TAAR1) is a G -protein coupled receptor activated by trace amines. The encoded protein responds little or not at all to dopamine, serotonin, epinephrine, or histamine, but responds well to beta-phenylethylamine, p-tyramine, octopamine, and tryptamine. This gene is thought to be intronless. TAAR1 agonists reduce the neurochemical effects of cocaine and amphetamines as well as attenuate addiction and abuse associated with these two psychostimulants. The mechanism involves blocking the firing rate of dopamine in the limbic system thereby decreasing a hyperdopaminergic trait/state, whereby the opposite is true for TAAR1 antagonists. Based on many studies, it is accepted that in Reward Deficiency Syndrome (RDS), there is weakened tonic and improved phasic dopamine discharge leading to a hypodopaminergic/glutamatergic trait. The dopamine pro-complex mixture KB220, following many clinical trials including neuroimaging studies, has been shown to enhance resting state functional connectivity in humans (abstinent heroin addicts), naïve rodent models, and regulates extensive theta action in the cingulate gyrus of abstinent psychostimulant abusers. In this article, we are hypothesizing that KB220 may induce its action on resting state functional connectivity, for example, by actually balancing (optimizing

  6. Hypothesizing that, A Pro-Dopamine Regulator (KB220Z) Should Optimize, but Not Hyper-Activate the Activity of Trace Amine-Associated Receptor 1 (TAAR-1) and Induce Anti-Craving of Psychostimulants in the Long-Term

    PubMed Central

    Blum, Kenneth; Badgaiyan, Rajendra D.; Braverman, Eric R.; Dushaj, Kristina; Li, Mona; Thanos, Peter K.; Demetrovics, Zsolt; Febo, Marcelo

    2017-01-01

    Unlike other drugs of abuse such as alcohol, nicotine, opiates/opioids, the FDA has not approved any agent to treat psychostimulant dependence. Certainly, it is widely acceptable that dopaminergic signaling is a key factor in both the initiation and continued motivation to abuse this class of stimulant substances. It is also well accepted that psychostimulants such as cocaine affect not only the release of neuronal dopamine at the nucleus accumbens (NAc), but also has powerful inhibitory actions on the dopamine transporter system. Understandably, certain individuals are at high risk and very vulnerable to abuse this class of substances. Trace-amine-associated receptor 1 (TAAR1) is a G -protein coupled receptor activated by trace amines. The encoded protein responds little or not at all to dopamine, serotonin, epinephrine, or histamine, but responds well to beta-phenylethylamine, p-tyramine, octopamine, and tryptamine. This gene is thought to be intronless. TAAR1 agonists reduce the neurochemical effects of cocaine and amphetamines as well as attenuate addiction and abuse associated with these two psychostimulants. The mechanism involves blocking the firing rate of dopamine in the limbic system thereby decreasing a hyperdopaminergic trait/state, whereby the opposite is true for TAAR1 antagonists. Based on many studies, it is accepted that in Reward Deficiency Syndrome (RDS), there is weakened tonic and improved phasic dopamine discharge leading to a hypodopaminergic/glutamatergic trait. The dopamine pro-complex mixture KB220, following many clinical trials including neuroimaging studies, has been shown to enhance resting state functional connectivity in humans (abstinent heroin addicts), naïve rodent models, and regulates extensive theta action in the cingulate gyrus of abstinent psychostimulant abusers. In this article, we are hypothesizing that KB220 may induce its action on resting state functional connectivity, for example, by actually balancing (optimizing

  7. The Major Leukocyte Chemotactic and Activating Factors in the Mouse Gut Lumen are not N-formylpeptide Receptor 1 (Fpr1) Agonists

    PubMed Central

    Ojode, Teresa; Schneider, Erich H.; Tiffany, H. Lee; Yung, Sunny; Gao, Ji-Liang; Murphy, Philip M.

    2013-01-01

    Cultured bacteria release N-formylpeptides, which are potent chemoattractants for phagocytic leukocytes acting at G protein-coupled receptors FPR1 and FPR2. However, the distribution and immunologic activity of these molecules at mucosal surfaces, where large numbers of bacteria are separated from the immune system by epithelium, remain undefined. To investigate this for the gut, we tested leukocyte responses to cell-free gut luminal contents from C57Bl/6 mice fed a chow diet. Small and large intestine contents were able to compete with labeled N-formylpeptide for binding to FPR1, indicating the presence of FPR1 ligands in the gut lumen. Material from both small and large intestine induced robust calcium flux responses by primary FPR1+ leukocytes (mouse bone marrow cells and splenocytes, and human peripheral blood neutrophils and mononuclear cells), as well as chemotactic responses by both mouse bone marrow cells and human peripheral blood neutrophils. However, unlike defined N-formylpeptides, calcium flux responses induced by gut luminal contents were insensitive both to pertussis toxin treatment of leukocytes and to proteinase K digestion of the samples. Moreover, the gut samples were fully active on neutrophils from mice lacking Fpr1, and the kinetics of the calcium flux response differed markedly for neutrophils and PBMCs. The active factor(s) could be dialyzed using a 3.5 kD pore size membrane. Thus, mouse intestinal lumen contains small, potent and highly efficacious leukocyte chemotactic and activating factors that may be distinct for neutrophils and PBMCs and distinct from Fpr1 agonists. PMID:22722599

  8. H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells.

    PubMed

    Song, Guohua; Tian, Hua; Liu, Jia; Zhang, Hongle; Sun, Xuejun; Qin, Shucun

    2011-09-01

    H(2) is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H(2) in endothelial cells. H(2) significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-L-cysteine. H(2) inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H(2) inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H(2) probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.

  9. Antitumor activity of 7RH, a discoidin domain receptor 1 inhibitor, alone or in combination with dasatinib exhibits antitumor effects in nasopharyngeal carcinoma cells.

    PubMed

    Lu, Qiu-Ping; Chen, Wen-Dan; Peng, Jie-Ren; Xu, Yao-Dong; Cai, Qian; Feng, Gong-Kan; Ding, Ke; Zhu, Xiao-Feng; Guan, Zhong

    2016-11-01

    Dysregulation of the discoidin domain receptors (DDRs) has been implicated in the development of numerous types of tumors, including head and neck cancer, and nasopharyngeal, breast, ovarian and esophageal carcinomas. Furthermore, agents that inhibit DDR1 activity are hypothesized to be useful for the treatment of nasopharyngeal carcinoma (NPC). The aim of the present study was to evaluate the effect of the DDR1 inhibitory (3-(2-(pyrazolo(1,5-a)pyrimidin-6-yl)-ethynyl)benzamide compound, 7RH, in NPC cells both in vitro and in vivo, and its effect when used in combination with dasatinib, a SRC family kinase (SFK) inhibitor. The effects of 7RH alone or in combination with dasatinib on cell viability were assessed using MTT assays and apoptosis was detected by flow cytometry. In addition, western blotting was performed to analyze the relative protein expression levels of cell cycle-associated genes in human NPC cell lines (CNE1, CNE2, HONE1 and SUNE1). Cell migration was also assessed using cell adhesion assays. Furthermore, tumor xenografts of CNE2 NPC cells were established in nude mice and the growth inhibitory effects of 7RH treatment alone or in combination with dasatinib were evaluated. Finally, knockdown of DDR1 protein expression was achieved by transfection of CNE2 cells with DDR1-specific small interfering RNA. Treatment with 7RH effectively suppressed the proliferation and induced the apoptosis of NPC cells. In addition, the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT3) signaling pathway was downregulated by 7RH, whereas the activities of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways were upregulated in response to 7RH treatment. Furthermore, the expression levels of phosphorylated SRC were increased in NPC cells treated with 7RH; thus indicating that SRC exhibits a vital function in the resistance of NPC cells

  10. Antitumor activity of 7RH, a discoidin domain receptor 1 inhibitor, alone or in combination with dasatinib exhibits antitumor effects in nasopharyngeal carcinoma cells

    PubMed Central

    Lu, Qiu-Ping; Chen, Wen-Dan; Peng, Jie-Ren; Xu, Yao-Dong; Cai, Qian; Feng, Gong-Kan; Ding, Ke; Zhu, Xiao-Feng; Guan, Zhong

    2016-01-01

    Dysregulation of the discoidin domain receptors (DDRs) has been implicated in the development of numerous types of tumors, including head and neck cancer, and nasopharyngeal, breast, ovarian and esophageal carcinomas. Furthermore, agents that inhibit DDR1 activity are hypothesized to be useful for the treatment of nasopharyngeal carcinoma (NPC). The aim of the present study was to evaluate the effect of the DDR1 inhibitory (3-(2-(pyrazolo(1,5-a)pyrimidin-6-yl)-ethynyl)benzamide compound, 7RH, in NPC cells both in vitro and in vivo, and its effect when used in combination with dasatinib, a SRC family kinase (SFK) inhibitor. The effects of 7RH alone or in combination with dasatinib on cell viability were assessed using MTT assays and apoptosis was detected by flow cytometry. In addition, western blotting was performed to analyze the relative protein expression levels of cell cycle-associated genes in human NPC cell lines (CNE1, CNE2, HONE1 and SUNE1). Cell migration was also assessed using cell adhesion assays. Furthermore, tumor xenografts of CNE2 NPC cells were established in nude mice and the growth inhibitory effects of 7RH treatment alone or in combination with dasatinib were evaluated. Finally, knockdown of DDR1 protein expression was achieved by transfection of CNE2 cells with DDR1-specific small interfering RNA. Treatment with 7RH effectively suppressed the proliferation and induced the apoptosis of NPC cells. In addition, the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT3) signaling pathway was downregulated by 7RH, whereas the activities of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways were upregulated in response to 7RH treatment. Furthermore, the expression levels of phosphorylated SRC were increased in NPC cells treated with 7RH; thus indicating that SRC exhibits a vital function in the resistance of NPC cells

  11. uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

    PubMed Central

    Kanno, Yosuke; Ishisaki, Akira; Kawashita, Eri; Kuretake, Hiromi; Ikeda, Kanako; Matsuo, Osamu

    2016-01-01

    Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases. PMID:26722218

  12. Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    PubMed Central

    Yu, Xinguang

    2016-01-01

    Among sphingosine 1-phosphate receptors (S1PRs) family, S1PR1 has been shown to be the most highly expressed subtype in neural stem cells (NSCs) and plays a crucial role in the migratory property of NSCs. Recent studies suggested that S1PR1 was expressed abundantly in the hippocampus, a specific neurogenic region in rodent brain for endogenous neurogenesis throughout life. However, the potential association between S1PR1 and neurogenesis in hippocampus following traumatic brain injury (TBI) remains unknown. In this study, the changes of hippocampal S1PR1 expression after TBI and their effects on neurogenesis and neurocognitive function were investigated, focusing on particularly the extracellular signal-regulated kinase (Erk) signaling pathway which had been found to regulate multiple properties of NSCs. The results showed that a marked upregulation of S1PR1 occurred with a peak at 7 days after trauma, revealing an enhancement of proliferation and neuronal differentiation of NSCs in hippocampus due to S1PR1 activation. More importantly, it was suggested that mitogen-activated protein kinase-Erk kinase (MEK)/Erk cascade was required for S1PR1-meidated neurogenesis and neurocognitive recovery following TBI. This study lays a preliminary foundation for future research on promoting hippocampal neurogenesis and improving TBI outcome. PMID:28018679

  13. Trait-associated sequence variation in the bovine growth hormone receptor 1A promoter does not affect promoter activity in vitro.

    PubMed

    Zhou, Y; Jiang, H

    2005-04-01

    Growth hormone (GH) plays a central role in growth and metabolism in cattle by binding to growth hormone receptor (GHR) and stimulating production of insulin-like growth factor 1 (IGF1). Two sequence variations in the promoter transcribing a major GHR mRNA variant, GHR 1A mRNA, have been reported to be associated with quantitative differences in growth rate or blood concentration of IGF1 in cattle. One such variation is in the length of a TG-repeat, being 11 or 16-20; the other variation is in the nucleotide 155 bp upstream from the transcription start site, being G or A. In this study, we determined whether these sequence variations would affect the activity of GHR 1A promoter. We cloned GHR 1A promoters bearing different sequence variations and linked each of them to a reporter gene. Transient transfection analyses revealed that these promoter-reporter constructs did not differ in reporter gene expression. Cotransfection analyses demonstrated that they also did not differ in activation by hepatocyte nuclear factor 4alpha, hepatocyte nuclear factor 4gamma and nuclear receptor subfamily 2 group F member 2, known transcription factors for bovine GHR 1A promoter. These in vitro results, together with a previous observation that neither the nucleotide 155 bp upstream from the transcription start site nor the TG-repeat was part of the GHR 1A promoter region interacting with nuclear proteins from bovine liver, do not support a cause-effect relationship between the reported sequence variations and the associated changes in growth rate or blood IGF1 concentration in cattle.

  14. Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

    PubMed

    Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-09-22

    Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

  15. Role of thrombin-PAR1-PKCθ/δ axis in brain pericytes in thrombin-induced MMP-9 production and blood-brain barrier dysfunction in vitro.

    PubMed

    Machida, Takashi; Dohgu, Shinya; Takata, Fuyuko; Matsumoto, Junichi; Kimura, Ikuya; Koga, Mariko; Nakamoto, Keiko; Yamauchi, Atsushi; Kataoka, Yasufumi

    2017-03-24

    Thrombin, an essential component in the coagulation cascade, participates in the pathogenesis of brain diseases, such as ischemic stroke, intracerebral hemorrhage, Alzheimer's disease and Parkinson's disease through blood-brain barrier (BBB) dysfunction. It is thought that the thrombin-matrix metalloproteinase (MMP)-9 axis is an important process in the pathogenesis of neurovascular disease, such as BBB dysfunction. We recently reported that brain pericytes are the most MMP-9-releasing cells in response to thrombin stimulation among the BBB-constituting cells. This thrombin-induced MMP-9 release is partially due to protease-activated receptor (PAR1), one of the specific thrombin receptors. Then, we evaluated the intracellular signaling pathways involved in MMP-9 release and the contribution of thrombin-reactive brain pericytes to BBB dysfunction. PKC activator evoked MMP-9 release from brain pericytes. The thrombin-induced MMP-9 release was inhibited by U0126, LY294002, Go6976, and Go6983. However, Go6976 decreased phosphorylation levels of PKCθ and Akt, and Go6983 decreased phosphorylation levels of PKCδ and extracellular signal-regulated kinase (ERK). Additionally, treatment of pericytes with thrombin or PAR1-activating peptide stimulated PKCδ/θ signaling. These substances impaired brain endothelial barrier function in the presence of brain pericytes. Brain pericytes function through two independent downstream signaling pathways via PAR1 activation to release MMP-9 in response to thrombin - the PKCθ-Akt pathway and the PKCδ-ERK1/2 pathway. These pathways participate in PAR1-mediated MMP-9 release from pericytes, which leads to BBB dysfunction. Brain pericytes and their specific signaling pathways could provide novel therapeutic targets for thrombin-induced neurovascular diseases.

  16. Repeated anabolic/androgenic steroid exposure during adolescence alters phosphate-activated glutaminase and glutamate receptor 1 (GluR1) subunit immunoreactivity in Hamster brain: correlation with offensive aggression.

    PubMed

    Fischer, Shannon G; Ricci, Lesley A; Melloni, Richard H

    2007-06-04

    Male Syrian hamsters (Mesocricetus auratus) treated with moderately high doses (5.0mg/kg/day) of anabolic/androgenic steroids (AAS) during adolescence (P27-P56) display highly escalated offensive aggression. The current study examined whether adolescent AAS-exposure influenced the immunohistochemical localization of phosphate-activated glutaminase (PAG), the rate-limiting enzyme in the synthesis of glutamate, a fast-acting neurotransmitter implicated in the modulation of aggression in various species and models of aggression, as well as glutamate receptor 1 subunit (GluR1). Hamsters were administered AAS during adolescence, scored for offensive aggression using the resident-intruder paradigm, and then examined for changes in PAG and GluR1 immunoreactivity in areas of the brain implicated in aggression control. When compared with sesame oil-treated control animals, aggressive AAS-treated hamsters displayed a significant increase in the number of PAG- and area density of GluR1-containing neurons in several notable aggression regions, although the differential pattern of expression did not appear to overlap across brain regions. Together, these results suggest that altered glutamate synthesis and GluR1 receptor expression in specific aggression areas may be involved in adolescent AAS-induced offensive aggression.

  17. Lectin-like oxidized low-density lipoprotein receptor-1 facilitates metastasis of gastric cancer through driving epithelial-mesenchymal transition and PI3K/Akt/GSK3β activation

    PubMed Central

    Li, Can; Zhang, Jie; Wu, Hao; Li, Lili; Yang, Caiting; Song, Shushu; Peng, Peike; Shao, Miaomiao; Zhang, Mingming; Zhao, Junjie; Zhao, Ran; Wu, Weicheng; Ruan, Yuanyuan; Wang, Lan; Gu, Jianxin

    2017-01-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern recognition receptor that plays a critical role in vascular diseases and host immune response. Recently, our research discovered that LOX-1 could facilitate the uptake of dying cells and cross-presentation of cellular antigen via binding with heat shock proteins, which have a close relationship with gastric neoplasia. Therefore, we speculated that LOX-1 may serve as an oncogene in gastric cancer (GC) development and progression. In this study, through immunohistochemistry staining assay and cancer-related databases, we found that LOX-1 expression was up-regulated in GC tissues and correlated with a poor prognosis in GC patients. The expression of LOX-1 was an independent prognostic factor for OS in GC patients, and the incorporation of LOX-1 with TNM stage is more accurate for predicting prognosis. Additionally, in vitro study by transwell assay and western blot analysis confirmed that LOX-1 could promote the migration and invasion of GC cells by driving epithelial-mesenchymal transition and PI3K/Akt/GSK3β activation. Taken together, we first explored the expression profiles, clinical significance and biological function of LOX-1 in GC, and these data suggest that LOX-1 may represent a promising prognostic biomarker for GC and offer a novel molecular target for GC therapies. PMID:28345638

  18. The TNF receptor 1: a split personality complex.

    PubMed

    Barnhart, Bryan C; Peter, Marcus E

    2003-07-25

    The tumor necrosis factor receptor 1 (TNFR1), a prototypic member of the death receptor family signals both cell survival and apoptosis. In this issue of Cell, report that apoptotic TNFR1 signaling proceeds via the sequential formation of two distinct complexes. Since the first complex can activate survival signals and influence the activity of the second complex, this mechanism provides a checkpoint to control the execution of apoptosis.

  19. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  20. Characterization of SB-705498, a potent and selective vanilloid receptor-1 (VR1/TRPV1) antagonist that inhibits the capsaicin-, acid-, and heat-mediated activation of the receptor.

    PubMed

    Gunthorpe, Martin J; Hannan, Sara Luis; Smart, Darren; Jerman, Jeffrey C; Arpino, Sandra; Smith, Graham D; Brough, Stephen; Wright, Jim; Egerton, Julie; Lappin, Sarah C; Holland, Vicky A; Winborn, Kim; Thompson, Mervyn; Rami, Harshad K; Randall, Andrew; Davis, John B

    2007-06-01

    Vanilloid receptor-1 (TRPV1) is a nonselective cation channel, predominantly expressed by sensory neurons, which plays a key role in the detection of noxious painful stimuli such as capsaicin, acid, and heat. TRPV1 antagonists may represent novel therapeutic agents for the treatment of a range of conditions including chronic pain, migraine, and gastrointestinal disorders. Here we describe the in vitro pharmacology of N-(2-bromophenyl)-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea (SB-705498), a novel TRPV1 antagonist identified by lead optimization of N-(2-bromophenyl)-N'-[2-[ethyl(3-methylphenyl)amino]ethyl]urea (SB-452533), which has now entered clinical trials. Using a Ca(2+)-based fluorometric imaging plate reader (FLIPR) assay, SB-705498 was shown to be a potent competitive antagonist of the capsaicin-mediated activation of the human TRPV1 receptor (pK(i) = 7.6) with activity at rat (pK(i) = 7.5) and guinea pig (pK(i) = 7.3) orthologs. Whole-cell patch-clamp electrophysiology was used to confirm and extend these findings, demonstrating that SB-705498 can potently inhibit the multiple modes of receptor activation that may be relevant to the pathophysiological role of TRPV1 in vivo: SB-705498 caused rapid and reversible inhibition of the capsaicin (IC(50) = 3 nM)-, acid (pH 5.3)-, or heat (50 degrees C; IC(50) = 6 nM)-mediated activation of human TRPV1 (at -70 mV). Interestingly, SB-705498 also showed a degree of voltage dependence, suggesting an effective enhancement of antagonist action at negative potentials such as those that might be encountered in neurons in vivo. The selectivity of SB-705498 was defined by broad receptor profiling and other cellular assays in which it showed little or no activity versus a wide range of ion channels, receptors, and enzymes. SB-705498 therefore represents a potent and selective multimodal TRPV1 antagonist, a pharmacological profile that has contributed to its definition as a suitable drug candidate for

  1. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    SciTech Connect

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  2. Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin.

    PubMed Central

    Festoff, B. W.; D'Andrea, M. R.; Citron, B. A.; Salcedo, R. M.; Smirnova, I. V.; Andrade-Gordon, P.

    2000-01-01

    BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar

  3. Protease-Activated Receptor 2 (PAR2) Is Upregulated by Acanthamoeba Plasminogen Activator (aPA) and Induces Proinflammatory Cytokine in Human Corneal Epithelial Cells

    PubMed Central

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2014-01-01

    Purpose. Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Methods. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Results. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells

  4. TRESK Background K+ Channel Is Inhibited by PAR-1/MARK Microtubule Affinity-Regulating Kinases in Xenopus Oocytes

    PubMed Central

    Braun, Gabriella; Nemcsics, Balázs; Enyedi, Péter; Czirják, Gábor

    2011-01-01

    TRESK (TWIK-related spinal cord K+ channel, KCNK18) is a major background K+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279) in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel. PMID:22145024

  5. Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

    PubMed Central

    Biernat, Jacek; Wu, Yong-Zhong; Timm, Thomas; Zheng-Fischhöfer, Qingyi; Mandelkow, Eckhard; Meijer, Laurent; Mandelkow, Eva-Maria

    2002-01-01

    Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites. PMID:12429843

  6. DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

    PubMed

    Kwan, Julian; Sczaniecka, Anna; Arash, Emad Heidary; Nguyen, Liem; Chen, Chia-Chun; Ratkovic, Srdjana; Klezovitch, Olga; Attisano, Liliana; McNeill, Helen; Emili, Andrew; Vasioukhin, Valeri

    2016-12-15

    Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5(-/-) tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

  7. Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature

    PubMed Central

    Benito-Sanz, Sara; Aza-Carmona, Miriam; Rodríguez-Estevez, Amaya; Rica-Etxebarria, Ixaso; Gracia, Ricardo; Campos-Barros, Ángel; Heath, Karen E

    2012-01-01

    Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ∼60% of Léri-Weill dyschondrosteosis (LWD) and ∼5–15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ∼286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS. PMID:22071895

  8. Immobilization of soluble complement receptor 1 on islets.

    PubMed

    Luan, Nguyen M; Teramura, Yuji; Iwata, Hiroo

    2011-07-01

    Transplantation of pancreatic islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus. Control of complement activation is necessary to improve graft survival in alloislet and xenoislet transplantation. In this study, human soluble complement receptor 1 (sCR1) was immobilized on the islet cell surface through poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) without loss of islet cell viability or insulin secretion ability. sCR1 on islets effectively inhibits complement activation and protects islets against attack by xenoreactive antibodies and complement. This method will be an efficient means to control early islet loss in clinical islet transplantation and realize xenoislet transplantation in the future.

  9. PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

    PubMed Central

    Elbert, Maya; Cohen, David

    2006-01-01

    Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes. PMID:16707567

  10. PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

    PubMed Central

    Auvergne, R; Wu, C; Connell, A; Au, S; Cornwell, A; Osipovitch, M; Benraiss, A; Dangelmajer, S; Guerrero-Cazares, H; Quinones-Hinojosa, A; Goldman, SA

    2016-01-01

    Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5+ TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5+ TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas. PMID:26616854

  11. Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling.

    PubMed

    Little, P J; Hollenberg, M D; Kamato, D; Thomas, W; Chen, J; Wang, T; Zheng, W; Osman, N

    2016-10-01

    Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and β-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different β-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  12. miR-29a suppresses MCF-7 cell growth by downregulating tumor necrosis factor receptor 1.

    PubMed

    Zhao, Yiling; Yang, Fenghua; Li, Wenyuan; Xu, Chunyan; Li, Li; Chen, Lifei; Liu, Yancui; Sun, Ping

    2017-02-01

    Tumor necrosis factor receptor 1 is the main receptor mediating many tumor necrosis factor-alpha-induced cellular events. Some studies have shown that tumor necrosis factor receptor 1 promotes tumorigenesis by activating nuclear factor-kappa B signaling pathway, while other studies have confirmed that tumor necrosis factor receptor 1 plays an inhibitory role in tumors growth by inducing apoptosis in breast cancer. Therefore, the function of tumor necrosis factor receptor 1 in breast cancer requires clarification. In this study, we first found that tumor necrosis factor receptor 1 was significantly increased in human breast cancer tissues and cell lines, and knockdown of tumor necrosis factor receptor 1 by small interfering RNA inhibited cell proliferation by arresting the cell cycle and inducing apoptosis. In addition, miR-29a was predicted as a regulator of tumor necrosis factor receptor 1 by TargetScan and was shown to be inversely correlated with tumor necrosis factor receptor 1 expression in human breast cancer tissues and cell lines. Luciferase reporter assay further confirmed that miR-29a negatively regulated tumor necrosis factor receptor 1 expression by binding to the 3' untranslated region. In our functional study, miR-29a overexpression remarkably suppressed cell proliferation and colony formation, arrested the cell cycle, and induced apoptosis in MCF-7 cell. Furthermore, in combination with tumor necrosis factor receptor 1 transfection, miR-29a significantly reversed the oncogenic role caused by tumor necrosis factor receptor 1 in MCF-7 cell. In addition, we demonstrated that miR-29a suppressed MCF-7 cell growth by inactivating the nuclear factor-kappa B signaling pathway and by decreasing cyclinD1 and Bcl-2/Bax protein levels. Taken together, our results suggest that miR-29a is an important regulator of tumor necrosis factor receptor 1 expression in breast cancer and functions as a tumor suppressor by targeting tumor necrosis factor receptor 1 to

  13. Par1b Induces Asymmetric Inheritance of Plasma Membrane Domains via LGN-Dependent Mitotic Spindle Orientation in Proliferating Hepatocytes

    PubMed Central

    Slim, Christiaan L.; Lázaro-Diéguez, Francisco; Bijlard, Marjolein; Toussaint, Mathilda J. M.; de Bruin, Alain; Du, Quansheng; Müsch, Anne; van IJzendoorn, Sven C. D.

    2013-01-01

    The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma. PMID:24358023

  14. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    SciTech Connect

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A.

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  15. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA

    PubMed Central

    Yu, Xuan; Zhang, Qiao; Zhao, Yan; Schwarz, Benjamin J.; Stallone, John N.; Heaps, Cristine L.; Han, Guichun

    2017-01-01

    Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM), or ESI-09 (20 μM), or CE3F4 (100 μM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3–3 μM); while concurrent administration of BFA and PKI (5 μM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1–100 μM), induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 μM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 μM) treatment, but not by 007 (50 μM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases. PMID:28278256

  16. Role of Fibrinogen and Protease-Activated Receptors in Acute Xenobiotic-Induced Cholestatic Liver Injury

    PubMed Central

    Luyendyk, James P.; Mackman, Nigel; Sullivan, Bradley P.

    2011-01-01

    Alpha-naphthylisothiocyanate (ANIT)–induced cholestatic liver injury causes tissue factor (TF)–dependent coagulation in mice, and TF deficiency reduces ANIT-induced liver injury. However, the mechanism whereby TF contributes to hepatotoxicity in this model is not known. Utilizing pharmacological and genetic strategies, we evaluated the contribution of fibrinogen and two distinct receptors for thrombin, protease-activated receptor-1 (PAR-1) and PAR-4, in a model of acute ANIT hepatotoxicity. ANIT administration (60 mg/kg, po) caused a marked induction of the genes encoding the three fibrinogen chains (α, β, and γ) in liver, an increase in plasma fibrinogen, and concurrent deposition of thrombin-cleaved fibrin in liver. Partial depletion of circulating fibrinogen with ancrod did not impact ANIT hepatotoxicity. However, complete fibrin(ogen) deficiency significantly reduced serum alanine aminotransferase activity and hepatocellular necrosis in ANIT-treated mice. ANIT-induced hepatocellular necrosis was similar in PAR-1−/− mice compared with PAR-1+/+ mice. Interestingly, the progression of ANIT-induced hepatocellular necrosis was significantly reduced in PAR-4−/− mice and by administration of an inhibitory PAR-4 pepducin (P4Pal-10, 0.5 mg/kg, sc) to wild-type mice 8 h after ANIT treatment. Interestingly, a distinct lesion, parenchymal-type peliosis, was also observed in PAR-4−/− mice treated with ANIT and in mice that were given P4Pal-10 prior to ANIT administration. The results suggest that fibrin(ogen), but not PAR-1, contributes to the progression of ANIT hepatotoxicity in mice. Moreover, the data suggest a dual role for PAR-4 in ANIT hepatotoxicity, both mediating an early protection against peliosis and contributing to the progression of hepatocellular necrosis. PMID:20974703

  17. Δ9-Tetrahydrocannabinol attenuates allogeneic host-versus-graft response and delays skin graft rejection through activation of cannabinoid receptor 1 and induction of myeloid-derived suppressor cells

    PubMed Central

    Sido, Jessica M.; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

    2015-01-01

    Immune cells have been shown to express cannabinoid receptors and to produce endogenous ligands. Moreover, activation of cannabinoid receptors on immune cells has been shown to trigger potent immunosuppression. Despite such studies, the role of cannabinoids in transplantation, specifically to prevent allograft rejection, has not, to our knowledge, been investigated previously. In the current study, we tested the effect of THC on the suppression of HvGD as well as rejection of skin allografts. To this end, we studied HvGD by injecting H-2k splenocytes into H-2b mice and analyzing the immune response in the draining ingLNs. THC treatment significantly reduced T cell proliferation and activation in draining LNs of the recipient mice and decreased early stage rejection-indicator cytokines, including IL-2 and IFN-γ. THC treatment also increased the allogeneic skin graft survival. THC treatment in HvGD mice led to induction of MDSCs. Using MDSC depletion studies as well as adoptive transfer experiments, we found that THC-induced MDSCs were necessary for attenuation of HvGD. Additionally, using pharmacological inhibitors of CB1 and CB2 receptors and CB1 and CB2 knockout mice, we found that THC was working preferentially through CB1. Together, our research shows, for the first time to our knowledge, that targeting cannabinoid receptors may provide a novel treatment modality to attenuate HvGD and prevent allograft rejection. PMID:26034207

  18. Δ⁹-Tetrahydrocannabinol attenuates allogeneic host-versus-graft response and delays skin graft rejection through activation of cannabinoid receptor 1 and induction of myeloid-derived suppressor cells.

    PubMed

    Sido, Jessica M; Nagarkatti, Prakash S; Nagarkatti, Mitzi

    2015-09-01

    Immune cells have been shown to express cannabinoid receptors and to produce endogenous ligands. Moreover, activation of cannabinoid receptors on immune cells has been shown to trigger potent immunosuppression. Despite such studies, the role of cannabinoids in transplantation, specifically to prevent allograft rejection, has not, to our knowledge, been investigated previously. In the current study, we tested the effect of THC on the suppression of HvGD as well as rejection of skin allografts. To this end, we studied HvGD by injecting H-2(k) splenocytes into H-2(b) mice and analyzing the immune response in the draining ingLNs. THC treatment significantly reduced T cell proliferation and activation in draining LNs of the recipient mice and decreased early stage rejection-indicator cytokines, including IL-2 and IFN-γ. THC treatment also increased the allogeneic skin graft survival. THC treatment in HvGD mice led to induction of MDSCs. Using MDSC depletion studies as well as adoptive transfer experiments, we found that THC-induced MDSCs were necessary for attenuation of HvGD. Additionally, using pharmacological inhibitors of CB1 and CB2 receptors and CB1 and CB2 knockout mice, we found that THC was working preferentially through CB1. Together, our research shows, for the first time to our knowledge, that targeting cannabinoid receptors may provide a novel treatment modality to attenuate HvGD and prevent allograft rejection.

  19. Trace amine-associated receptor 1-Family archetype or iconoclast?

    PubMed

    Grandy, David K

    2007-12-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, noncatecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the "trace amines" (TA) beta-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of 2 publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers Borowsky et al. as TA1 and Bunzow et al. as TA receptor 1 (TAR1). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines, recombinant rodent and human TAR dose-dependently couple to the stimulation of adenosine 3',5'-monophosphate (cAMP) production. Structure-activity profiling based on this functional response has revealed that in addition to the TA, other biologically active compounds containing a 2-carbon aliphatic side chain linking an amino group to at least 1 benzene ring are potent and efficacious TA receptor agonists with amphetamine (AMPH), methamphetamine, 3-iodothyronamine, thyronamine, and dopamine (DA) among the most notable. Almost 100 years after the search for TAR began, numerous TA1/TAR1-related sequences, now called TA-associated receptors (TAAR), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TAR, a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCR. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast.

  20. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity.

    PubMed

    Lewandowski, Katherine T; Piwnica-Worms, Helen

    2014-01-15

    The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

  1. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity

    PubMed Central

    Lewandowski, Katherine T.; Piwnica-Worms, Helen

    2014-01-01

    ABSTRACT The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell–cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell–ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell–ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell–ECM contacts required for epithelial cells to establish apical-basal cell polarity. PMID:24259665

  2. The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20.

    PubMed Central

    Couvineau, A; Maoret, J J; Rouyer-Fessard, C; Carrero, I; Laburthe, M

    2000-01-01

    The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2

  3. Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): Association with aryl hydrocarbon receptor 1 and 2 isoforms

    SciTech Connect

    Lee, Jin-Seon; Kim, Eun-Young Iwata, Hisato

    2009-01-01

    The present study focuses on the molecular mechanism and interspecies differences in susceptibility of avian aryl hydrocarbon receptor (AHR)-cytochrome P4501A (CYP1A) signaling pathway. By the cloning of 5'-flanking regions of CYP1A5 gene from common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus), seven putative xenobiotic response elements (XREs) were identified within 2.7 kb upstream region of common cormorant CYP1A5 (ccCYP1A5), and six XREs were found within 0.9 kb of chicken CYP1A5 (ckCYP1A5). Analysis of sequential deletion and mutagenesis of the binding sites in avian CYP1A5 genes by in vitro reporter gene assays revealed that two XREs at -613 bp and -1585 bp in ccCYP1A5, and one XRE at -262 bp in ckCYP1A5 conferred TCDD-responsiveness. The binding of AHR1 with AHR nuclear translocator 1 (ARNT1) to the functional XRE in a TCDD-dependent manner was verified with gel shift assays, suggesting that avian CYP1A5 is induced by TCDD through AHR1/ARNT1 signaling pathway as well as mammalian CYP1A1 but through a distinct pathway from mammalian CYP1A2, an ortholog of the CYP1A5. TCDD-EC{sub 50} for the transcriptional activity in both cormorant AHR1- and AHR2-ccCYP1A5 reporter construct was 10-fold higher than that in chicken AHR1-ckCYP1A5 reporter construct. In contrast, chicken AHR2 showed no TCDD-dependent response. The TCDD-EC{sub 50} for CYP1A5 transactivation was altered by switching AHR1 between the two avian species, irrespective of the species from which the regulatory region of CYP1A5 gene originates. Therefore, the structural difference in AHR, not the CYP1A5 regulatory region may be a major factor to account for the dioxin susceptibility in avian species.

  4. Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms.

    PubMed

    Lee, Jin-Seon; Kim, Eun-Young; Iwata, Hisato

    2009-01-01

    The present study focuses on the molecular mechanism and interspecies differences in susceptibility of avian aryl hydrocarbon receptor (AHR)-cytochrome P4501A (CYP1A) signaling pathway. By the cloning of 5'-flanking regions of CYP1A5 gene from common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus), seven putative xenobiotic response elements (XREs) were identified within 2.7 kb upstream region of common cormorant CYP1A5 (ccCYP1A5), and six XREs were found within 0.9 kb of chicken CYP1A5 (ckCYP1A5). Analysis of sequential deletion and mutagenesis of the binding sites in avian CYP1A5 genes by in vitro reporter gene assays revealed that two XREs at -613 bp and -1585 bp in ccCYP1A5, and one XRE at -262 bp in ckCYP1A5 conferred TCDD-responsiveness. The binding of AHR1 with AHR nuclear translocator 1 (ARNT1) to the functional XRE in a TCDD-dependent manner was verified with gel shift assays, suggesting that avian CYP1A5 is induced by TCDD through AHR1/ARNT1 signaling pathway as well as mammalian CYP1A1 but through a distinct pathway from mammalian CYP1A2, an ortholog of the CYP1A5. TCDD-EC(50) for the transcriptional activity in both cormorant AHR1- and AHR2-ccCYP1A5 reporter construct was 10-fold higher than that in chicken AHR1-ckCYP1A5 reporter construct. In contrast, chicken AHR2 showed no TCDD-dependent response. The TCDD-EC(50) for CYP1A5 transactivation was altered by switching AHR1 between the two avian species, irrespective of the species from which the regulatory region of CYP1A5 gene originates. Therefore, the structural difference in AHR, not the CYP1A5 regulatory region may be a major factor to account for the dioxin susceptibility in avian species.

  5. Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.

    PubMed

    De Esch, I J; Vollinga, R C; Goubitz, K; Schenk, H; Appelberg, U; Hacksell, U; Lemstra, S; Zuiderveld, O P; Hoffmann, M; Leurs, R; Menge, W M; Timmerman, H

    1999-04-08

    Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.

  6. Trace Amine Associated Receptor 1 Modulates Behavioral Effects of Ethanol

    PubMed Central

    Lynch, Laurie J.; Sullivan, Katherine A.; Vallender, Eric J.; Rowlett, James K.; Platt, Donna M.; Miller, Gregory M.

    2013-01-01

    Background Few treatment options for alcohol use disorders (AUDs) exist and more are critically needed. Here, we assessed whether trace amine associated receptor 1 (TAAR1), a modulator of brain monoamine systems, is involved in the behavioral and reinforcement-related effects of ethanol and whether it could potentially serve as a therapeutic target. Methods Wild-type (WT) and TAAR1 knockout (KO) mice (75% C57J/BL6 and 25% 129S1/Sv background) were compared in tests of ethanol consumption (two-bottle choice [TBC]), motor impairment (loss of righting reflex, [LORR], locomotor activity) and ethanol clearance (blood ethanol level [BEL]). Results As compared with WT mice, KO mice displayed (1) significantly greater preference for and consumption of ethanol in a TBC paradigm (3%–11% vol/vol escalating over 10 weeks), with no significant difference observed in TBC with sucrose (1%–3%); (2) significantly greater sedative-like effects of acute ethanol (2.0 or 2.5 g/kg, intraperitoneal [i.p.]) manifested as LORR observed at a lower dose and for longer time, with similar BELs and rates of ethanol clearance; and (3) lower cumulative locomotor activity over 60 minutes in response to an acute ethanol challenge (1.0–2.5 g/kg, i.p.). Conclusions The present findings are the first to implicate TAAR1 in the behavioral and reinforcement-related effects of ethanol and raise the question of whether specific drugs that target TAAR1 could potentially reduce alcohol consumption in humans with AUDs. PMID:23861588

  7. Attenuation of synaptic toxicity and MARK4/PAR1-mediated Tau phosphorylation by methylene blue for Alzheimer’s disease treatment

    PubMed Central

    Sun, Wenchao; Lee, Seongsoo; Huang, Xiaoran; Liu, Song; Inayathullah, Mohammed; Kim, Kwang-Min; Tang, Hongxiang; Ashford, J. Wesson; Rajadas, Jayakumar

    2016-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease characterized by genotypic and phenotypic heterogeneity. Critical components of the two AD pathological pathways, Aβ-amyloidosis and Tauopathy, have been considered as therapeutic targets. Among them, much effort is focused on aberrant Tau phosphorylation and targeting Tau-phosphorylating kinases. Methylene blue (MB), a phenothiazine dye that crosses the blood-brain barrier, has been shown to hit multiple molecular targets involved in AD and have beneficial effects in clinical studies. Here we present evidence that microtubule affinity-regulating kinase (MARK4) is a novel target of MB. MB partially rescued the synaptic toxicity in Drosophila larva overexpressing PAR1 (MARK analog). In 293T culture, MB decreased MARK4-mediated Tau phosphorylation in a dose dependent manner. Further studies revealed a two-fold mechanism by MB including down-regulation of MARK4 protein level through ubiquitin-proteasome pathway and inhibition of MARK4 kinase activity in vitro. This study highlights the importance of MARK4 as a viable target for Tauopathy and provides fresh insight into the complex mechanism used by MB to treat AD. PMID:27708431

  8. Influence of Genetic Ancestry on INDEL Markers of NFKβ1, CASP8, PAR1, IL4 and CYP19A1 Genes in Leprosy Patients

    PubMed Central

    Pinto, Pablo; Salgado, Claudio; Santos, Ney Pereira Carneiro; Santos, Sidney; Ribeiro-dos-Santos, Ândrea

    2015-01-01

    Background Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria Mycobacterium leprae, and host genetic factors can modulate the immune response and generate distinct categories of leprosy susceptibility that are also influenced by genetic ancestry. Methodology/Principal Findings We investigated the possible effects of CYP19A1 [rs11575899], NFKβ1 [rs28362491], IL1α [rs3783553], CASP8 [rs3834129], UGT1A1 [rs8175347], PAR1 [rs11267092], CYP2E1 [INDEL 96pb] and IL4 [rs79071878] genes in a group of 141 leprosy patients and 180 healthy individuals. The INDELs were typed by PCR Multiplex in ABI PRISM 3130 and analyzed with GeneMapper ID v3.2. The NFKβ1, CASP8, PAR1 and IL4 INDELs were associated with leprosy susceptibility, while NFKβ1, CASP8, PAR1 and CYP19A1 were associated with the MB (Multibacilary) clinical form of leprosy. Conclusions/Significance NFKβ1 [rs28362491], CASP8 [rs3834129], PAR1 [rs11267092] and IL4 [rs79071878] genes are potential markers for susceptibility to leprosy development, while the INDELs in NFKβ1, CASP8, PAR1 and CYP19A1 (rs11575899) are potential markers for the severe clinical form MB. Moreover, all of these markers are influenced by genetic ancestry, and European contribution increases the risk to leprosy development, in other hand an increase in African contribution generates protection against leprosy. PMID:26367014

  9. Arginase-1 is a more sensitive marker than HepPar-1 and AFP in differential diagnosis of hepatocellular carcinoma from nonhepatocellular carcinoma.

    PubMed

    Sang, Wei; Zhang, Wei; Cui, Wenli; Li, Xinxia; Abulajiang, Gulinar; Li, Qiaoxin

    2015-05-01

    Distinguishing hepatocellular carcinoma (HCC) from metastatic tumors is a challenging issue, especially in differential diagnosis between poorly differentiated HCC and metastasis tumors. Expression of Arg-1, HepPar-1, and α-fetoprotein (AFP) in 78 cases of HCC, 34 cases of metastatic tumors, and 228 cases of nonhepatocellular tumors of surgical specimens is measured by immunohistochemistry. Arg-1 immunoreactivity was detected in 75 of 78 (96.1 %) cases of HCC, whereas HepPar-1 and AFP immunoreactivity was detected in 63 of 78 (80.7 %) and 40 of 78 (51.3 %) cases of HCC, respectively. HepPar-1 and AFP expression was observed in three of 34 (8.8 %) cases and one of 34 (2.9 %) cases of metastatic tumors, respectively. In contrast, Arg-1 expression was absent in all 34 (0 %) cases of metastatic tumors. The sensitivity, specificity, positive predictive value, and negative predictive value of Arg-1 in distinguishing HCC from metastatic tumors and nonhepatocellular tumors are 96.1, 99.6, 98.7, and 98.8 % compared with 80.7, 92.0, 75.0, and 94.1 % for HepPar-1 and 51.3, 97.7, 87.0, and 87.1 % for AFP, respectively. Arg-1 is a more sensitive and better specific marker for HCC compared with HepPar-1 and AFP, indicating that Arg-1 can be easily applied in distinguishing HCC from metastatic tumors.

  10. Interaction of lipids with the neurotensin receptor 1.

    PubMed

    Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

    2016-06-01

    Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs.

  11. Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells.

    PubMed

    Yuan, Hebao; Chiang, C-Y Ason; Cheng, Jun; Salzmann, Viktoria; Yamashita, Yukiko M

    2012-01-01

    Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. We recently demonstrated that GSCs possess a checkpoint ("the centrosome orientation checkpoint") that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. Here, we show that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. We propose that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint.

  12. PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium †

    PubMed Central

    London, Fredda S.; Marcinkiewicz, Mariola; Walsh, Peter N.

    2008-01-01

    We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (Kd 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (Kd 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients. PMID:16752917

  13. Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1.

    PubMed

    Vacchelli, Erika; Ma, Yuting; Baracco, Elisa E; Sistigu, Antonella; Enot, David P; Pietrocola, Federico; Yang, Heng; Adjemian, Sandy; Chaba, Kariman; Semeraro, Michaela; Signore, Michele; De Ninno, Adele; Lucarini, Valeria; Peschiaroli, Francesca; Businaro, Luca; Gerardino, Annamaria; Manic, Gwenola; Ulas, Thomas; Günther, Patrick; Schultze, Joachim L; Kepp, Oliver; Stoll, Gautier; Lefebvre, Céline; Mulot, Claire; Castoldi, Francesca; Rusakiewicz, Sylvie; Ladoire, Sylvain; Apetoh, Lionel; Bravo-San Pedro, José Manuel; Lucattelli, Monica; Delarasse, Cécile; Boige, Valérie; Ducreux, Michel; Delaloge, Suzette; Borg, Christophe; André, Fabrice; Schiavoni, Giovanna; Vitale, Ilio; Laurent-Puig, Pierre; Mattei, Fabrizio; Zitvogel, Laurence; Kroemer, Guido

    2015-11-20

    Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses.

  14. Trace amine-associated receptor 1: A promising target for the treatment of psychostimulant addiction.

    PubMed

    Jing, Li; Li, Jun-Xu

    2015-08-15

    Abuse of and addiction to psychostimulants remains a challenging clinical issue; yet no effective pharmacotherapy is available. Trace amine associated receptor 1 (TAAR 1) is increasingly recognized as a novel drug target that participates in the modulation of drug abuse. This review analyzed existing preclinical evidence from electrophysiological, biochemical to behavioral aspects regarding the functional interactions between TAAR 1 and dopaminergic system. TAAR 1 knockout mice demonstrate increased sensitivity to dopaminergic activation while TAAR 1 agonists reduce the neurochemical effects of cocaine and amphetamines, attenuate abuse- and addiction-related behavioral effects of cocaine and methamphetamine. It is concluded that TAAR 1 activation functionally modulates the dopaminergic activity and TAAR 1 agonists appear to be promising pharmacotherapies against psychostimulant addiction.

  15. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    PubMed Central

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. PMID:27314054

  16. Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

    PubMed Central

    Kugler, Eva M.; Mazzuoli, Gemma; Demir, Ihsan E.; Ceyhan, Güralp O.; Zeller, Florian; Schemann, Michael

    2012-01-01

    Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system. PMID:22988431

  17. par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans

    PubMed Central

    Jiu, Yaming; Hasygar, Kiran; Tang, Lois; Liu, Yanbo; Holmberg, Carina I.; Bürglin, Thomas R.; Hietakangas, Ville; Jäntti, Jussi

    2013-01-01

    The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process. PMID:24192838

  18. Central Administration of Galanin Receptor 1 Agonist Boosted Insulin Sensitivity in Adipose Cells of Diabetic Rats

    PubMed Central

    Zhang, Zhenwen; Fang, Penghua; He, Biao; Guo, Lili; Runesson, Johan; Langel, Ülo; Shi, Mingyi; Zhu, Yan; Bo, Ping

    2016-01-01

    Our previous studies testified the beneficial effect of central galanin on insulin sensitivity of type 2 diabetic rats. The aim of the study was further to investigate whether central M617, a galanin receptor 1 agonist, can benefit insulin sensitivity. The effects of intracerebroventricular administration of M617 on insulin sensitivity and insulin signaling were evaluated in adipose tissues of type 2 diabetic rats. The results showed that central injection of M617 significantly increased plasma adiponectin contents, glucose infusion rates in hyperinsulinemic-euglycemic clamp tests, GLUT4 mRNA expression levels, GLUT4 contents in plasma membranes, and total cell membranes of the adipose cells but reduced the plasma C-reactive protein concentration in nondiabetic and diabetic rats. The ratios of GLUT4 contents were higher in plasma membranes to total cell membranes in both nondiabetic and diabetic M617 groups than each control. In addition, the central administration of M617 enhanced the ratios of pAkt/Akt and pAS160/AS160, but not phosphorylative cAMP response element-binding protein (pCREB)/CREB in the adipose cells of nondiabetic and diabetic rats. These results suggest that excitation of central galanin receptor 1 facilitates insulin sensitivity via activation of the Akt/AS160 signaling pathway in the fat cells of type 2 diabetic rats. PMID:27127795

  19. A pivotal role for enhanced brainstem Orexin receptor 1 signaling in the central cannabinoid receptor 1-mediated pressor response in conscious rats.

    PubMed

    Ibrahim, Badr Mostafa; Abdel-Rahman, Abdel A

    2015-10-05

    Orexin receptor 1 (OX1R) signaling is implicated in cannabinoid receptor 1 (CB1R) modulation of feeding. Further, our studies established the dependence of the central CB1R-mediated pressor response on neuronal nitric oxide synthase (nNOS) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation in the RVLM. Here, we tested the novel hypothesis that brainstem orexin-A/OX1R signaling plays a pivotal role in the central CB1R-mediated pressor response. Our multiple labeling immunofluorescence findings revealed co-localization of CB1R, OX1R and the peptide orexin-A within the C1 area of the rostral ventrolateral medulla (RVLM). Activation of central CB1R following intracisternal (i.c.) WIN55,212-2 (15μg/rat) in conscious rats caused significant increases in BP and orexin-A level in RVLM neuronal tissue. Additional studies established a causal role for orexin-A in the central CB1R-mediated pressor response because (i) selective blockade of central CB1R (AM251, 30μg/rat; i.c.) abrogated WIN55,212-2-evoked increases in RVLM orexin-A level, (ii) the selective OX1R antagonist SB-408124 (10nmol/rat; i.c.) attenuated orexin-A (3nmol/rat; i.c.) or WIN55,212-2 (15μg/rat; i.c.)-evoked pressor response while selective CB1R blockade (AM251) had no effect on orexin-A (3nmol/rat; i.c.)-evoked pressor response, (iii) direct CB1R activation in the RVLM (WIN55,212-2; 0.1μg/rat) increased RVLM orexin-A and BP. Finally, SB-408124 attenuated WIN55,212-2-evoked increases in RVLM nNOS and ERK1/2 phosphorylation and BP. Our findings suggest that orexin-A/OX1R dependent activation of the RVLM nNOS/ERK1/2 cascade is essential neurochemical mechanism for the central CB1R-mediated pressor response in conscious rats.

  20. A pivotal role for enhanced brainstem Orexin receptor 1 signaling in the central cannabinoid receptor 1-mediated pressor response in conscious rats

    PubMed Central

    Ibrahim, Badr Mostafa; Abdel-Rahman, Abdel A.

    2015-01-01

    Orexin receptor 1 (OX1R) signaling is implicated in cannabinoid receptor 1 (CB1R) modulation of feeding. Further, our studies established the dependence of the central CB1R-mediated pressor response on neuronal nitric oxide synthase (nNOS) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation in the RVLM. We tested the novel hypothesis that brainstem orexin-A/OX1R signaling plays a pivotal role in the central CB1R-mediated pressor response. Our multiple labeling immunofluorescence findings revealed co-localization of CB1R, OX1R and the peptide orexin-A within the C1 area of the rostral ventrolateral medulla (RVLM). Activation of central CB1R following intracisternal (i.c.) WIN55,212-2 (15 µg/rat) in conscious rats caused significant increases in BP and orexin-A level in RVLM neuronal tissue. Additional studies established a causal role for orexin-A in the central CB1R-mediated pressor response because (i) selective blockade of central CB1R (AM251, 30 µg/rat; i.c.) abrogated WIN55,212-2-evoked increases in RVLM orexin-A level, (ii) the selective OX1R antagonist SB-408124 (10 nmol/rat; i.c.) attenuated orexin-A (3 nmol/rat; i.c.) or WIN55,212-2 (15 µg/rat; i.c.)-evoked pressor response while selective CB1R blockade (AM251) had no effect on orexin-A (3 nmol/rat; i.c.)-evoked pressor response, (iii) direct CB1R activation in the RVLM (WIN55,212-2; 0.1 µg/rat) increased RVLM orexin-A and BP. Finally, SB-408124 attenuated WIN55,212-2-evoked increases in RVLM nNOS and ERK1/2 phosphorylation and BP. Our findings suggest that orexin-A/OX1R dependent activation of the RVLM nNOS/ERK1/2 cascade is essential neurochemical mechanism for the central CB1R–mediated pressor response in conscious rats. PMID:26096126

  1. Conformational thermostabilisation of corticotropin releasing factor receptor 1

    PubMed Central

    Kean, James; Bortolato, Andrea; Hollenstein, Kaspar; Marshall, Fiona H.; Jazayeri, Ali

    2015-01-01

    Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation. PMID:26159865

  2. Multiple Roles for Nogo Receptor 1 in Visual System Plasticity.

    PubMed

    Stephany, Céleste-Élise; Frantz, Michael G; McGee, Aaron W

    2016-12-01

    During the developmental critical period for visual plasticity, discordant vision alters the responsiveness of neurons in visual cortex. The subsequent closure of the critical period not only consolidates neural function but also limits recovery of acuity from preceding abnormal visual experience. Despite species-specific differences in circuitry of the visual system, these characteristics are conserved. The nogo-66 receptor 1 (ngr1) is one of only a small number of genes identified thus far that is essential to closing the critical period. Mice lacking a functional ngr1 gene retain developmental visual plasticity as adults and their visual acuity spontaneously improves after prolonged visual deprivation. Experiments employing conditional mouse genetics have revealed that ngr1 restricts plasticity within distinct circuits for ocular dominance and visual acuity. However, the mechanisms by which NgR1 limits plasticity have not been elucidated, in part because the subcellular localization and signal transduction of the protein are only partially understood. Here we explore potential mechanisms for NgR1 function in relation to manipulations that reactivate visual plasticity in adults and propose lines of investigation to address relevant gaps in knowledge.

  3. Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1.

    PubMed

    Tonniges, Jeffrey R; Albert, Benjamin; Calomeni, Edward P; Roy, Shuvro; Lee, Joan; Mo, Xiaokui; Cole, Susan E; Agarwal, Gunjan

    2016-06-01

    The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

  4. Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1

    PubMed Central

    Tonniges, Jeffrey R.; Albert, Benjamin; Calomeni, Edward P.; Roy, Shuvro; Lee, Joan; Mo, Xiaokui; Cole, Susan E.; Agarwal, Gunjan

    2016-01-01

    The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson’s trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM. PMID:27329311

  5. Chimpanzee Personality and the Arginine Vasopressin Receptor 1A Genotype.

    PubMed

    Wilson, V A D; Weiss, A; Humle, T; Morimura, N; Udono, T; Idani, G; Matsuzawa, T; Hirata, S; Inoue-Murayama, M

    2017-03-01

    Polymorphisms of the arginine vasopressin receptor 1a (AVPR1a) gene have been linked to various measures related to human social behavior, including sibling conflict and agreeableness. In chimpanzees, AVPR1a polymorphisms have been associated with traits important for social interactions, including sociability, joint attention, dominance, conscientiousness, and hierarchical personality dimensions named low alpha/stability, disinhibition, and negative emotionality/low dominance. We examined associations between AVPR1a and six personality domains and hierarchical personality dimensions in 129 chimpanzees (Pan troglodytes) living in Japan or in a sanctuary in Guinea. We fit three linear and three animal models. The first model included genotype, the second included sex and genotype, and the third included genotype, sex, and sex × genotype. All personality phenotypes were heritable. Chimpanzees possessing the long form of the allele were higher in conscientiousness, but only in models that did not include the other predictors; however, additional analyses suggested that this may have been a consequence of study design. In animal models that included sex and sex × genotype, chimpanzees homozygous for the short form of the allele were higher in extraversion. Taken with the findings of previous studies of chimpanzees and humans, the findings related to conscientiousness suggest that AVPR1a may be related to lower levels of impulsive aggression. The direction of the association between AVPR1a genotype and extraversion ran counter to what one would expect if AVPR1a was related to social behaviors. These results help us further understand the genetic basis of personality in chimpanzees.

  6. Corneal avascularity is due to soluble VEGF receptor-1.

    PubMed

    Ambati, Balamurali K; Nozaki, Miho; Singh, Nirbhai; Takeda, Atsunobu; Jani, Pooja D; Suthar, Tushar; Albuquerque, Romulo J C; Richter, Elizabeth; Sakurai, Eiji; Newcomb, Michael T; Kleinman, Mark E; Caldwell, Ruth B; Lin, Qing; Ogura, Yuichiro; Orecchia, Angela; Samuelson, Don A; Agnew, Dalen W; St Leger, Judy; Green, W Richard; Mahasreshti, Parameshwar J; Curiel, David T; Kwan, Donna; Marsh, Helene; Ikeda, Sakae; Leiper, Lucy J; Collinson, J Martin; Bogdanovich, Sasha; Khurana, Tejvir S; Shibuya, Masabumi; Baldwin, Megan E; Ferrara, Napoleone; Gerber, Hans-Peter; De Falco, Sandro; Witta, Jassir; Baffi, Judit Z; Raisler, Brian J; Ambati, Jayakrishna

    2006-10-26

    Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

  7. Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase.

    PubMed

    Buttery, Shawnna M; Kono, Keiko; Stokasimov, Ema; Pellman, David

    2012-10-01

    Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.

  8. Human maternal behaviour is associated with arginine vasopressin receptor 1A gene.

    PubMed

    Avinun, Reut; Ebstein, Richard P; Knafo, Ariel

    2012-10-23

    Parenting is one of the main influences on children's early development, and yet its underlying genetic mechanisms have only recently begun to be explored, with many studies neglecting to control for possible child effects. This study focuses on maternal behaviour and on an allele at the RS3 promoter region of the arginine vasopressin receptor 1A (AVPR1A) gene, previously associated with autism and with higher amygdala activation in a face-matching task. Mothers were observed during a free-play session with each of their 3.5-year-old twins. Multilevel regression analyses revealed that mothers who are carriers of the AVPR1A RS3 allele tend to show less structuring and support throughout the interaction independent of the child's sex and RS3 genotype. This finding advances our understanding of the genetic influences on human maternal behaviour.

  9. Levels of plasma soluble complement receptor 1 (sCR1) in normal Indian adult population.

    PubMed

    Sivasankar, B; Raju, K R; Anand, V; Malu, S; Padmanabhan, S; Tiwari, S C; Das, N; Srivastava, L M

    1999-07-01

    A decrease in the membrane anchored erythrocyte complement receptor 1 (CR1) is reported as an acquired phenomenon in a number of inflammatory and autoimmune diseases with concomitant rise in soluble CR1 (sCR1) levels in plasma. There is a need to establish the normal range of sCR1 in Indian adults to assess the function and disease association of this protein. The plasma sCR1 levels of 50 healthy individuals have been estimated by an indigenously developed sandwich ELISA. sCR1 levels from 26 patients suffering from nephropathies had also been assayed which was much higher than the normal controls. This observation suggests sCR1 as a potential market for the assessment of disease activity in nephropathies.

  10. Soluble complement receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis.

    PubMed

    Piddlesden, S J; Jiang, S; Levin, J L; Vincent, A; Morgan, B P

    1996-12-01

    The loss of muscle function seen in myasthenia gravis and in the animal model of the disease, experimental autoimmune myasthenia gravis (EAMG) is in part due to the activation of complement by anti-acetylcholine receptor (AChR) antibodies at the motor end-plate. In this study we describe the effects of a soluble recombinant form of human complement receptor 1 (sCR1) on the development of clinical disease and receptor loss in EAMG induced passively by administration of anti-AChR antibodies. Daily intraperitoneal injection of sCR1 significantly reduced the weight loss and severity of clinical symptoms seen and allowed treated animals to recover normal muscle function. These data suggest that sCR1 could provide a useful additional therapeutic agent in myasthenia.

  11. Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury.

    PubMed

    Ramaglia, Valeria; Wolterman, Ruud; de Kok, Maryla; Vigar, Miriam Ann; Wagenaar-Bos, Ineke; King, Rosalind Helen Mary; Morgan, Brian Paul; Baas, Frank

    2008-04-01

    Complement activation is a crucial early event in Wallerian degeneration. In this study we show that treatment of rats with soluble complement receptor 1 (sCR1), an inhibitor of all complement pathways, blocked both systemic and local complement activation after crush injury of the sciatic nerve. Deposition of membrane attack complex (MAC) in the nerve was inhibited, the nerve was protected from axonal and myelin breakdown at 3 days after injury, and macrophage infiltration and activation was strongly reduced. We show that both classical and alternative complement pathways are activated after acute nerve trauma. Inhibition of the classical pathway by C1 inhibitor (Cetor) diminished, but did not completely block, MAC deposition in the injured nerve, blocked myelin breakdown, inhibited macrophage infiltration, and prevented macrophage activation at 3 days after injury. However, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. We conclude that sCR1 protects the nerve from early axon loss after injury and propose complement inhibition as a potential therapy for the treatment of diseases in which axon loss is the main cause of disabilities.

  12. Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42–Tuba pathway

    PubMed Central

    Juin, Amélie; Di Martino, Julie; Leitinger, Birgit; Henriet, Elodie; Gary, Anne-Sophie; Paysan, Lisa; Bomo, Jeremy; Baffet, Georges; Gauthier-Rouvière, Cécile; Rosenbaum, Jean

    2014-01-01

    Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. We previously found that collagen I fibrils induced the formation of peculiar linear invadosomes in an unexpected integrin-independent manner. Here, we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer, colocalizes with linear invadosomes in tumor cells and is required for their formation and matrix degradation ability. Unexpectedly, DDR1 kinase activity is not required for invadosome formation or activity, nor is Src tyrosine kinase. We show that the RhoGTPase Cdc42 is activated on collagen in a DDR1-dependent manner. Cdc42 and its specific guanine nucleotide-exchange factor (GEF), Tuba, localize to linear invadosomes, and both are required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasion in a collagen gel. Altogether, our data uncover an important role for DDR1, acting through Tuba and Cdc42, in proteolysis-based cell invasion in a collagen-rich environment. PMID:25422375

  13. Design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor 1 antagonist.

    PubMed

    Hwang, Tsong-Long; Hung, Chih-Hao; Hsu, Ching-Yun; Huang, Yin-Ting; Tsai, Yu-Chi; Hsieh, Pei-Wen

    2013-06-14

    Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selectively inhibited neutrophil elastase release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-containing dipeptides were synthesized and their pharmacological activities were investigated in human neutrophils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on FMLP-induced superoxide anion (O2˙(-)) generation and neutrophil elastase release in neutrophils with IC50 values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of structure-activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most suitable as a core structure for interaction with FPR1, and may be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized compounds exhibited separable conformational isomers, and showed diverse bioactivities, the conformation analysis of these compounds is also discussed herein.

  14. Layer-by-layer co-immobilization of soluble complement receptor 1 and heparin on islets.

    PubMed

    Luan, Nguyen Minh; Teramura, Yuji; Iwata, Hiroo

    2011-09-01

    Early graft loss due to instant blood-mediated inflammatory reactions (IBMIRs) is a major obstacle of clinical islet transplantation; inhibition of blood coagulation and complement activation is necessary to inhibit IBMIRs. Here, human soluble form complement receptor 1 (sCR1) and heparin were co-immobilized onto the surfaces of islet cells. sCR1 molecules carrying thiol groups were immobilized through maleimide-poly(ethylene glycol)-phospholipids anchored in the lipid bilayers of islet cells. Heparin was immobilized on the sCR1 layer via the affinity between sCR1 and heparin, and additional layers of sCR1 and heparin were formed layer-by-layer. The sCR1 and heparin molecules in these layers maintained anti-complement activation and anti-coagulation activities, respectively. This promising method could be employed to reduce the number of islet cells required to reverse hyperglycemia and prolong graft survival in both allo- and xeno-islet transplantation.

  15. Regulation of brachyury by fibroblast growth factor receptor 1 in lung cancer

    PubMed Central

    Hu, Yunping; Feng, Xin; Mintz, Akiva; Petty, W. Jeffrey; Hsu, Wesley

    2016-01-01

    Recent evidence suggests that T-box transcription factor brachyury plays an important role in lung cancer development and progression. However, the mechanisms underlying brachyury-driven cellular processes remain unclear. Here we found that fibroblast growth factor receptor 1/mitogen-activated protein kinase (FGFR1/MAPK) signaling regulated brachyury in lung cancer. Analysis of FGFR1-4 and brachyury expression in human lung tumor tissue and cell lines found that only expression of FGFR1 was positively correlated with brachyury expression. Specific knockdown of FGFR1 by siRNA suppressed brachyury expression and epithelial–mesenchymal transition (EMT) (upregulation of E-cadherin and β-catenin and downregulation of Snail and fibronectin), whereas forced overexpression of FGFR1 induced brachyury expression and promoted EMT in lung cancer cells. Activation of fibroblast growth factor (FGF)/FGFR1 signaling promoted phosphorylated MAPK extracellular signal-regulated kinase (ERK) 1/2 translocation from cytoplasm to nucleus, upregulated brachyury expression, and increased cell growth and invasion. In addition, human lung cancer cells with higher brachyury expression were more sensitive to inhibitors targeting FGFR1/MAPK pathway. These findings suggest that FGFR1/MAPK may be important for brachyury activation in lung cancer, and this pathway may be an appealing therapeutic target for a subset of brachyury-driven lung cancer. PMID:27893433

  16. Sleep architecture of the melanin-concentrating hormone receptor 1-knockout mice.

    PubMed

    Adamantidis, Antoine; Salvert, Denise; Goutagny, Romain; Lakaye, Bernard; Gervasoni, Damien; Grisar, Thierry; Luppi, Pierre-Hervé; Fort, Patrice

    2008-04-01

    Growing amounts of data indicate involvement of the posterior hypothalamus in the regulation of sleep, especially paradoxical sleep (PS). Accordingly, we previously showed that the melanin-concentrating hormone (MCH)-producing neurons of the rat hypothalamus are selectively activated during a PS rebound. In addition, intracerebroventricular infusion of MCH increases total sleep duration, suggesting a new role for MCH in sleep regulation. To determine whether activation of the MCH system promotes sleep, we studied spontaneous sleep and its homeostatic regulation in mice with deletion of the MCH-receptor 1 gene (MCH-R1-/- vs. MCH-R1+/+) and their behavioural response to modafinil, a powerful antinarcoleptic drug. Here, we show that the lack of functional MCH-R1 results in a hypersomniac-like phenotype, both in basal conditions and after total sleep deprivation, compared to wild-type mice. Further, we found that modafinil was less potent at inducing wakefulness in MCH-R1-/- than in MCH-R1+/+ mice. We report for the first time that animals with genetically inactivated MCH signaling exhibit altered vigilance state architecture and sleep homeostasis. This study also suggests that the MCH system may modulate central pathways involved in the wake-promoting effect of modafinil.

  17. Cysteinyl leukotriene receptor-1 antagonists as modulators of innate immune cell function.

    PubMed

    Theron, A J; Steel, H C; Tintinger, G R; Gravett, C M; Anderson, R; Feldman, C

    2014-01-01

    Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8(+) cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5'-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

  18. Association of the arginine vasopressin receptor 1A (AVPR1A) haplotypes with listening to music.

    PubMed

    Ukkola-Vuoti, Liisa; Oikkonen, Jaana; Onkamo, Päivi; Karma, Kai; Raijas, Pirre; Järvelä, Irma

    2011-04-01

    Music is listened in all cultures. We hypothesize that willingness to produce and perceive sound and music is social communication that needs musical aptitude. Here, listening to music was surveyed using a web-based questionnaire and musical aptitude using the auditory structuring ability test (Karma Music test) and Carl Seashores tests for pitch and for time. Three highly polymorphic microsatellite markers (RS3, RS1 and AVR) of the arginine vasopressin receptor 1A (AVPR1A) gene, previously associated with social communication and attachment, were genotyped and analyzed in 31 Finnish families (n=437 members) using family-based association analysis. A positive association between the AVPR1A haplotype (RS1 and AVR) and active current listening to music (permuted P=0.0019) was observed. Other AVPR1A haplotype (RS3 and AVR) showed association with lifelong active listening to music (permuted P=0.0022). In addition to AVPR1A, two polymorphisms (5-HTTLPR and variable number of tandem repeat) of human serotonin transporter gene (SLC6A4), a candidate gene for many neuropsychiatric disorders and previously associated with emotional processing, were analyzed. No association between listening to music and the polymorphisms of SLC6A4 were detected. The results suggest that willingness to listen to music is related to neurobiological pathways affecting social affiliation and communication.

  19. Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging.

    PubMed

    Qian, Feng; Guo, Xiuyang; Wang, Xiaomei; Yuan, Xiaoling; Chen, Shu; Malawista, Stephen E; Bockenstedt, Linda K; Allore, Heather G; Montgomery, Ruth R

    2014-02-01

    Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

  20. Discoidin domain receptor 1: isoform expression and potential functions in cirrhotic human liver.

    PubMed

    Song, Sunmi; Shackel, Nicholas A; Wang, Xin M; Ajami, Katerina; McCaughan, Geoffrey W; Gorrell, Mark D

    2011-03-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

  1. Signaling through hepatocyte vasopressin receptor 1 protects mouse liver from ischemia-reperfusion injury

    PubMed Central

    Jiang, Jingbo; Ma, Tonghui; Lin, Xiaozhu; Jiang, Liping; Cheng, Jilin; Tao, Ran

    2016-01-01

    Terlipressin has been used extensively in the management of certain complications associated with end-stage liver diseases (ESLDs). In our pilot study, terlipressin treatment showed beneficial effects on liver function in patients with decompensated cirrhosis, however whether it plays a role in liver ischemia-reperfusion injury (IRI) remains unknown. Using a mouse nonlethal hepatic IR model, we found terlipressin administration significantly ameliorated IR-induced liver apoptosis, necrosis and inflammation. Furthermore, despite its known effect on visceral vasoconstriction, hemodynamic evaluation of murine hepatic tissue after IR revealed no change of overall hepatic blood flow after terlipressin treatment. Further studies identified the upregulation of vasopressin receptor 1 (V1R) expression on hepatocytes upon IR. In isolated hepatocyte hypoxia/reoxygenation model, the active component of terlipressin, lysine vasopressin, conferred hepatocytes resistant to oxidative stress-induced apoptosis. Mechanistic studies revealed the V1R engagement activated the Wnt/β-catenin/FoxO3a/AKT pathway, which subsequently circumvented the proapoptotic events, thus ameliorated hepatocyte apoptosis. Furthermore, genetic knockdown of V1R expression in hepatocyte cell lines or blockade of this signaling pathway abrogated such protective effect. Conclusion: These data highlight the functional importance of the hepatocyte V1R/Wnt/β-catenin/FoxO3a/AKT pathway in protecting liver from oxidative stress-induced injury. PMID:27713143

  2. Genetic variability in the human cannabinoid receptor 1 is associated with resting state EEG theta power in humans.

    PubMed

    Heitland, I; Kenemans, J L; Böcker, K B E; Baas, J M P

    2014-11-01

    It has long been postulated that exogenous cannabinoids have a profound effect on human cognitive functioning. These cannabinoid effects are thought to depend, at least in parts, on alterations of phase-locking of local field potential neuronal firing. The latter can be measured as activity in the theta frequency band (4-7Hz) by electroencephalogram. Theta oscillations are supposed to serve as a mechanism in neural representations of behaviorally relevant information. However, it remains unknown whether variability in endogenous cannabinoid activity is involved in theta rhythms and therefore, may serve as an individual differences index of human cognitive functioning. To clarify this issue, we recorded resting state EEG activity in 164 healthy human subjects and extracted EEG power across frequency bands (δ, θ, α, and β). To assess variability in the endocannabinoid system, two genetic polymorphisms (rs1049353, rs2180619) within the cannabinoid receptor 1 (CB1) were determined in all participants. As expected, we observed significant effects of rs1049353 on EEG power in the theta band at frontal, central and parietal electrode regions. Crucially, these effects were specific for the theta band, with no effects on activity in the other frequency bands. Rs2180619 showed no significant associations with theta power after Bonferroni correction. Taken together, we provide novel evidence in humans showing that genetic variability in the cannabinoid receptor 1 is associated with resting state EEG power in the theta frequency band. This extends prior findings of exogenous cannabinoid effects on theta power to the endogenous cannabinoid system.

  3. Modulators of the Sphingosine 1-phosphate receptor 1.

    PubMed

    Urbano, Mariangela; Guerrero, Miguel; Rosen, Hugh; Roberts, Edward

    2013-12-01

    The Sphingosine 1-phosphate receptor (S1P-R) signaling system has proven to be of biological and medical importance in autoimmune settings. S1P1-R is a validated drug target for multiple sclerosis (MS) for which FTY720 (Fingolimod), a S1P1,3-5-R pan-agonist, was recently approved as the first orally active drug for the treatment of relapsing-remitting MS. Transient bradycardia and long half-life are the FTY720 critical pitfalls. This review provides the latest advances on next-generation S1P1-R modulators from 2012 up to date, with an overview of the chemical structures, structure-activity relationships, and relevant biological and clinical properties.

  4. Modulation of Pilocarpine-Induced Seizures by Cannabinoid Receptor 1

    PubMed Central

    Kow, Rebecca L.; Jiang, Kelly; Naydenov, Alipi V.; Le, Joshua H.; Stella, Nephi; Nathanson, Neil M.

    2014-01-01

    Administration of the muscarinic agonist pilocarpine is commonly used to induce seizures in rodents for the study of epilepsy. Activation of muscarinic receptors has been previously shown to increase the production of endocannabinoids in the brain. Endocannabinoids act at the cannabinoid CB1 receptors to reduce neurotransmitter release and the severity of seizures in several models of epilepsy. In this study, we determined the effect of CB1 receptor activity on the induction in mice of seizures by pilocarpine. We found that decreased activation of the CB1 receptor, either through genetic deletion of the receptor or treatment with a CB1 antagonist, increased pilocarpine seizure severity without modifying seizure-induced cell proliferation and cell death. These results indicate that endocannabinoids act at the CB1 receptor to modulate the severity of pilocarpine-induced seizures. Administration of a CB1 agonist produced characteristic CB1-dependent behavioral responses, but did not affect pilocarpine seizure severity. A possible explanation for the lack of effect of CB1 agonist administration on pilocarpine seizures, despite the effects of CB1 antagonist administration and CB1 gene deletion, is that muscarinic receptor-stimulated endocannabinoid production is acting maximally at CB1 receptors to modulate sensitivity to pilocarpine seizures. PMID:24752144

  5. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis.

    PubMed

    Couser, W G; Johnson, R J; Young, B A; Yeh, C G; Toth, C A; Rudolph, A R

    1995-05-01

    Complement is a major mediator of tissue injury in several types of glomerulonephritis. However, no therapeutic agents that inhibit complement activation are available for human use. sCR1 (TP10, BRL 55736) is a recombinant, soluble human complement receptor 1 (CR1) molecule lacking transmembrane and cytoplasmic domains that inhibits C3 and C5 convertase activity by preferentially binding C4b and C3b. To test the efficacy of sCR1 on complement-mediated glomerulonephritis, rats were pretreated with sCR1 (60 mg/kg per day) before and during the induction of three models of complement-dependent glomerulonephritis (concanavalin A and antithymocyte serum models of proliferative glomerulonephritis, passive Heyman nephritis). Daily sCR1 and complement hemolytic activity levels were measured, and renal histology and urine protein excretion were examined. Mean serum sCR1 levels of 100 to 200 micrograms/mL were maintained with a reduction in complement hemolytic activity to less than 15% in most animals. In the antithymocyte serum model, sCR1-treated animals had significant reductions in mesangiolysis, glomerular platelet and macrophage infiltrates, and proteinuria at 48 h. In the concanavalin A model, sCR1 significantly reduced glomerular C3 and fibrin deposits, platelet infiltrates, and proteinuria at 48 h. In passive Heymann nephritis, proteinuria was also significantly reduced (199 +/- 8.5 versus 125 +/- 16 mg/day, P < 0.002) at 5 days. It was concluded that sCR1 significantly reduces both morphologic and functional consequences of several different types of complement-mediated glomerulonephritis and deserves evaluation as a potential therapeutic agent in complement-mediated immune glomerular disease in humans.

  6. Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

    PubMed Central

    Benz, Alexander H.; Renné, Christoph; Maronde, Erik; Koch, Marco; Grabiec, Urszula; Kallendrusch, Sonja; Rengstl, Benjamin; Newrzela, Sebastian; Hartmann, Sylvia; Hansmann, Martin-Leo; Dehghani, Faramarz

    2013-01-01

    Background Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date. Design and Methods We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated. Results A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells. Conclusions The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma. PMID:24349109

  7. Enterovirus 71 Disrupts Interferon Signaling by Reducing the Level of Interferon Receptor 1

    PubMed Central

    Lu, Jing; Yi, Lina; Zhao, Jin; Yu, Jun; Chen, Ying; Lin, Marie C.; Kung, Hsiang-Fu

    2012-01-01

    The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-β production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies. PMID:22258259

  8. Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development

    PubMed Central

    Jung, Bongnam; Obinata, Hideru; Galvani, Sylvain; Mendelson, Karen; Ding, Bisen; Skoura, Athanasia; Kinzel, Bernd; Brinkmann, Volker; Rafii, Shahin; Evans, Todd; Hla, Timothy

    2012-01-01

    SUMMARY During angiogenesis, nascent vascular sprouts fuse to form vascular networks enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis. PMID:22975328

  9. Nogo Receptor 1 Confines a Disinhibitory Microcircuit to the Critical Period in Visual Cortex.

    PubMed

    Stephany, Céleste-Élise; Ikrar, Taruna; Nguyen, Collins; Xu, Xiangmin; McGee, Aaron W

    2016-10-26

    A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the "critical period," when the circuitry of primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal nogo-66-receptor 1 (ngr1/rtn4r) is required to close the critical period. Here we combined mouse genetics, electrophysiology, and circuit mapping with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1 We demonstrate that ngr1 mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a "lower PV network configuration" in both critical-period wild-type mice and adult ngr1(-/-) mice. We propose that ngr1 limits disinhibition to close the critical period for OD plasticity and that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.

  10. Recombinant pigment epithelium-derived factor PEDF binds vascular endothelial growth factor receptors 1 and 2.

    PubMed

    Johnston, Erin K; Francis, Mary K; Knepper, Janice E

    2015-08-01

    Angiogenesis, or the formation of new blood vessels, is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis. To explore the mechanism by which PEDF acts, recombinant PEDF was expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The PEDF fusion protein was confirmed to be active in inhibition of endothelial cell proliferation and migration. Direct binding of PEDF to both vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 was demonstrated in an in vitro assay similar to an enzyme-linked immunosorbent assay (ELISA). PEDF was shown by immune-confocal microscopy to be localized within treated endothelial cells. When VEGF-stimulated endothelial cells were incubated with PEDF the VEGF receptors showed intracellular localization. These data suggest that the interaction between PEDF and VEGFR-1 or VEGFR-2 may be a possible mechanism for inhibiting angiogenesis. PEDF may be binding to the VEGF receptors to promote their internalization and/or degradation to limit VEGF responses in treated cells.

  11. Cannabinoid receptor 1 ligands revisited: Pharmacological assessment in the ACTOne system.

    PubMed

    Presley, Chaela S; Abidi, Ammaar H; Moore, Bob M

    2016-04-01

    In vitro cannabinoid pharmacology has evolved over time from simple receptor binding to include [(35)S]GTPγ, β-arrestin, and cAMP assays. Each assay has benefits and drawbacks; however, no single functional system has been used for high-throughput evaluation of compounds from binding to pharmacological functionality and antagonist assessment in a well-characterized human cell line. In this study, we evaluated and validated one system-ACTOne human embryonic kidney cells transfected with a cyclic nucleotide gated channel and cannabinoid receptor 1 (CB1)-and compared human CB1 affinity, functional, and antagonistic effects on cAMP with previously published results. The study was conducted on a diverse group of CB1 ligands, including endocannabinoids and related compounds, 2-AG, AEA, MAEA, and ACEA, the phytocannabinoid Δ(9) THC, and synthetic cannabinoids CP 55,940, WIN 55,212-2, SR 141716A, CP 945,598, and WIN 55,212-3. Our results were compared with literature values where human CB1 was used for affinity determination and cAMP was used as a functional readout. Here we report the first detailed evaluation of the ACTOne assay for the pharmacological evaluation of CB1 ligands. The results from the study reveal some interesting deviations from previously reported functional activities of the aforementioned ligands.

  12. Xenotransplantation of islets enclosed in agarose microcapsule carrying soluble complement receptor 1.

    PubMed

    Luan, Nguyen Minh; Iwata, Hiroo

    2012-11-01

    Strong immunological reactions remain a major barrier to treating diabetic patients using xenogeneic islets. In a previous study, we developed a method for enclosing islets with agarose microbeads carrying soluble complement receptor 1 (sCR1-Mics), a potent complement inhibitor in both classical and alternative complement activation pathways. This is the follow-up in vivo study to evaluate the protective effect of these sCR1-Mics using a xenotransplantation model (rats to mice). ACI/NSIc rat islets enclosed in sCR1-Mics were transplanted into the intraperitoneal cavity of diabetic C57BL/6 mice without immunosuppression therapy. Transplantation of islets in plain agarose microbeads (Mics) was used as a reference. While islets enclosed in plain Mics were rapidly destroyed (graft survival in recipients of 1000 islets: 11.6±3.8 days), transplantation of islets in sCR1-Mics significantly prolonged graft survival (34.1±3.2 days). Moreover, intraperitoneal glucose tolerance tests revealed that islets in sCR1-Mics normalized blood glucose levels in a similar manner as islets in pancreas of normal mice. In conclusion, sCR1 immobilized onto agarose microbeads exerted some protective effect in xenogeneic islets resulting in prolonged graft survival.

  13. Splicing alterations in human renal allografts: detection of a new splice variant of protein kinase Par1/Emk1 whose expression is associated with an increase of inflammation in protocol biopsies of transplanted patients.

    PubMed

    Hueso, Miguel; Beltran, Violeta; Moreso, Francesc; Ciriero, Eva; Fulladosa, Xavier; Grinyó, Josep Maria; Serón, Daniel; Navarro, Estanis

    2004-05-24

    Protein kinase Emk1/Par1 (GenBank accession no. X97630) has been identified as a regulator of the immune system homeostasis. Since immunological factors are critical for the development of chronic allograft nephropathy (CAN), we reasoned that expression of Par1/Emk1 could be altered in kidney allografts undergoing CAN. In this paper, we have analysed the association among renal allograft lesions and expression of Par1/Emk1, studied by RT-PCR on total RNA from 51 protocol biopsies of transplanted kidneys, five normal kidneys, and five dysfunctional allografts. The most significant result obtained has been the detection of alterations in the normal pattern of alternative splicing of the Par1/Emk1 transcript, alterations that included loss of expression of constitutively expressed isoforms, and the inclusion of a cryptic exon to generate a new Emk1 isoform (Emk1C). Expression of Emk1C was associated with an increase in the extension of the interstitial infiltrate (0.88+/-0.33 in Emk1C([+]) vs. 0.41+/-0.50 in Emk1C([-]); P<0.011), and with a trend to display higher interstitial scarring (0.66+/-0.70 vs. 0.29+/-0.52; P=0.09) in protocol biopsies when evaluated according to the Banff schema. Moreover, a higher mean arterial pressure (MAP) was also observed (110+/-11 vs. 99+/-11 mm Hg; P=0.012). From these results we propose that Par1/Emk1 could have a role in the development of CAN in kidney allografts.

  14. Disruption of PTH Receptor 1 in T Cells Protects against PTH-Induced Bone Loss

    PubMed Central

    Tawfeek, Hesham; Bedi, Brahmchetna; Li, Jau-Yi; Adams, Jonathan; Kobayashi, Tatsuya; Weitzmann, M. Neale; Kronenberg, Henry M.; Pacifici, Roberto

    2010-01-01

    Background Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs. Methodology/Principal Findings Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor α (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio. Conclusions/Significance These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism. PMID:20808842

  15. Hypocretin receptor 1 blockade preferentially reduces high effort responding for cocaine without promoting sleep

    PubMed Central

    Brodnik, Zachary D.; Bernstein, David L.; Prince, Courtney D.; España, Rodrigo A.

    2015-01-01

    Recent evidence suggests that blockade of the hypocretin receptor 1 may act as a useful pharmacotherapy for cocaine abuse. Here we investigated the extent to which various doses of a hypocretin receptor 1 antagonist, SB-334867, affect cocaine self-administration at varying doses of cocaine and across a range of effort requirements, and tested if these SB-334867 doses produce sedative effects. First, we trained animals to self-administer one of three doses of cocaine on a progressive ratio schedule, and then tested the effects of three doses of SB-334867. Responding for cocaine was then analyzed to segregate features of relatively high and low effort requirements across the progressive ratio session. In another set of experiments we tested the sleep-promoting effects of the same doses of SB-334867. Our data indicate that blockade of hypocretin receptor 1 preferentially reduces high effort responding for cocaine at levels that do not promote sedation. PMID:26049058

  16. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses.

  17. Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function

    PubMed Central

    Shi, Xiao; Walter, Nicole A. R.; Harkness, John H.; Neve, Kim A.; Williams, Robert W.; Lu, Lu; Belknap, John K.; Eshleman, Amy J.; Phillips, Tamara J.; Janowsky, Aaron

    2016-01-01

    Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and β-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30–40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options. PMID:27031617

  18. Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1.

    PubMed

    Speyer, Cecilia L; Nassar, Mahdy A; Hachem, Ali H; Bukhsh, Miriam A; Jafry, Waris S; Khansa, Rafa M; Gorski, David H

    2016-06-01

    Riluzole, the only drug approved by the FDA for treating amyotrophic lateral sclerosis, inhibits melanoma proliferation through its inhibitory effect on glutamatergic signaling. We demonstrated that riluzole also inhibits the growth of triple-negative breast cancer (TNBC) and described a role for metabotropic glutamate receptor-1 (GRM1) in regulating TNBC cell growth and progression. However, the role of GRM1 in mediating riluzole's effects in breast cancer has not been fully elucidated. In this study, we seek to determine how much of riluzole's action in breast cancer is mediated through GRM1. We investigated anti-tumor properties of riluzole in TNBC and ER+ cells using cell growth, invasion, and soft-agar assays and compared riluzole activity with GRM1 levels. Using Lentiviral vectors expressing GRM1 or shGRM1, these studies were repeated in cells expressing high or low GRM1 levels where the gene was either silenced or overexpressed. Riluzole inhibited proliferation, invasion, and colony formation in both TNBC and ER+ cells. There was a trend between GRM1 expression in TNBC cells and their response to riluzole in both cell proliferation and invasion assays. However, silencing and overexpression studies had no effect on cell sensitivity to riluzole. Our results clearly suggest a GRM1-independent mechanism through which riluzole mediates its effects on breast cancer cells. Understanding the mechanism by which riluzole mediates breast cancer progression will be useful in identifying new therapeutic targets for treating TNBC and in facilitating stratification of patients in clinical trials using riluzole in conjunction with conventional therapy.

  19. Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical Implications.

    PubMed

    Pei, Yue; Asif-Malik, Aman; Canales, Juan J

    2016-01-01

    Biogenic amines are a collection of endogenous molecules that play pivotal roles as neurotransmitters and hormones. In addition to the "classical" biogenic amines resulting from decarboxylation of aromatic acids, including dopamine (DA), norepinephrine, epinephrine, serotonin (5-HT), and histamine, other biogenic amines, present at much lower concentrations in the central nervous system (CNS), and hence referred to as "trace" amines (TAs), are now recognized to play significant neurophysiological and behavioral functions. At the turn of the century, the discovery of the trace amine-associated receptor 1 (TAAR1), a phylogenetically conserved G protein-coupled receptor that is responsive to both TAs, such as β-phenylethylamine, octopamine, and tyramine, and structurally-related amphetamines, unveiled mechanisms of action for TAs other than interference with aminergic pathways, laying the foundations for deciphering the functional significance of TAs and its mammalian CNS receptor, TAAR1. Although, its molecular interactions and downstream targets have not been fully elucidated, TAAR1 activation triggers accumulation of intracellular cAMP, modulates PKA and PKC signaling and interferes with the β-arrestin2-dependent pathway via G protein-independent mechanisms. TAAR1 is uniquely positioned to exert direct control over DA and 5-HT neuronal firing and release, which has profound implications for understanding the pathophysiology of, and therefore designing more efficacious therapeutic interventions for, a range of neuropsychiatric disorders that involve aminergic dysregulation, including Parkinson's disease, schizophrenia, mood disorders, and addiction. Indeed, the recent development of novel pharmacological tools targeting TAAR1 has uncovered the remarkable potential of TAAR1-based medications as new generation pharmacotherapies in neuropsychiatry. This review summarizes recent developments in the study of TAs and TAAR1, their intricate neurochemistry and

  20. Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical Implications

    PubMed Central

    Pei, Yue; Asif-Malik, Aman; Canales, Juan J.

    2016-01-01

    Biogenic amines are a collection of endogenous molecules that play pivotal roles as neurotransmitters and hormones. In addition to the “classical” biogenic amines resulting from decarboxylation of aromatic acids, including dopamine (DA), norepinephrine, epinephrine, serotonin (5-HT), and histamine, other biogenic amines, present at much lower concentrations in the central nervous system (CNS), and hence referred to as “trace” amines (TAs), are now recognized to play significant neurophysiological and behavioral functions. At the turn of the century, the discovery of the trace amine-associated receptor 1 (TAAR1), a phylogenetically conserved G protein-coupled receptor that is responsive to both TAs, such as β-phenylethylamine, octopamine, and tyramine, and structurally-related amphetamines, unveiled mechanisms of action for TAs other than interference with aminergic pathways, laying the foundations for deciphering the functional significance of TAs and its mammalian CNS receptor, TAAR1. Although, its molecular interactions and downstream targets have not been fully elucidated, TAAR1 activation triggers accumulation of intracellular cAMP, modulates PKA and PKC signaling and interferes with the β-arrestin2-dependent pathway via G protein-independent mechanisms. TAAR1 is uniquely positioned to exert direct control over DA and 5-HT neuronal firing and release, which has profound implications for understanding the pathophysiology of, and therefore designing more efficacious therapeutic interventions for, a range of neuropsychiatric disorders that involve aminergic dysregulation, including Parkinson's disease, schizophrenia, mood disorders, and addiction. Indeed, the recent development of novel pharmacological tools targeting TAAR1 has uncovered the remarkable potential of TAAR1-based medications as new generation pharmacotherapies in neuropsychiatry. This review summarizes recent developments in the study of TAs and TAAR1, their intricate neurochemistry and

  1. MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a

    PubMed Central

    Man, Xiao-Fei; Tan, Shu-Wen; Tang, Hao-Neng; Guo, Yue; Tang, Chen-Yi; Tang, Jun; Zhou, Ci-La; Zhou, Hou-De

    2016-01-01

    Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy. PMID:27398155

  2. Disruption of the melanin-concentrating hormone receptor 1 (MCH1R) affects thyroid function.

    PubMed

    Chung, Shinjae; Liao, Xiao-Hui; Di Cosmo, Caterina; Van Sande, Jacqueline; Wang, Zhiwei; Refetoff, Samuel; Civelli, Olivier

    2012-12-01

    Melanin-concentrating hormone (MCH) is a peptide produced in the hypothalamus and the zona incerta that acts on one receptor, MCH receptor 1 (MCH1R), in rodents. The MCH system has been implicated in the regulation of several centrally directed physiological responses, including the hypothalamus-pituitary-thyroid axis. Yet a possible direct effect of the MCH system on thyroid function has not been explored in detail. We now show that MCH1R mRNA is expressed in thyroid follicular cells and that mice lacking MCH1R [MCH1R-knockout (KO)] exhibit reduced circulating iodothyronine (T(4), free T(4), T(3), and rT(3)) levels and high TRH and TSH when compared with wild-type (WT) mice. Because the TSH of MCH1R-KO mice displays a normal bioactivity, we hypothesize that their hypothyroidism may be caused by defective thyroid function. Yet expression levels of the genes important for thyroid hormones synthesis or secretion are not different between the MCH1R-KO and WT mice. However, the average thyroid follicle size of the MCH1R-KO mice is larger than that of WT mice and contained more free and total T(4) and T(3) than the WT glands, suggesting that they are sequestered in the glands. Indeed, when challenged with TSH, the thyroids of MCH1R-KO mice secrete lower amounts of T(4). Similarly, secretion of iodothyronines in the plasma upon (125)I administration is significantly reduced in MCH1R-KO mice. Therefore, the absence of MCH1R affects thyroid function by disrupting thyroid hormone secretion. To our knowledge, this study is the first to link the activity of the MCH system to the thyroid function.

  3. Folding and conformational studies on SCR1-3 domains of human complement receptor 1.

    PubMed

    Clark, N S; Dodd, I; Mossakowska, D E; Smith, R A; Gore, M G

    1996-10-01

    Short consensus repeats SCR3 and SCR1-3 are soluble recombinant proteins, consisting of the third and first three N-terminal domains of complement receptor 1, respectively, which retain some anti-complement activity. The conformational stabilities and folding/unfolding of SCR3 and SCR1-3 have been studied using circular dichroism and equilibrium and pre-equilibrium fluorescence spectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl) is rapid and completely reversible. Reduction of disulphide bridges in the folded proteins by beta-mercaptoethanol leads to an increase in fluorescence intensity. The fluorescence intensity of the folded proteins is approximately 7.5% of that of the respective unfolded proteins. The data can be approximated to a two-state transition between native and denatured forms of the proteins. SCR3 has a conformational stability in water of 12-13 kJ/mol whereas that of SCR1-3 is 19.5-19.9 kJ/mol depending upon the technique utilized. The heat capacity change associated with the unfolding of SCR1-3 was obtained by a series of GdnHCl unfolding experiments over a range of temperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol(residue). The refolding process of SCR3 was found to be simple, described by a single exponential equation, whereas that of SCR1-3 was found to be complex and could be fitted to a double exponential equation indicating the presence of folding intermediates.

  4. Soluble complement receptor 1 preserves endothelial barrier function and microcirculation in postischemic pancreatitis in the rat.

    PubMed

    von Dobschuetz, E; Bleiziffer, O; Pahernik, S; Dellian, M; Hoffmann, T; Messmer, K

    2004-05-01

    Components of the activated complement cascade are considered to play a pivotal role in ischemia-reperfusion-induced organ injury. With the use of intravital epifluorescence microscopy, we investigated the effect of complement inhibition by the recombinant soluble complement receptor 1 (sCR1; TP10) on the effect of macromolecular microvascular permeability, functional capillary perfusion, and leukocyte endothelium interaction in postischemic pancreatitis. Anaesthetized Sprague-Dawley rats were subjected to 60 min of normothermic pancreatic ischemia induced by microclipping of the blood-supplying arteries of the organ. Rats who received sCR1 (15 mg/kg body wt iv; n = 7) during reperfusion showed a significant reduction of permeability (1.77 +/- 1.34 x 10(-8) cm/s; n = 7) of tetramethylrhodamine isothiocyanate-labeled albumin injected 90 min after the onset of reperfusion compared with vehicle-treated animals (6.95 +/- 1.56 x 10(-8) cm/s; n = 7). At 120 min after the onset of reperfusion, the length of red blood cell-perfused capillaries (functional capillary density) was significantly improved (from 279 +/- 15.7 to 330 +/- 3.7 cm(-1); n = 7) and the number of leukocytes adherent to postcapillary venules was significantly reduced (from 314 +/- 87 to 163 +/- 71 mm(-2); n = 7) by sCR1 compared with vehicle treatment. Complement inhibition by sCR1 effectively ameliorates pancreatic ischemia-reperfusion-induced microcirculatory disturbances and might be considered for treatment of postischemic pancreatitis.

  5. Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

    PubMed Central

    Amdahl, Hanne; Tan, Lydia; Meri, Taru; Kuusela, Pentti I.; van Strijp, Jos A.

    2017-01-01

    Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus. PMID:28273167

  6. Hypoxia-inducible factor-1alpha suppresses the expression of macrophage scavenger receptor 1.

    PubMed

    Shirato, Ken; Kizaki, Takako; Sakurai, Takuya; Ogasawara, Jun-Etsu; Ishibashi, Yoshinaga; Iijima, Takehiko; Okada, Chikako; Noguchi, Izumi; Imaizumi, Kazuhiko; Taniguchi, Naoyuki; Ohno, Hideki

    2009-11-01

    Macrophages are distributed in all peripheral tissues and play a critical role in the first line of the innate immune defenses against bacterial infection by phagocytosis of bacterial pathogens through the macrophage scavenger receptor 1 (MSR1). Within tissues, the partial pressure of oxygen (pO2) decreases depending on the distance of cells from the closest O2-supplying blood vessel. However, it is not clear how the expression of MSR1 in macrophages is regulated by low pO2. On the other hand, hypoxia-inducible factor (HIF)-1alpha is well known to control hypoxic responses through regulation of hypoxia-inducible genes. Therefore, we investigated the effects of hypoxia and HIF-1alpha on MSR1 expression and function in the macrophage cell line RAW264. Exposure to 1% O2 or treatment with the hypoxia-mimetic agent cobalt chloride (CoCl2) significantly suppressed the expression of MSR1 mRNA, accompanied by a markedly increase in levels of nuclear HIF-1alpha protein. The overexpression of HIF-1alpha in RAW264 cells suppressed the expression of MSR1 mRNA and protein, transcriptional activity of the MSR1 gene, and phagocytic capacity against the Gram-positive bacteria Listeria monocytogenes. The suppression of MSR1 mRNA by hypoxia or CoCl2 was inhibited by YC-1, an inhibitor of HIF-1alpha, or by the depletion of HIF-1alpha expression by small interference RNA. These results indicate that hypoxia transcriptionally suppresses MSR1 expression through HIF-1alpha.

  7. Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-α-induced NF-κB signaling pathway.

    PubMed

    Hirano, Seiya; Kataoka, Takao

    2013-02-15

    Trichothecene mycotoxins are known to inhibit eukaryotic translation and to trigger the ribotoxic stress response, which regulates gene expression via the activation of the mitogen-activated protein (MAP) kinase superfamily. In this study, we found that deoxynivalenol induced the ectodomain shedding of tumor necrosis factor (TNF) receptor 1 (TNFRSF1A) and thereby inhibited the TNF-α-induced signaling pathway. In human lung carcinoma A549 cells, deoxynivalenol and 3-acetyldeoxynivalenol inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-α more strongly than that induced by interleukin 1α (IL-1α), whereas T-2 toxin and verrucarin A exerted nonselective inhibitory effects. Deoxynivalenol and 3-acetyldeoxynivalenol also inhibited the nuclear factor κB (NF-κB) signaling pathway induced by TNF-α, but not that induced by IL-1α. Consistent with these findings, deoxynivalenol and 3-acetyldeoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TNF-α-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17). In addition to the TACE inhibitor TAPI-2, the MAP kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol and reversed its selective inhibition of TNF-α-induced ICAM-1 expression. Our results demonstrate that deoxynivalenol induces the TACE-dependent ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, and thereby inhibits the TNF-α-induced NF-κB signaling pathway.

  8. Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.

    PubMed

    Shirakawa, Hisashi; Sakimoto, Shinya; Nakao, Kenji; Sugishita, Aiko; Konno, Masakazu; Iida, Shota; Kusano, Ayaka; Hashimoto, Emina; Nakagawa, Takayuki; Kaneko, Shuji

    2010-09-29

    Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own

  9. Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells

    PubMed Central

    Teh, Jessica L. F.; Shah, Raj; La Cava, Stephanie; Dolfi, Sonia C.; Mehta, Madhura S.; Kongara, Sameera; Price, Sandy; Ganesan, Shridar; Reuhl, Kenneth R.; Hirshfield, Kim M.

    2016-01-01

    Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60–80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential

  10. Multiorgan chronic inflammatory hepatobiliary pancreatic murine model deficient in tumor necrosis factor receptors 1 and 2

    PubMed Central

    Oz, Helieh S

    2016-01-01

    AIM: To provoke persistent/chronic multiorgan inflammatory response and to contribute to stones formation followed by fibrosis in hepatobiliary and pancreatic tissues. METHODS: Tumor necrosis factor receptors 1 and 2 (TNFR1/R2) deficient mice reared in-house were given dibutyltin dichloride (DBTC) twice within 10 d by oral gavage delivery. Sham control animals received vehicle treatment and naïve animals remained untreated throughout the study. Animals were monitored daily for symptoms of pain and discomfort. The abdominal and hindpaw hypersensitivity were assessed with von Frey microfilaments. Exploratory behaviors were recorded at the baseline, after initiation of treatment, and before study termination. Histopathological changes were examined postmortem in tissues. Collagen accumulation and fibrosis were confirmed with Sirius Red staining. RESULTS: Animals lost weight after oral administration of DBTC and developed persistent inflammatory abdominal and hindpaw hypersensitivity compared to sham-treated controls (P < 0.0001). These pain related secondary mechanical hypersensitivity responses increased more than 2-fold in DBTC-treated animals. The drastically diminished rearing and grooming rates persisted after DBTC administration throughout the study. Gross as well as micropathology at one month confirmed that animals treated with DBTC developed chronic hepatobiliary injuries evidenced with activation of stellate cells, multifocal necrosis, fatty degeneration of hepatocytes, periportal infiltration of inflammatory cells, and prominent biliary ductal dilation. The severity of hepatitis was scored 3.7 ± 0.2 (severe) in DBTC-treated animals vs score 0 (normal) in sham-treated animals. Fibrotic thickening was extensive around portal ducts, in hepatic parenchyma as well as in lobular pancreatic structures and confirmed with Sirius Red histopathology. In addition, pancreatic microarchitecture was presented with distortion of islets, and parenchyma, infiltration of

  11. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  12. Evidence for association between polymorphisms in the Cannabinoid Receptor 1 (CNR1) gene and cannabis dependence

    PubMed Central

    Agrawal, Arpana; Wetherill, Leah; Dick, Danielle M.; Xuei, Xiaoling; Hinrichs, Anthony; Hesselbrock, Victor; Kramer, John; Nurnberger, John I.; Schuckit, Marc; Bierut, Laura J.; Edenberg, Howard J.; Foroud, Tatiana

    2009-01-01

    Genomic studies of cannabis use disorders have been limited. The cannabinoid receptor 1 gene (CNR1) on chromosome 6q14–15 is an excellent candidate gene for cannabis dependence due to the important role of the G-protein coupled receptor encoded by this gene in the rewarding effects of Δ9-tetrahydrocannabinol. Previous studies have found equivocal evidence for an association between SNPs in CNR1 and a general vulnerability to substance use disorders. We investigate the association between 9 SNPs spanning CNR1 and cannabis dependence in 1,923 individuals. Two SNPs that were previously associated with cannabis dependence in other studies were also significant with this phenotype in our analyses [rs806368 (p = 0.05) and rs806380 (p = 0.009)]. Haplotype analyses revealed the association to be largely driven by the SNP rs806380. These results suggest a role for the cannabinoid receptor 1 gene in cannabis dependence. PMID:19016476

  13. Lectin-like oxidized LDL receptor-1 is an enhancer of tumor angiogenesis in human prostate cancer cells.

    PubMed

    González-Chavarría, Iván; Cerro, Rita P; Parra, Natalie P; Sandoval, Felipe A; Zuñiga, Felipe A; Omazábal, Valeska A; Lamperti, Liliana I; Jiménez, Silvana P; Fernandez, Edelmira A; Gutiérrez, Nicolas A; Rodriguez, Federico S; Onate, Sergio A; Sánchez, Oliberto; Vera, Juan C; Toledo, Jorge R

    2014-01-01

    Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.

  14. Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function.

    PubMed

    Ramachandran, Rithwik; Mihara, Koichiro; Thibeault, Pierre; Vanderboor, Christina M; Petri, Björn; Saifeddine, Mahmoud; Bouvier, Michel; Hollenberg, Morley D

    2017-04-01

    Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and β-arrestin interactions. By disrupting this PAR4 calcium/β-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.

  15. Failure to extinguish fear and genetic variability in the human cannabinoid receptor 1.

    PubMed

    Heitland, I; Klumpers, F; Oosting, R S; Evers, D J J; Leon Kenemans, J; Baas, J M P

    2012-09-25

    Failure to extinguish fear can lead to persevering anxiety and has been postulated as an important mechanism in the pathogenesis of human anxiety disorders. In animals, it is well documented that the endogenous cannabinoid system has a pivotal role in the successful extinction of fear, most importantly through the cannabinoid receptor 1. However, no human studies have reported a translation of this preclinical evidence yet. Healthy medication-free human subjects (N=150) underwent a fear conditioning and extinction procedure in a virtual reality environment. Fear potentiation of the eyeblink startle reflex was measured to assess fear-conditioned responding, and subjective fear ratings were collected. Participants were genotyped for two polymorphisms located within the promoter region (rs2180619) and the coding region (rs1049353) of cannabinoid receptor 1. As predicted from the preclinical literature, acquisition and expression of conditioned fear did not differ between genotypes. Crucially, whereas both homozygote (G/G, N=23) and heterozygote (A/G, N=68) G-allele carriers of rs2180619 displayed robust extinction of fear, extinction of fear-potentiated startle was absent in A/A homozygotes (N=51). Additionally, this resistance to extinguish fear left A/A carriers of rs2180619 with significantly higher levels of fear-potentiated startle at the end of the extinction training. No effects of rs1049353 genotype were observed regarding fear acquisition and extinction. These results suggest for the first time involvement of the human endocannabinoid system in fear extinction. Implications are that genetic variability in this system may underlie individual differences in anxiety, rendering cannabinoid receptor 1 a potential target for novel pharmacological treatments of anxiety disorders.

  16. Receptor for Advanced Glycation End Products Regulates Leukotriene B4 Receptor 1 Signaling.

    PubMed

    Ichiki, Takako; Koga, Tomoaki; Yokomizo, Takehiko

    2016-12-01

    Leukotriene B4 receptor 1 (BLT1), a high-affinity G protein-coupled receptor (GPCR) for leukotriene B4 (LTB4), plays important roles in inflammatory and immune reactions. Although the LTB4-BLT1 axis is known to promote inflammation, the binding proteins that modulate LTB4-BLT1 signaling have not been identified. Recently, we discovered that receptor for advanced glycation end products (RAGE) interacts with BLT1 and modulates LTB4-BLT1 signaling. We propose RAGE as a new class of GPCR modulator and a new target of future GPCR studies.

  17. Diagnostic and therapeutic challenges in a child with complete Interferon-γ Receptor 1 deficiency

    PubMed Central

    Olbrich, Peter; Martínez-Saavedra, Maria Teresa; Hurtado, José Maria Perez; Sanchez, Cristina; Sanchez, Berta; Deswarte, Carolina; Obando, Ignacio; Casanova, Jean-Laurent; Speckmann, Carsten; Bustamante, Jacinta; Rodriguez-Gallego, Carlos; Neth, Olaf

    2015-01-01

    Autosomal recessive (AR) complete Interferon-γ Receptor1 (IFN-γR1) deficiency is a rare variant of Mendelian susceptibility to mycobacterial disease (MSMD). Whilst hematopoietic stem cell transplantation (HSCT) remains the only curative treatment, outcomes are heterogeneous; delayed engraftment and/or graft rejection being commonly observed. This case report and literature review expands the knowledge about this rare but potentially fatal pathology, providing details regarding diagnosis, antimicrobial treatment, transplant performance and outcome that may help to guide physicians caring for patients with AR complete IFN-γR1 or IFN-γR2 deficiency. PMID:26173802

  18. Nonhuman Transferrin Receptor 1 Is an Efficient Cell Entry Receptor for Ocozocoautla de Espinosa Virus

    PubMed Central

    Caì, Yíngyún; Yú, Shuĭqìng; Mazur, Steven; Dŏng, Lián; Janosko, Krisztina; Zhāng, Téngfēi; Müller, Marcel A.; Hensley, Lisa E.; Bavari, Sina; Jahrling, Peter B.

    2013-01-01

    Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor. PMID:24109228

  19. Inhibition of leukotriene B4 receptor 1 attenuates lipopolysaccharide-induced cardiac dysfunction: role of AMPK-regulated mitochondrial function

    PubMed Central

    Sun, Meng; Wang, Rui; Han, Qinghua

    2017-01-01

    Leukotriene B4 (LTB4)-mediated leukocyte recruitment and inflammatory cytokine production make crucial contributions to chronic inflammation and sepsis; however, the role of LTB4 in lipopolysaccharide (LPS)-induced cardiac dysfunction remains unclear. Therefore, the present study addressed this issue using an LTB4 receptor 1 (BLT1) inhibitor. Administration of LPS to mice resulted in decreased cardiovascular function. Inhibition of LTB4/BLT1 with the BLT1 inhibitor U75302 significantly improved survival and attenuated the LPS-induced acute cardiac dysfunction. During LPS challenge, the phosphorylated AMPK/ACC signaling pathway was slightly activated, and this effect was enhanced by U75302. Additionally, pNF-κB, Bax and cleaved caspase-3 were upregulated by LPS, and Bcl-2, IκB-α, mitochondrial complex I, complex II, and OPA1 were downregulated; however, these effects were reversed by U75302. The results indicated that the BLT1 antagonist suppressed cardiac apoptosis, inflammation, and mitochondrial impairment. Furthermore, the protection provided by the BLT1 inhibitor against LPS-induced cardiac dysfunction was significantly reversed by the AMPK inhibitor Compound C. In conclusion, inhibiting the LTB4/BLT1 signaling pathway via AMPK activation is a potential treatment strategy for septic cardiac dysfunction because it efficiently attenuates cardiac apoptosis, which may occur via the inhibition of inflammation and mitochondrial dysfunction. PMID:28290498

  20. Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

    PubMed

    Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

    2016-06-01

    Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target.

  1. A partial trace amine-associated receptor 1 agonist exhibits properties consistent with a methamphetamine substitution treatment.

    PubMed

    Pei, Yue; Asif-Malik, Aman; Hoener, Marius; Canales, Juan J

    2016-05-19

    Recent evidence suggests that the trace amine-associated receptor 1 (TAAR1) plays a pivotal role in the regulation of dopamine (DA) transmission and psychostimulant action. Several selective TAAR1 agonists have previously shown efficacy in models of cocaine addiction. However, the effects of TAAR1 activation on methamphetamine (METH)-induced behaviours are less well understood, as indeed are the underlying neurochemical mechanisms mediating potential interactions between TAAR1 and METH. Here, in a progressive ratio schedule of reinforcement the partial TAAR1 agonist, RO5263397, reduced the break-point for METH self-administration, while significantly increasing responding maintained by food reward. Following self-administration and extinction training, RO5263397 completely blocked METH-primed reinstatement of METH seeking. Moreover, when used as a substitute, unlike a low dose of METH, which sustained vigorous responding when substituting for the training dose of METH, RO5263397 was not self-administered at any dose, thus exhibiting no apparent abuse liability. Fast-scan cyclic voltammetry experiments showed that RO5263397 prevented METH-induced DA overflow in slices of the nucleus accumbens, while having no effect on DA transmission in its own right. Collectively, the present observations demonstrate that partial TAAR1 activation decreases the motivation to self-administer METH, blocks METH-primed reinstatement of METH seeking and prevents METH-induced DA elevations in the nucleus accumbens, and strongly support the candidacy of TAAR1-based medications as potential substitute treatment in METH addiction.

  2. Monoclonal Antibody Targeting of Fibroblast Growth Factor Receptor 1c Ameliorates Obesity and Glucose Intolerance via Central Mechanisms

    PubMed Central

    Lelliott, Christopher J.; Ahnmark, Andrea; Admyre, Therese; Ahlstedt, Ingela; Irving, Lorraine; Keyes, Feenagh; Patterson, Laurel; Mumphrey, Michael B.; Bjursell, Mikael; Gorman, Tracy; Bohlooly-Y, Mohammad; Buchanan, Andrew; Harrison, Paula; Vaughan, Tristan; Berthoud, Hans-Rudolf; Lindén, Daniel

    2014-01-01

    We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation. PMID:25427253

  3. DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD

    PubMed Central

    Nissen, Judith Becker; Hansen, Christine Søholm; Starnawska, Anna; Mattheisen, Manuel; Børglum, Anders Dupont; Buttenschøn, Henriette Nørmølle; Hollegaard, Mads

    2016-01-01

    Obsessive–compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline

  4. Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).

    PubMed

    Mayerhofer, Hubert; Panneerselvam, Saravanan; Kaljunen, Heidi; Tuukkanen, Anne; Mertens, Haydyn D T; Mueller-Dieckmann, Jochen

    2015-01-30

    Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.

  5. KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

    PubMed

    Kamimura, Daisuke; Katsunuma, Kokichi; Arima, Yasunobu; Atsumi, Toru; Jiang, Jing-jing; Bando, Hidenori; Meng, Jie; Sabharwal, Lavannya; Stofkova, Andrea; Nishikawa, Naoki; Suzuki, Hironao; Ogura, Hideki; Ueda, Naoko; Tsuruoka, Mineko; Harada, Masaya; Kobayashi, Junya; Hasegawa, Takanori; Yoshida, Hisahiro; Koseki, Haruhiko; Miura, Ikuo; Wakana, Shigeharu; Nishida, Keigo; Kitamura, Hidemitsu; Fukada, Toshiyuki; Hirano, Toshio; Murakami, Masaaki

    2015-06-17

    KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

  6. The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

    PubMed Central

    Yossef, Rami; Hadad, Uzi; Elkabets, Moshe; Vallon-Eberhard, Alexandra; Hulihel, Luai; Jung, Steffen; Ghadially, Hormas; Braiman, Alex; Apte, Ron N.; Mandelboim, Ofer; Dagan, Ron; Mizrachi-Nebenzahl, Yaffa; Porgador, Angel

    2011-01-01

    Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC. PMID:21887255

  7. Binding of Hepatitis A Virus to its Cellular Receptor 1 Inhibits T-Regulatory Cell Functions in Humans

    PubMed Central

    Manangeeswaran, Mohanraj; Jacques, Jérôme; Tami, Cecilia; Konduru, Krishnamurthy; Amharref, Nadia; Perrella, Oreste; Casasnovas, Jose M.; Umetsu, Dale T.; DeKruyff, Rosemarie H.; Freeman, Gordon J.; Perrella, Alessandro; Kaplan, Gerardo G.

    2012-01-01

    Background & Aims CD4+ T regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. Methods We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. Results Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-β (TGF–β), which limited leukocyte recruitment and survival, and high levels of interleukin-22, which prevented liver damage. Conclusions Interaction between HAV and its receptor HAVCR1 inhibits Treg cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection—a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anti-cancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection. PMID:22430395

  8. The natural cytotoxicity receptor 1 contribution to early clearance of Streptococcus pneumoniae and to natural killer-macrophage cross talk.

    PubMed

    Elhaik-Goldman, Shirin; Kafka, Daniel; Yossef, Rami; Hadad, Uzi; Elkabets, Moshe; Vallon-Eberhard, Alexandra; Hulihel, Luai; Jung, Steffen; Ghadially, Hormas; Braiman, Alex; Apte, Ron N; Mandelboim, Ofer; Dagan, Ron; Mizrachi-Nebenzahl, Yaffa; Porgador, Angel

    2011-01-01

    Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligand(high) lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-ligand(dull) macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.

  9. Antidepressant/anxiolytic potential and adverse effect liabilities of melanin-concentrating hormone receptor 1 antagonists in animal models.

    PubMed

    Chaki, Shigeyuki; Shimazaki, Toshiharu; Nishiguchi, Mariko; Funakoshi, Takeo; Iijima, Michihiko; Ito, Akie; Kanuma, Kosuke; Sekiguchi, Yoshinori

    2015-08-01

    Melanin-concentrating hormone receptor 1 (MCH1 receptor) is known to be involved in the control of mood and stress, in addition to the regulation of feeding. Here, we report further evidence that the blockade of the MCH1 receptor exhibits antidepressant and anxiolytic-like effects in a variety of animal models using TASP0382650 and TASP0489838, newly synthesized MCH1 receptor antagonists, with different scaffolds. Both TASP0382650 and TASP0489838 exhibited high affinities for human MCH1 receptor with IC50 values of 7.13 and 3.80nM, respectively. Both compounds showed potent antagonist activities at the MCH1 receptor, as assessed using MCH-increased [(35)S]GTPγS binding to human MCH1 receptor and an MCH-induced [Ca(2+)]i assay in rat MCH1 receptor expressing cells. In contrast, neither TASP0382650 nor TASP0489838 showed an affinity for the MCH2 receptor, another MCH receptor subtype. The oral administration of TASP0382650 or TASP0489838 significantly reduced the immobility time during the forced swimming test in rats, and reduced hyperemotionality induced by an olfactory bulbectomy, both of which are indicative of an antidepressant-like potential. In the olfactory bulbectomy model, the antidepressant effect of TASP0382650 appeared following a single administration, suggesting a faster onset of action, compared with current medications. Moreover, both TASP0382650 and TASP0489838 exhibited anxiolytic effects in several animal models of anxiety. In contrast, both TASP0382650 and TASP0489838 did not affect spontaneous locomotor activity, motor function, spatial memory during the Morris water maze task, or the convulsion threshold to pentylenetetrazole. These findings provide additional evidence that the blockade of the MCH1 receptor exhibits antidepressant- and anxiolytic activities with no adverse effects in experimental animal models.

  10. Novel Mutation of Interferon-γ Receptor 1 Gene Presenting as Early Life Mycobacterial Bronchial Disease

    PubMed Central

    Gutierrez, Maria J.; Kalra, Neelu; Horwitz, Alexandra; Nino, Gustavo

    2016-01-01

    Mendelian susceptibility to mycobacterial diseases (MSMD) are a spectrum of inherited disorders characterized by localized or disseminated infections caused by atypical mycobacteria. Interferon-γ receptor 1 (IFNGR1) deficiency was the first identified genetic disorder recognized as MSMD. Mutations in the genes encoding IFNGR1 can be recessive or dominant and cause complete or partial receptor deficiency. We present the case of a 2½-year-old boy with a history of recurrent wheezing, diagnosed with endobronchial mycobacterial infection. Immunological workup revealed a homozygous nonsense mutation in the IFNGR1 gene, a novel mutation predicted in silico to cause complete IFNGR1 deficiency. This case demonstrates that (a) Interferon-γ receptor deficiency can present resembling common disorders of the lung; (b) mycobacterial infections should be suspected when parenchymal lung disease, hilar lymphadenopathy, and endobronchial disease are present; and (c) high index of suspicion for immunodeficiency should be maintained in patients with disseminated nontubercular mycobacterial infection. PMID:27868075

  11. Novel Mutation of Interferon-γ Receptor 1 Gene Presenting as Early Life Mycobacterial Bronchial Disease.

    PubMed

    Gutierrez, Maria J; Kalra, Neelu; Horwitz, Alexandra; Nino, Gustavo

    2016-01-01

    Mendelian susceptibility to mycobacterial diseases (MSMD) are a spectrum of inherited disorders characterized by localized or disseminated infections caused by atypical mycobacteria. Interferon-γ receptor 1 (IFNGR1) deficiency was the first identified genetic disorder recognized as MSMD. Mutations in the genes encoding IFNGR1 can be recessive or dominant and cause complete or partial receptor deficiency. We present the case of a 2½-year-old boy with a history of recurrent wheezing, diagnosed with endobronchial mycobacterial infection. Immunological workup revealed a homozygous nonsense mutation in the IFNGR1 gene, a novel mutation predicted in silico to cause complete IFNGR1 deficiency. This case demonstrates that (a) Interferon-γ receptor deficiency can present resembling common disorders of the lung; (b) mycobacterial infections should be suspected when parenchymal lung disease, hilar lymphadenopathy, and endobronchial disease are present; and (c) high index of suspicion for immunodeficiency should be maintained in patients with disseminated nontubercular mycobacterial infection.

  12. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome.

    PubMed

    Muenke, M; Schell, U; Hehr, A; Robin, N H; Losken, H W; Schinzel, A; Pulleyn, L J; Rutland, P; Reardon, W; Malcolm, S

    1994-11-01

    Pfeiffer syndrome (PS) is one of the classic autosomal dominant craniosynostosis syndromes with craniofacial anomalies and characteristic broad thumbs and big toes. We have previously mapped one of the genes for PS to the centromeric region of chromosome 8 by linkage analysis. Here we present evidence that mutations in the fibroblast growth factor receptor-1 (FGFR1) gene, which maps to 8p, cause one form of familial Pfeiffer syndrome. A C to G transversion in exon 5, predicting a proline to arginine substitution in the putative extracellular domain, was identified in all affected members of five unrelated PS families but not in any unaffected individuals. FGFR1 therefore becomes the third fibroblast growth factor receptor to be associated with an autosomal dominant skeletal disorder.

  13. Involvement of Prokineticin 2 and Prokineticin Receptor 1 in Lipopolysaccharide-Induced Testitis in Rats.

    PubMed

    Chen, Biao; Yu, Lili; Wang, Jiaojiao; Li, Cuiling; Zhao, Kai; Zhang, Huiping

    2016-04-01

    Prokineticin 2, a newly discovered proinflammatory peptide, has been amply evidenced to be involved in the occurrence and progress of local and systematical inflammation. Although the presence of Prokineticn 2 in mammal testis has been documented clearly, research targeting the involvement of prokineticin 2 in testicular pathology, especially testitis, is rather scarce. Employing a lipopolysaccharide-induced testitis rat model, we for the first time demonstrated the expression and upregulation of prokineticin 2 in orchitis at several levels. Our effort also addressed the differential expression patterns of prokineticin 2 and interleukin-1β, a key inflammation indicator, during testitis suggesting Prokineticn 2 serves more than a proinflammatory factor in the context of testitis. Given one of the cognate receptors of prokineticin 2, prokineticin receptor 1 (PKR1) was also significantly upregulated in orchitis as discussed in the current study, it is very likely that PK2/PKR1 signaling contribute to the development of inflammation-related testicular diseases.

  14. Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction.

    PubMed

    Lynskey, Nicola N; Banerji, Suneale; Johnson, Louise A; Holder, Kayla A; Reglinski, Mark; Wing, Peter A C; Rigby, David; Jackson, David G; Sriskandan, Shiranee

    2015-09-01

    The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.

  15. Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

    PubMed Central

    Johnson, Louise A.; Holder, Kayla A.; Reglinski, Mark; Wing, Peter A. C.; Rigby, David; Jackson, David G.; Sriskandan, Shiranee

    2015-01-01

    The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology. PMID:26352587

  16. Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors.

    PubMed

    Wang, Zhen; Zhang, Yali; Bartual, Sergio G; Luo, Jinfeng; Xu, Tingting; Du, Wenting; Xun, Qiuju; Tu, Zhengchao; Brekken, Rolf A; Ren, Xiaomei; Bullock, Alex N; Liang, Guang; Lu, Xiaoyun; Ding, Ke

    2017-03-09

    Acute lung injury (ALI) is a deadly symptom for serious lung inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-inflammatory drug discovery. A new selective tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to tightly bind the DDR1 protein and potently inhibit its kinase function with a Kd value of 2.2 nM and an IC50 value of 6.6 nM, respectively. The compound dose-dependently inhibited lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) release in mouse primary peritoneal macrophages (MPMs). In addition, 7ae also exhibited promising in vivo anti-inflammatory effects in a LPS-induced mouse ALI model. To the best of our knowledge, this is the first "proof of concept" investigation on the potential application of a small molecule DDR1 inhibitor to treat ALI.

  17. Methamphetamine induces trace amine-associated receptor 1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.

    PubMed

    Sriram, Uma; Cenna, Jonathan M; Haldar, Bijayesh; Fernandes, Nicole C; Razmpour, Roshanak; Fan, Shongshan; Ramirez, Servio H; Potula, Raghava

    2016-01-01

    The novel transmembrane G protein-coupled receptor, trace amine-associated receptor 1 (TAAR1), represents a potential, direct target for drugs of abuse and monoaminergic compounds, including amphetamines. For the first time, our studies have illustrated that there is an induction of TAAR1 mRNA expression in resting T lymphocytes in response to methamphetamine. Methamphetamine treatment for 6 h significantly increased TAAR1 mRNA expression (P < 0.001) and protein expression (P < 0.01) at 24 h. With the use of TAAR1 gene silencing, we demonstrate that methamphetamine-induced cAMP, a classic response to methamphetamine stimulation, is regulated via TAAR1. We also show by TAAR1 knockdown that the down-regulation of IL-2 in T cells by methamphetamine, which we reported earlier, is indeed regulated by TAAR1. Our results also show the presence of TAAR1 in human lymph nodes from HIV-1-infected patients, with or without a history of methamphetamine abuse. TAAR1 expression on lymphocytes was largely in the paracortical lymphoid area of the lymph nodes with enhanced expression in lymph nodes of HIV-1-infected methamphetamine abusers rather than infected-only subjects. In vitro analysis of HIV-1 infection of human PBMCs revealed increased TAAR1 expression in the presence of methamphetamine. In summary, the ability of methamphetamine to activate trace TAAR1 in vitro and to regulate important T cell functions, such as cAMP activation and IL-2 production; the expression of TAAR1 in T lymphocytes in peripheral lymphoid organs, such as lymph nodes; and our in vitro HIV-1 infection model in PBMCs suggests that TAAR1 may play an important role in methamphetamine -mediated immune-modulatory responses.

  18. Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy.

    PubMed

    Yazdanbakhsh, Karina; Kang, Stanley; Tamasauskas, Daniel; Sung, Dorothy; Scaradavou, Andromachi

    2003-06-15

    Activation of complement cascade via the antibody-mediated classical pathway can initiate red blood cell (RBC) destruction, causing transfusion reactions and hemolytic anemia. In the present study, we have assessed the ability of a human recombinant soluble form of complement receptor 1 (sCR1) to inhibit complement-mediated RBC destruction in vitro and in vivo. Using an in vitro alloimmune incompatibility model, sCR1 inhibited complement activation and prevented hemolysis. Following transfusion of human group O RBCs into mice lacking detectable pre-existing antibodies against the transfused RBCs, systemic coadministration of 10 mg/kg sCR1, a dose well tolerated in human subjects for prevention of tissue injury, completely inhibited the in vivo clearance of the transfused RBCs and surface C3 deposition in the first hour after transfusion, correlating with the half-life of sCR1 in the circulation. Treatment with sCR1 increased the survival of transfused human group A RBCs in the circulation of mice with pre-existing anti-A for 2 hours after transfusion by 50%, reduced intravascular hemolysis, and lowered the levels of complement deposition (C3 and C4), but not immunoglobulin G (IgG) or IgM, on the transfused cells by 100-fold. We further identified potential functional domains in CR1 that can act to limit complement-mediated RBC destruction in vitro and in vivo. Collectively, our data highlight a potential use of CR1-based inhibitors for prevention of complement-dependent immune hemolysis.

  19. Linkage and association analysis of candidate genes for TB and TNFalpha cytokine expression: evidence for association with IFNGR1, IL-10, and TNF receptor 1 genes.

    PubMed

    Stein, Catherine M; Zalwango, Sarah; Chiunda, Allan B; Millard, Christopher; Leontiev, Dmitry V; Horvath, Amanda L; Cartier, Kevin C; Chervenak, Keith; Boom, W Henry; Elston, Robert C; Mugerwa, Roy D; Whalen, Christopher C; Iyengar, Sudha K

    2007-07-01

    Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor necrosis factor-alpha (TNFalpha) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes related to TNFalpha regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFalpha receptor 1 (TNFR1) genes were linked and associated to both TB and TNFalpha. We also show that these associations are with progression to active disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study further illustrates the utility of such a model for disentangling complex traits.

  20. Losartan attenuates human monocyte-derived dendritic cell immune maturation via downregulation of lectin-like oxidized low-density lipoprotein receptor-1.

    PubMed

    Huang, Dong; Lu, Hao; Liu, Hongying; Yao, Kang; Sun, Aijun; Zou, Yunzeng; Ge, Junbo

    2012-08-01

    The angiotensin II receptor-1 blockers have generally been shown to have antiatherogenic effects, and dendritic cells (DCs) are the most efficient antigen presenting cells that play an active role in the development of atherosclerosis through inflammatory-immune responses. Here, we tested the hypothesis that the antiatherogenic effect of losartan, the first angiotensin II receptor-1 blockers, might partly be mediated by attenuating DCs maturation. In this study, we showed that oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) could induce the maturation of human monocyte-derived DCs, stimulate CD83, HLA-DR expressions and IL-12, interferon-gamma secretions and increase the capacity of DCs to stimulate T-cell proliferation, which were suppressed by losartan. OxLDL could promote the autocrine secretion of Ang II by DCs and upregulate the expressions of 3 scavenger receptors SR-A, CD36, and LOX-1. Losartan reduced oxLDL-induced LOX-1 expression but not SR-A and CD36 expressions. Ang II could only upregulate the LOX-1 expression, which was reduced by losartan. OxLDL- and Ang II-induced upregulation of CD83 and secretion of IL-12 were all attenuated by LOX-1 neutralizing antibody. In conclusion, losartan could attenuate the oxLDL- and Ang II-induced immune maturation of human monocyte-derived DCs partly through downregulation of the LOX-1 expression.

  1. Variation in the corticotropin-releasing hormone receptor 1 (CRHR1) gene modulates age effects on working memory.

    PubMed

    Grimm, Simone; Gärtner, Matti; Fuge, Philipp; Fan, Yan; Weigand, Anne; Feeser, Melanie; Aust, Sabine; Heekeren, Hauke R; Jacobs, Arthur; Heuser, Isabella; Bajbouj, Malek

    2015-02-01

    Decline in working memory (WM) functions during aging has been associated with hippocampal dysfunction mediated by age-related changes to the corticotropin-releasing hormone (CRH) system. Recent reports suggest that GG-homozygous individuals of single nucleotide polymorphisms (rs110402 and rs242924) in the CRH receptor 1 (CRHR1) gene show increased stress vulnerability and decreased BOLD responses in WM relevant regions. However, until now, no study investigated the interaction effects of variation in the CRHR1 gene and age on individual differences in WM. Here, young, middle-aged and old subjects (N = 466) were genotyped for rs110402 and rs242924 within the CRHR1 gene and an n-back task was used to investigate the hypothesis that vulnerable genotypes (GG-homozygotes) would show impaired WM functions that might be magnified by increased CRH production with advancing age. Our results show an impact of genotype already in middle-age with significantly better performance in AT-carriers. Working memory performance in AT-carriers did not differ between young and middle-aged subjects, but was significantly impaired in old age. In GG-homozygotes, severe working memory dysfunction occurred already in middle age. Our data indicate that GG-homozygotes of CRHR1 rs110402 and rs242924 represent a genetically driven subtype of early WM impairments due to alterations in hippocampal CRHR1 activation. Early interventions that have proven effective in delaying cognitive decline appear to be particularly important for these subjects at risk for premature memory decline, who are in the prime of their personal and professional lives.

  2. Association of single nucleotide polymorphism in melatonin receptor 1A gene with egg production traits in Yangzhou geese.

    PubMed

    Alsiddig, M A; Yu, S G; Pan, Z X; Widaa, H; Badri, T M; Chen, J; Liu, H L

    2017-04-01

    In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5' regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34-week egg-laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real-time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.

  3. Molecular expression of vascular endothelial growth factor, prokineticin receptor-1 and other biomarkers in infiltrating canalicular carcinoma of the breast

    PubMed Central

    Morales, Angélica; Morimoto, Sumiko; Vilchis, Felipe; Taniyama, Natsuko; Bautista, Claudia J.; Robles, Carlos; Bargalló, Enrique

    2016-01-01

    Vascular endothelial growth factor (VEGF) is important in the growth and metastasis of cancer cells. In 2001, another angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), was characterized and sequenced. EG-VEGF activity appears to be restricted to endothelial cells derived from endocrine glands. At the molecular level, its expression is regulated by hypoxia and steroid hormones. Although VEGF and EG-VEGF are structurally different, they function in a coordinated fashion. Since the majority of mammary tumors are hormone-dependent, it was hypothesized that EG-VEGF would be expressed in these tumors, and therefore, represent a potential target for anti-angiogenic therapy. The aim of the present study was to assess the expression of VEGF, EG-VEGF and its receptor (prokineticin receptor-1), as well as that of breast cancer resistant protein, estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, in 50 breast samples of infiltrating canalicular carcinoma (ICC) and their correlation with tumor staging. The samples were analyzed using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Both angiogenic growth factors were identified in all samples. However, in 90% of the samples, the expression level of VEGF was significantly higher than that of EG-VEGF (P=0.024). There was no association between the expression of VEGF, EG-VEGF or its receptor with tumor stage. In ICC, the predominant angiogenic factor expressed was VEGF. The expression level of either factor was not correlated with the tumor-node-metastasis stage. Although ICC is derived from endothelial cells, EG-VEGF expression was not the predominant angiogenic/growth factor in ICC. PMID:27703528

  4. The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB(1)).

    PubMed

    Grabiec, Urszula; Koch, Marco; Kallendrusch, Sonja; Kraft, Robert; Hill, Kerstin; Merkwitz, Claudia; Ghadban, Chalid; Lutz, Beat; Straiker, Alex; Dehghani, Faramarz

    2012-03-01

    Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N-methyl-d-aspartate (NMDA, 50 μM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM-50 μM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a slight reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 μM NADA (p < 0.05). To identify the responsive receptor type of NADA mediated neuroprotection, we applied the cannabinoid (CB) receptor 1 (CB(1)) inverse agonist/antagonist AM251, CB(2) inverse agonist/antagonist AM630, abnormal-cannabidiol (abn-CBD)-sensitive receptor antagonist O-1918, transient receptor potential channel V1 (TRPV1) antagonist 6-iodonordihydrocapsaicin and A1 (TRPA1) antagonist HC-030031. Neuroprotective properties of low (1 nM) but not high (10 μM) NADA concentrations were solely blocked by AM251 and were absent in CB(1)(-/-) mice. AM630, O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all NADA concentrations applied. Our findings demonstrate that NADA protects dentate gyrus granule cells by acting via CB(1). NADA reduced the number of microglial cells at distinct concentrations. TRPV1 and TRPA1 were not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA mediated activation of neuronal CB(1) may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.

  5. G protein-coupled oestrogen receptor 1 (GPER1)/GPR30: a new player in cardiovascular and metabolic oestrogenic signalling

    PubMed Central

    Nilsson, Bengt-Olof; Olde, Björn; Leeb-Lundberg, LM Fredrik

    2011-01-01

    Oestrogens are important sex hormones central to health and disease in both genders that have protective effects on the cardiovascular and metabolic systems. These hormones act in complex ways via both genomic and non-genomic mechanisms. The genomic mechanisms are relatively well characterized, whereas the non-genomic ones are only beginning to be explored. Two oestrogen receptors (ER), ERα and ERβ, have been described that act as nuclear transcription factors but can also associate with the plasma membrane and influence cytosolic signalling. ERα has been shown to mediate both anti-atherogenic effects and pro-survival effects in pancreatic β-cells. In recent years, a third membrane-bound ER has emerged, G protein-coupled receptor 30 or G protein-coupled oestrogen receptor 1 (GPER1), which mediates oestrogenic responses in cardiovascular and metabolic regulation. Both GPER1 knock-out models and pharmacological agents are now available to study GPER1 function. These tools have revealed that GPER1 activation may have several beneficial effects in the cardiovascular system including vasorelaxation, inhibition of smooth muscle cell proliferation, and protection of the myocardium against ischaemia/reperfusion injury, and in the metabolic system including stimulation of insulin release and protection against pancreatic β-cell apoptosis. Thus, GPER1 is emerging as a candidate therapeutic target in both cardiovascular and metabolic disease. LINKED ARTICLES This article is one of a set of reviews submitted to BJP in connection with talks given at the September 2010 meeting of the International Society of Hypertension in Vancouver, Canada. To view the other articles in this collection visit http://dx.doi.org/10.1111/j.1476-5381.2010.01167.x, http://dx.doi.org/10.1111/j.1476-5381.2011.01260.x and http://dx.doi.org/10.1111/j.1476-5381.2011.01366.x PMID:21250980

  6. Tissue-Specific Expression of Estrogen Receptor 1 Is Regulated by DNA Methylation in a T-DMR.

    PubMed

    Maekawa, Ryo; Sato, Shun; Okada, Maki; Lee, Lifa; Tamura, Isao; Jozaki, Kosuke; Kajimura, Takuya; Asada, Hiromi; Yamagata, Yoshiaki; Tamura, Hiroshi; Yamamoto, Shigeru; Sugino, Norihiro

    2016-03-01

    The mechanism controlling tissue-specific expression of estrogen receptor 1 (ESR1) is unclear. In other genes, DNA methylation of a region called the tissue-dependent and differentially methylated region (T-DMR) has been associated with tissue-specific gene expression. This study investigated whether human ESR1 has a T-DMR and whether DNA methylation of the T-DMR regulates its expression. ESR1 expression was tissue-specific, being high in the endometrium and mammary gland and low/nil in the placenta and skin. Therefore, DNA methylation profiles of the promoter of ESR1 were analyzed in these tissues and in breast cancer tissues. In all of the normal tissues, the proximal promoter regions were unmethylated. On the other hand, the distal regions (T-DMR) were unmethylated in the endometrium and mammary gland, but were moderately methylated and hypermethylated in the placenta and skin, respectively. T-DMR-methylated reporter assay was performed to examine whether DNA methylation at the T-DMR suppresses ESR1 transcription. T-DMR, but not the promoter region, had transcriptional activities and DNA methylation of the T-DMR suppressed ESR1 transcription. Early growth response protein 1 was shown to be a possible transcription factor to bind the T-DMR and up-regulate ESR1 expression. ESR1 has several upstream exons, and each upstream exon, Exon-A/Exon-B/Exon-C, had its own T-DMR. In some breast cancer cases and breast cancer cell lines, ESR1 expression was not regulated by DNA methylation at T-DMR as it is in normal tissues. In conclusion, ESR1 has a T-DMR. DNA methylation status at the T-DMR is involved in tissue-specific ESR1 expression in normal tissues but not always in breast cancer.

  7. Angiotensin II receptor 1 gene variants are associated with high-altitude pulmonary edema risk.

    PubMed

    Jin, Tianbo; Ren, Yongchao; Zhu, Xikai; Li, Xun; Ouyang, Yongri; He, Xue; Zhang, Zhiying; Zhang, Yuan; Kang, Longli; Yuan, Dongya

    2016-11-22

    Previous studies demonstrated that Angiotensin II Receptor 1 (AGTR1) may play an important role in the development of high-altitude pulmonary edema. We envisaged a role for AGTR1 gene variants in the pathogenesis of HAPE and investigated their potential associations with HAPE in a Han Chinese population. We genotyped seven AGTR1 polymorphisms in 267 patients with diagnosed HAPE and 304 controls and evaluated their association with risk of HAPE. Statistically significant associations were found for the single nucleotide polymorphisms (SNPs) rs275651 (p = 0.017; odds ratio [OR] = 0.65) and rs275652 (p = 0.016; OR = 0.64). Another SNP rs10941679 showed a marginally significant association after adjusting for age and sex in the additive genetic model (adjusted OR = 1.44, 95% CI = 1.01-2.04, p = 0.040). Haplotype analysis confirmed that the haplotype "AG" was associated with a 35% reduction in the risk of developing HAPE, while the haplotype "AA" increased the risk of developing HAPE by 44%. These results provide the first evidence linking genetic variations in AGTR1 with HAPE risk in Han Chinese individuals.

  8. Clinical significance of leukocyte-associated immunoglobulin-like receptor-1 expression in human cervical cancer

    PubMed Central

    Wang, Yue; Zhang, Xueshan; Miao, Fang; Cao, Yanning; Xue, Jiangnan; Cao, Qizhi; Zhang, Xiaoshu

    2016-01-01

    Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is broadly expressed on the majority of immune cells; however, the biological role of LAIR in solid tumors has yet to be elucidated. In the present study, using immunohistochemical staining analysis, the expression of LAIR-1 in human cervical cancer (HCC) and nontumor-adjacent tissue specimens was determined, and the results indicated that the expression of LAIR-1 in HCC tissue was higher compared with that in noncancerous tissue. The χ2 test was used to analyze the correlation between the expression of LAIR-1 in tumor tissues with clinicopathological parameters. The results showed that the expression of LAIR-1 in the cancer cell nucleus was significantly associated with tumor size, pathological differentiation, T classification and clinical stage. In addition, the expression in the cytoplasm was evidently associated with the number of positive lymph nodes. The HCC cell line, ME-180, which does not express LAIR-1, was stably transfected using LAIR-1 cDNA. Cell Counting Kit-8 and an annexin V assay showed that the overexpression of LAIR-1 in ME-180 cells suppressed the proliferation and anti-apoptosis capacity of the cells. These findings demonstrated that LAIR-1 is markedly overexpressed in HCC tissue, and that its expression status is associated with tumor progression. LAIR-1 may be a biomarker and target in the diagnosis and treatment of patients with HCC. PMID:28105100

  9. Endosomal recycling regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-dependent cell spreading.

    PubMed

    Gu, Jingsheng; Faundez, Victor; Werner, Erica

    2010-07-15

    Mechanisms for receptor-mediated anthrax toxin internalization and delivery to the cytosol are well understood. However, far less is known about the fate followed by anthrax toxin receptors prior and after cell exposure to the toxin. We report that Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8 (TEM8) localized at steady state in Rab11a-positive and transferrin receptor-containing recycling endosomes. TEM8 followed a slow constitutive recycling route of approximately 30min as determined by pulsed surface biotinylation and chase experiments. A Rab11a dominant negative mutant and Myosin Vb tail expression impaired TEM8 recycling by sequestering TEM8 in intracellular compartments. Sequestration of TEM8 in intracellular compartments with monensin coincided with increased TEM8 association with a multi-protein complex isolated with antibodies against transferrin receptor. Addition of the cell-binding component of anthrax toxin, Protective Antigen, reduced TEM8 half-life from 7 to 3 hours, without preventing receptor recycling. Pharmacological and molecular perturbation of recycling endosome function using monensin, dominant negative Rab11a, or myosin Vb tail, reduced PA binding efficiency and TEM8-dependent cell spreading on PA-coated surfaces without affecting toxin delivery to the cytosol. These results indicate that the intracellular fate of TEM8 differentially affect its cell adhesion and cell intoxication functions.

  10. Blockade of hypocretin receptor-1 preferentially prevents cocaine seeking: comparison with natural reward seeking

    PubMed Central

    Martin-Fardon, Rémi; Weiss, Friedbert

    2014-01-01

    Hypothalamic orexin/hypocretin (Orx/Hcrt) peptides participate in the regulation of a wide range of physiological processes and are recruited by drugs of abuse. To advance our understanding of the potential of the Orx/Hcrt receptor-1 (Hcrt-r1) as a treatment target for cocaine addiction, the effect of SB334867, a specific Hcrt-r1 antagonist, on reinstatement elicited by cocaine-associated stimuli vs. stimuli associated with a highly palatable conventional reinforcer (sweetened condensed milk [SCM]) was tested. Two separate groups of male Wistar rats were trained to associate a discriminative stimulus (S+) with the response-contingent availability of cocaine (0.25 mg/0.1 ml/infusion) or SCM (2/1 [v/v]) and subjected to reinstatement tests following extinction, during which the reinforcers and S+ were withheld, of cocaine or SCM-reinforced behavior. Following extinction, presentation of the cocaine or SCM S+ produced comparable recovery of responding. Hcrt-r1 blockade by SB334867 (1–10 mg/kg, IP) dose-dependently and selectively reversed conditioned reinstatement induced by cocaine-related stimuli, without interfering with reward seeking produced by the same stimulus when conditioned to SCM. The findings implicate an important role for Hcrt-r1 in appetitive behavior controlled by reward-related stimuli with selectivity for cocaine seeking and identify Hcrt-r1 as a potential treatment target for cocaine relapse prevention. PMID:24407199

  11. Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease.

    PubMed

    Kerroch, Monique; Alfieri, Carlo; Dorison, Aude; Boffa, Jean-Jacques; Chatziantoniou, Christos; Dussaule, Jean-Claude

    2016-02-16

    Chronic kidney disease is a progressive incurable pathology affecting millions of people. Intensive investigations aim to identify targets for therapy. We have previously demonstrated that abnormal expression of the Discoidin Domain Receptor 1 (DDR1) is a key factor of renal disease by promoting inflammation and fibrosis. The present study investigates whether blocking the expression of DDR1 after the initiation of renal disease can delay or arrest the progression of this pathology. Severe renal disease was induced by either injecting nephrotoxic serum (NTS) or performing unilateral ureteral obstruction in mice, and the expression of DDR1 was inhibited by administering antisense oligodeoxynucleotides either at 4 or 8 days after NTS (corresponding to early or more established phases of disease, respectively), or at day 2 after ligation. DDR1 antisense administration at day 4 stopped the increase of proteinuria and protected animals against the progression of glomeruloneprhitis, as evidenced by functional, structural and cellular indexes. Antisense administration at day 8 delayed progression -but to a smaller degree- of renal disease. Similar beneficial effects on renal structure and inflammation were observed with the antisense administration of DDR1 after ureteral ligation. Thus, targeting DDR1 can be a promising strategy in the treatment of chronic kidney disease.

  12. Uremic Pruritus Is Not Associated with Endocannabinoid Receptor 1 Gene Polymorphisms

    PubMed Central

    Heisig, Monika; Łaczmański, Łukasz; Reich, Adam; Lwow, Felicja

    2016-01-01

    Uremic pruritus (UP) is a frequent and bothersome symptom in hemodialysis patients. Its etiology is not fully understood and that is why there is no specific treatment. The endocannabinoid system plays a role in many pathological conditions. There is reliable evidence on the association between cannabinoid system and pruritus. In our study, we aimed to evaluate whether genetic variations in the endocannabinoid receptor 1 (CNR1) gene can affect UP. The rs12720071, rs806368, rs1049353, rs806381, rs10485170, rs6454674, and rs2023239 polymorphisms of the CNR1 gene were genotyped in 159 hemodialysis patients and 150 healthy controls using two multiplex polymerase chain reactions and the minisequencing technique. No statistically significant relationship was found in any of the evaluated genotypes between patients with and without UP, even after excluding patients with diabetes and dyslipidemia. There were no differences between patients with UP and the control group. However, in the group of all HD patients, a significantly higher incidence of GA genotype and lower incidence in GG genotype in the polymorphism rs806381s were revealed versus the control group (p = 0.04). It seems that polymorphisms of the CNR1 gene are not associated with uremic pruritus. PMID:27034934

  13. Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

    SciTech Connect

    Eto, Hideyuki; Miyata, Masaaki . E-mail: miyatam@m3.kufm.kagoshima-u.ac.jp; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

    2006-03-10

    Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 {mu}g/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

  14. Alpha-2-macroglobulin gene, oxidized low-density lipoprotein receptor-1 locus, and sporadic Alzheimer's disease.

    PubMed

    Colacicco, Anna Maria; Solfrizzi, Vincenzo; D'Introno, Alessia; Capurso, Cristiano; Kehoe, Patrick G; Seripa, Davide; Pilotto, Alberto; Santamato, Andrea; Capurso, Antonio; Panza, Francesco

    2009-09-01

    A total sample of 169 AD patients, and 264 age- and sex-matched unrelated caregivers from Apulia, southern Italy, were genotypized for alpha-2-macroglobulin (A2M) Val1000/Ile single-nucleotide polymorphism (SNP) (rs669), apolipoprotein E (APOE), and SNPs (+1073 and +1071) in the oxidized low-density lipoprotein receptor-1 (OLR1) gene on chromosome 12. A2M allele and genotype frequencies were similar between AD patients and controls, also after stratification for late onset (>/=70 years) and early onset (<70 years) or APOE varepsilon4 status. However, there was evidence in support of LD between the OLR1+1071, the OLR1+1073, and the rs669 SNPs, with T-C-A haplotype being associated with significant increased risk of AD in both the whole sample and when we stratified according to early and late onset AD subjects, with the allelic association with AD predominantly from the OLR1+1073 SNP, further supporting the role of OLR1 as a candidate risk gene for sporadic AD.

  15. Polymorphisms in the oxidized low-density lipoprotein receptor-1 gene and risk of Alzheimer's disease.

    PubMed

    D'Introno, Alessia; Solfrizzi, Vincenzo; Colacicco, Anna M; Capurso, Cristiano; Torres, Francesco; Capurso, Sabrina A; Capurso, Antonio; Panza, Francesco

    2005-03-01

    The +1073 C/T polymorphism of the oxidized low-density lipoprotein receptor-1 (OLR1) gene has been reported to be associated with late-onset Alzheimer's disease, whereas for the +1071 T/A polymorphism no association was found. We genotyped 169 sporadic Alzheimer's disease patients and 264 sex- and age-matched nondemented controls from Southern Italy for OLR1 +1073 C/T and +1071 T/A polymorphisms and for apolipoprotein E and LBP-1c/CP2/LSF. We also performed haplotype analysis. For the +1073 C/T polymorphism, the C allele and the CC genotype have been associated with a higher risk for Alzheimer's disease without apolipoprotein E or CP2 interaction. The two polymorphisms were in linkage disequilibrium, with the haplotype T-C at significant increased risk of developing Alzheimer's disease in the whole sample and in elderly persons 70 years or older. In our population, the +1073 C/T OLR1 polymorphism exhibited a significant association with Alzheimer's disease, further supporting the role of OLR1 as a candidate risk gene for sporadic Alzheimer's disease.

  16. Expression of Nogo receptor 1 in microglia during development and following traumatic brain injury.

    PubMed

    Liu, Gaoxiang; Ni, Jie; Mao, Lei; Yan, Ming; Pang, Tao; Liao, Hong

    2015-11-19

    As the receptor of myelin associated inhibitory factors Nogo receptor 1 (NgR1) plays an important role in central nervous system (CNS) injury and regeneration. It is found that NgR1 complex acts in neurons to transduce the signals intracelluarly including induction of growth cone collapse, inhibition of axonal regeneration and regulation of nerve inflammation. In recent studies, NgR1 has also been found to be expressed in the microglia. However, NgR1 expressed in microglia in the developing nervous systems and following CNS injury have not been widely investigated. In this study, we detected the expression and cellular localization of NgR1 in microglia during development and following traumatic brain injury (TBI) in mice. The results showed that NgR1 was mainly expressed in microglia during embryonic and postnatal periods. The expression levels peaked at P4 and decreased thereafter into adulthood, while increased significantly with aging representatively at 17 mo. On the other hand, there was no significant difference in the number of double positive NgR1(+)Iba1(+) cells between normal and TBI group. In summary, we first detected the expression of NgR1 in microglia during development and found that NgR1 protein expression increased significantly in microglia with aging. These findings will contribute to make a foundation for subsequent study about the role of NgR1 expressed in microglia on the CNS disorders.

  17. Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats

    SciTech Connect

    Yamada, Takanori; Obo, Yumi; Furukawa, Mami; Hotta, Mayuko; Yamasaki, Ayako; Honoki, Kanya; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2009-01-16

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 out of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP.

  18. Brain structural and clinical changes after first episode psychosis: Focus on cannabinoid receptor 1 polymorphisms.

    PubMed

    Suárez-Pinilla, Paula; Roiz-Santiañez, Roberto; Ortiz-García de la Foz, Víctor; Guest, Paul C; Ayesa-Arriola, Rosa; Córdova-Palomera, Aldo; Tordesillas-Gutierrez, Diana; Crespo-Facorro, Benedicto

    2015-08-30

    Cannabinoid receptor 1 (CNR1) gene polymorphisms have been associated with central and peripheral effects of cannabis and schizophrenia pathophysiology. Here, we have tested whether three CNR1 variants (rs1049353, rs1535255 and rs2023239) are associated with changes in brain volumes, body mass index (BMI) or psychopathological scores in a 3-year longitudinal study of 65 first-episode psychosis patients. The rs1049353 at-risk allele was significantly associated with a greater reduction of caudate volume, and the rs2023239 T/C polymorphism showed a significant decrease in thalamic volume after the 3-year period. For those who were not cannabis users, the rs1535255 and rs2023239 polymorphisms had effects in lateral ventricle (LV), and LV and white matter, respectively. The rs2023239 variant also was associated with significant improvements in positive and negative symptoms of schizophrenia. There was no significant effect of any of the variants on changes in BMI over the 3-year study. Finally, an interaction between all three polymorphisms was found involving evolution of positive symptoms. These findings suggest that the cannabinoid pathway is associated with schizophrenia evolution over time. However, further studies using larger cohorts are needed to confirm these results. If confirmed, the present findings could lead in subsequent investigations for identification of novel drug targets for improved treatment of patients suffering from schizophrenia.

  19. Recent advances in the medicinal chemistry of the metabotropic glutamate receptor 1 (mGlu₁).

    PubMed

    Owen, Dafydd R

    2011-08-17

    This Review summarizes the medicinal chemistry found in publications on both orthosteric and allosteric modulators of the metabotropic glutamate receptor 1 (mGlu(1)) from 2005 to the present. The time period covered by the scope of this current review has been particularly rich in mGlu(1)-related publications with numbers quadrupling when compared to the preceding five year period of 2000-2005. Publications in the field peaked in 2007 with over 35 articles appearing in the peer reviewed literature in the course of that year. Given that glutamate is one of the primary excitatory neurotransmitters in the mammalian central nervous system (CNS), it is unsurprising that it acts upon several receptors that are considered to be of potential therapeutic interest for many indications. Orthosteric and allosteric modulation of the receptor is possible, with a logical extrapolation to the chemotypes used for each strategy. The last five years of publications have yielded many mGlu(1) selective antagonist chemotypyes, most of which have shown efficacy in pain in vivo models. However, the primary impact of these compounds has been to highlight the mechanistic safety risks of mGlu(1) antagonism, independent of chemotype. As a review in medicinal chemistry, the primary focus of this paper will be on the design and, to a lesser degree, synthetic strategies for the delivery of subtype selective, CNS penetrant, druglike compounds through a "medchem" program, targeting modulators of the mGlu(1) receptor.

  20. Musashi RNA-binding protein 2 regulates estrogen receptor 1 function in breast cancer.

    PubMed

    Kang, M-H; Jeong, K J; Kim, W Y; Lee, H J; Gong, G; Suh, N; Győrffy, B; Kim, S; Jeong, S-Y; Mills, G B; Park, Y-Y

    2017-03-23

    Musashi RNA-binding protein 2 (MSI2) has important roles in human cancer. However, the regulatory mechanisms by which MSI2 alters breast cancer pathophysiology have not been clearly identified. Here we demonstrate that MSI2 directly regulates estrogen receptor 1 (ESR1), which is a well-known therapeutic target and has been shown to reflect clinical outcomes in breast cancer. Based on gene expression data analysis, we found that MSI2 expression was highly enriched in estrogen receptor (ER)-positive breast cancer and that MSI2 expression was significantly correlated with ESR1 expression, including expression of ESR1 downstream target genes. In addition, MSI2 levels were associated with clinical outcomes. MSI2 influenced breast cancer cell growth by altering ESR1 function. MSI2 alters ESR1 by binding specific sites in ESR1 RNA and by increasing ESR1 protein stability. Taken together, our findings identified a novel regulatory mechanism of MSI2 as an upstream regulator of ESR1 and revealed the clinical relevance of the RNA-binding protein MSI2 in breast cancer.

  1. Characterization of Chronic Cutaneous Lesions from TNF-Receptor-1-Deficient Mice Infected by Leishmania major

    PubMed Central

    Oliveira, Carolina Ferreira; Manzoni-de-Almeida, Daniel; Mello, Paula Seixas; Natale, Caio Cotta; Santiago, Helton da Costa; Miranda, Luíza da Silva; Ferraz, Fernanda Oliveira; dos Santos, Liliane Martins; Teixeira, Mauro Martins; Arantes, Rosa Maria Esteves; Vieira, Leda Quercia

    2012-01-01

    Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis. PMID:22203861

  2. Ganglioside mediate the interaction between Nogo receptor 1 and LINGO-1.

    PubMed

    Saha, Nayanendu; Kolev, Momchil V; Semavina, Mariya; Himanen, Juha; Nikolov, Dimitar B

    2011-09-16

    Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.

  3. Prolonging Survival of Corneal Transplantation by Selective Sphingosine-1-Phosphate Receptor 1 Agonist

    PubMed Central

    Gao, Min; Liu, Yong; Xiao, Yang; Han, Gencheng; Jia, Liang; Wang, Liqiang; Lei, Tian; Huang, Yifei

    2014-01-01

    Corneal transplantation is the most used therapy for eye disorders. Although the cornea is somewhat an immune privileged organ, immune rejection is still the major problem that reduces the success rate. Therefore, effective chemical drugs that regulate immunoreactions are needed to improve the outcome of corneal transplantations. Here, a sphingosine-1-phosphate receptor 1 (S1P1) selective agonist was systematically evaluated in mouse allogeneic corneal transplantation and compared with the commonly used immunosuppressive agents. Compared with CsA and the non-selective sphingosine 1-phosphate (S1P) receptor agonist FTY720, the S1P1 selective agonist can prolong the survival corneal transplantation for more than 30 days with a low immune response. More importantly, the optimal dose of the S1P1 selective agonist was much less than non-selective S1P receptor agonist FTY720, which would reduce the dose-dependent toxicity in drug application. Then we analyzed the mechanisms of the selected S1P1 selective agonist on the immunosuppression. The results shown that the S1P1 selective agonist could regulate the distribution of the immune cells with less CD4+ T cells and enhanced Treg cells in the allograft, moreover the expression of anti-inflammatory cytokines TGF-β1 and IL-10 unregulated which can reduce the immunoreactions. These findings suggest that S1P1 selective agonist may be a more appropriate immunosuppressive compound to effectively prolong mouse allogeneic corneal grafts survival. PMID:25216235

  4. Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells

    PubMed Central

    Kim, Ji Tae; Li, Jing; Song, Jun; Lee, Eun Y.; Weiss, Heidi L.; Townsend, Courtney M.; Evers, B. Mark

    2015-01-01

    Neurotensin (NTS), localized predominantly to the small bowel, stimulates the growth of a variety of cancers, including neuroendocrine tumors (NETs), mainly through its interaction with the high-affinity NTS receptor 1 (NTSR1). Here, we observed increased expression of NTSR1 in almost all tested clinical NET samples, but not in normal tissues. Through RT-PCR analysis, we found that the expression of NTSR1 and NTSR2 was either variable (NTSR1) or absent (NTSR2) in human NET cell lines. In contrast, NTSR3 and NTS were expressed in all NET cells. Treatment with 5-aza-2′-deoxycytidine, a demethylating agent, increased levels of NTSR1 and NTSR2 suggesting that DNA methylation contributes to NTSR1/2 expression patterns, which was confirmed by methylation analyses. In addition, we found that knockdown of NTSR1 decreased proliferation, expression levels of growth-related proteins, and anchorage-independent growth of BON human carcinoid cells. Moreover, stable silencing of NTSR1 suppressed BON cell growth, adhesion, migration and invasion. Our results show that high expression of NTSR1 is found in clinical NETs and that promoter methylation is an important mechanism controlling the differential expression of NTSR1 and silencing of NTSR2 in NET cells. Furthermore, knockdown of NTSR1 in BON cells suppressed oncogenic functions suggesting that NTSR1 contributes to NET tumorigenesis. PMID:26298774

  5. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival

    PubMed Central

    Rice, Dennis S.; Calandria, Jorgelina M.; Gordon, William C.; Jun, Bokkyoo; Zhou, Yongdong; Gelfman, Claire M.; Li, Songhua; Jin, Minghao; Knott, Eric J.; Chang, Bo; Abuin, Alex; Issa, Tawfik; Potter, David; Platt, Kenneth A.; Bazan, Nicolas G.

    2015-01-01

    The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity. PMID:25736573

  6. Synthesis and evaluation of novel angiotensin II receptor 1 antagonists as anti-hypertension drugs.

    PubMed

    Bao, Xiaolu; Zhu, Weibo; Zhang, Ruijing; Wen, Caihong; Wang, Li; Yan, Yijia; Tang, Hesheng; Chen, Zhilong

    2016-05-01

    Three new angiotensin II receptor 1 antagonists, 1, 2 and 3 were designed, synthesized and evaluated. The AT1 receptor-binding assays in vitro showed that all the synthesized compounds had nanomolar affinity for the AT1 receptor. From which compound 3 was found to be the most potent ligands with an IC50 value of 2.67±0.23 nM. Biological evaluation in vivo revealed that all the compounds could cause significant decrease on MBP in a dose dependent manner in spontaneously hypertensive rats, and compound 3 especially showed an efficient and long-lasting effect in reducing blood pressure, whose maximal response lowered 41 mmHg of MBP at 10mg/kg and 62 mmHg at 15 mg/kg after oral administration, the significant anti-hypertensive effect lasted beyond 12 h, which is better than the reference compound losartan. The pharmacokinetic experiments showed that compound 3 could be absorbed efficiently and metabolized smoothly both in blood and in tissues in Wistar rats. The acute toxicity assay suggested that it has low toxicity with the LD50 value of 2974.35 mg/kg. These results demonstrate that compound 3 is a potent angiotensin AT1 receptor antagonist which could be considered as a novel anti-hypertension candidate and deserved for further investigation.

  7. Relationship between estrogen receptor 1 gene polymorphisms and postmenopausal osteoporosis of the spine in Chinese women.

    PubMed

    Shang, D P; Lian, H Y; Fu, D P; Wu, J; Hou, S S; Lu, J M

    2016-06-03

    The purpose of this study was to evaluate single nucleotide polymorphism (SNP) variants of the estrogen receptor 1 gene (ESR1) at rs2234693 and rs9340799, as well as to investigate the relationship between ESR gene polymorphisms and postmenopausal osteoporosis (OP) of the spine in Chinese women. We recruited 198 postmenopausal women with OP and 276 healthy women between May 2012 and September 2015 in Zhongshan Hospital. Dual energy x-ray absorptiometry was used to measure the bone mineral density (BMD) of the lumbar vertebrae in all subjects. In addition, PCR-restriction fragment length polymorphism based analysis was conducted to identify the genotypes of ESR1. The distribution of ESR1 in the osteoporosis group and the control group was determined; the relationship between ESR polymorphisms and BMD was analyzed. The distributions of BMD were: TT < TC < CC, GG < AG < AA. The TT, TTGG, and TCGG genotypes were found to be lower as compared to the other genotypes. Stratified analysis suggested that the TT genotype and the combined genotypes TTGG and TCGG were significantly higher in the OP group as compared to the control group (P < 0.01). Therefore, ESR1 polymorphisms at rs2234693 and rs9340799 may be associated with OP, and could be used as markers to screen those with high risks to postmenopausal OP in Chinese women.

  8. Expression of Adiponectin Receptor-1 and Prognosis of Epithelial Ovarian Cancer Patients

    PubMed Central

    Li, Xiahui; Yu, Zhe; Fang, Liping; Liu, Fang; Jiang, Kui

    2017-01-01

    Background Adiponectin receptor-1 (AdipoR1) has been reported to be associated with the risk of obesity-associated malignancies, including epithelial ovarian cancer (EOC). The aim of this study was to determine if AdipoR1 could serve as a prognosis indicator for patients with EOC. Material/Methods In this study, expression of AdipoR1 in 73 EOC patients consecutively admitted to our hospital was detected by immunohistochemical staining. Univariate and multivariate analyses were performed to assess the relationship between AdipoR1 expression level and progression-free survival (PFS) and overall survival (OS) rates in patients. Results A relatively lower expression of AdipoR1 in the cancerous tissues was detected compared to normal ovarian tissues, but the difference was not significant (p>0.05). AdipoR1 expression level in EOC patients was negatively correlated with advanced FIGO stages in patients and tumor differentiation, but had no correlation with pathological types, presenting of ascites, shorter platinum-free interval (PFI), diabetes, preoperative and postoperative body mass index (BMI), or platelet counts (p>0.05). Moreover, patients with AdipoR1 expression had a significantly longer PFS and OS compared to the negative expression group (p<0.001). Conclusions Our findings suggest that AdipoR1 expression level in cancerous tissues might serve as an independent prognostic indicator in EOC patients and is associated with longer PFS and OS. PMID:28356549

  9. Angiotensin II receptor 1 gene variants are associated with high-altitude pulmonary edema risk

    PubMed Central

    Zhu, Xikai; Li, Xun; Ouyang, Yongri; He, Xue; Zhang, Zhiying; Zhang, Yuan; Kang, Longli; Yuan, Dongya

    2016-01-01

    Previous studies demonstrated that Angiotensin II Receptor 1 (AGTR1) may play an important role in the development of high-altitude pulmonary edema. We envisaged a role for AGTR1 gene variants in the pathogenesis of HAPE and investigated their potential associations with HAPE in a Han Chinese population. We genotyped seven AGTR1 polymorphisms in 267 patients with diagnosed HAPE and 304 controls and evaluated their association with risk of HAPE. Statistically significant associations were found for the single nucleotide polymorphisms (SNPs) rs275651 (p = 0.017; odds ratio [OR] = 0.65) and rs275652 (p = 0.016; OR = 0.64). Another SNP rs10941679 showed a marginally significant association after adjusting for age and sex in the additive genetic model (adjusted OR = 1.44, 95% CI = 1.01-2.04, p = 0.040). Haplotype analysis confirmed that the haplotype “AG” was associated with a 35% reduction in the risk of developing HAPE, while the haplotype “AA” increased the risk of developing HAPE by 44%. These results provide the first evidence linking genetic variations in AGTR1 with HAPE risk in Han Chinese individuals. PMID:27732943

  10. A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

    PubMed

    Jabara, Haifa H; Boyden, Steven E; Chou, Janet; Ramesh, Narayanaswamy; Massaad, Michel J; Benson, Halli; Bainter, Wayne; Fraulino, David; Rahimov, Fedik; Sieff, Colin; Liu, Zhi-Jian; Alshemmari, Salem H; Al-Ramadi, Basel K; Al-Dhekri, Hasan; Arnaout, Rand; Abu-Shukair, Mohammad; Vatsayan, Anant; Silver, Eli; Ahuja, Sanjay; Davies, E Graham; Sola-Visner, Martha; Ohsumi, Toshiro K; Andrews, Nancy C; Notarangelo, Luigi D; Fleming, Mark D; Al-Herz, Waleed; Kunkel, Louis M; Geha, Raif S

    2016-01-01

    Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

  11. Artificial selection of the melanocortin receptor 1 gene in Chinese domestic pigs during domestication.

    PubMed

    Li, J; Yang, H; Li, J-r; Li, H-p; Ning, T; Pan, X-R; Shi, P; Zhang, Y-P

    2010-09-01

    Black coat colour is common in Chinese indigenous domestic pigs, but not among their wild ancestors, and it is thus presumed to be a 'domestication trait.' To determine whether artificial interference contributes to morphological diversification, we examined nucleotide variation from 157 Chinese domestic pigs and 40 wild boars in the melanocortin receptor 1 (MC1R) gene, which has a key role in the coat pigmentation of Sus scrofa. Compared with a pseudogene GPIP, our results showed that the joint effects of demography and selection have resulted in markedly low genetic diversity of MC1R in Chinese domestic pigs. Coalescent simulation and selection tests further suggest that the fixation of two non-synonymous substitutions associated with black colour is the result of artificial selection. In contrast, a much higher genetic diversity and only a single non-synonymous substitution were found among the wild boars, suggesting a strong functional constraint. Moreover, our conclusion is consistent with the preference for black colour in the ancient Chinese sacrificial culture. This case provides an interesting example of a molecular evaluation of artificial livestock selection and its associated cultural impact in ancient China.

  12. [Circadian changes of the density of melatonin receptors 1A in the neurons of the suprachiasmatic nuclei of the rat hypothalamus under conditions of diverse functional activiity of the pineal gland].

    PubMed

    Pishak, V P; Bulyk, R Ie

    2008-01-01

    An immunohistochemical study of the density of melatonin receptors 1A in the neurons of the rat suprachiasmatic nuclei with diverse functional activity of the pineal gland has been carried out. The density of melatonin receptors 1A under conditions of the physiological function of the pineal gland was characterized by clear-cut diurnal variations. Simultaneously, a dysfunction of the gland results in their marked disturbance. In case of a hypofunction of the pineal body the density of the structures was reliably lower than in case of hyperfunction. It has been demonstrated that in case of a suppressed activity of the pineal body the maximum number of melatonin receptors 1A in the neurons of the hypothalamic suprachiasmatic nuclei shifts from 02.00 a.m. to 02.00 p.m. and constitutes 0.35+/-0.012 conventional units (c.u.) of density, whereas a larger index is noticed at 20 hours making up 0.43+/-0.015 c.u. of density when the gland is activated.

  13. Possible role of rare variants in Trace amine associated receptor 1 in schizophrenia.

    PubMed

    John, Jibin; Kukshal, Prachi; Bhatia, Triptish; Chowdari, K V; Nimgaonkar, V L; Deshpande, S N; Thelma, B K

    2017-02-24

    Schizophrenia (SZ) is a chronic mental illness with behavioral abnormalities. Recent common variant based genome wide association studies and rare variant detection using next generation sequencing approaches have identified numerous variants that confer risk for SZ, but etiology remains unclear propelling continuing investigations. Using whole exome sequencing, we identified a rare heterozygous variant (c.545G>T; p.Cys182Phe) in Trace amine associated receptor 1 gene (TAAR1 6q23.2) in three affected members in a small SZ family. The variant predicted to be damaging by 15 prediction tools, causes breakage of a conserved disulfide bond in this G-protein-coupled receptor. On screening this intronless gene for additional variant(s) in ~800 sporadic SZ patients, we identified six rare protein altering variants (MAF<0.001) namely p.Ser47Cys, p.Phe51Leu, p.Tyr294Ter, p.Leu295Ser in four unrelated north Indian cases (n=475); p.Ala109Thr and p.Val250Ala in two independent Caucasian/African-American patients (n=310). Five of these variants were also predicted to be damaging. Besides, a rare synonymous variant was observed in SZ patients. These rare variants were absent in north Indian healthy controls (n=410) but significantly enriched in patients (p=0.036). Conversely, three common coding SNPs (rs8192621, rs8192620 and rs8192619) and a promoter SNP (rs60266355) tested for association with SZ in the north Indian cohort were not significant (P>0.05). TAAR1 is a modulator of monoaminergic pathways and interacts with AKT signaling pathways. Substantial animal model based pharmacological and functional data implying its relevance in SZ are also available. However, this is the first report suggestive of the likely contribution of rare variants in this gene to SZ.

  14. Antibodies to inositol 1,4,5-triphosphate receptor 1 in patients with cerebellar disease

    PubMed Central

    Fouka, Penelope; Alexopoulos, Harry; Chatzi, Ioanna; Dedos, Skarlatos G.; Samiotaki, Martina; Panayotou, George; Politis, Panagiotis; Tzioufas, Athanasios

    2016-01-01

    Objective: To describe newly identified autoantibodies associated with cerebellar disorders. Design/Methods: We first screened the sera of 15 patients with cerebellar ataxia, without any known associated autoantibodies, with immunocytochemistry on mouse brain. After characterization and validation of a newly identified antibody, 85 additional patients with suspected autoimmune cerebellar disease were screened using a cell-based assay. Results: Immunoglobulin G from one of the first 15 patients demonstrated a distinct staining pattern on Purkinje neurons. This autoantibody, as characterized further by immunoprecipitation and mass spectrometry, was binding inositol 1,4,5-triphosphate receptor 1 (IP3R1), an intracellular channel that mediates the release of Ca2+ from intracellular stores. Anti-IP3R1 specificity was then validated with a cell-based assay. On this basis, screening of 85 other patients with cerebellar disease revealed 2 additional IP3R1-positive patients. All 3 patients presented with cerebellar ataxia; the first was eventually diagnosed with primary progressive multiple sclerosis, the second had a homozygous CAG insertion at the gene TBP, and the third was thought to have a neurodegenerative disease. Conclusions: We independently identified an autoantibody against IP3R1, a protein highly expressed in Purkinje neurons, confirming an earlier report. Because a mouse knockout model for IP3R1 exhibits ataxia and epilepsy, this autoantibody may have a functional role. The heterogeneity of the antibody-positive patients suggests that this antibody may either have a direct involvement in disease pathogenesis or it is a surrogate marker secondary to cerebellar injury. Anti-IP3R1 antibodies should be further explored in various ataxic and epileptic syndromes as they may denote a marker of response to immunotherapies. PMID:27957507

  15. Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction.

    PubMed

    Fisch, Adam S; Yerges-Armstrong, Laura M; Backman, Joshua D; Wang, Hong; Donnelly, Patrick; Ryan, Kathleen A; Parihar, Ankita; Pavlovich, Mary A; Mitchell, Braxton D; O'Connell, Jeffrey R; Herzog, William; Harman, Christopher R; Wren, Jonathan D; Lewis, Joshua P

    2015-01-01

    Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.

  16. Trace Amine-Associated Receptor 1 Regulation of Methamphetamine Intake and Related Traits

    PubMed Central

    Harkness, John H; Shi, Xiao; Janowsky, Aaron; Phillips, Tamara J

    2015-01-01

    Continued methamphetamine (MA) use is dependent on a positive MA experience and is likely attenuated by sensitivity to the aversive effects of MA. Bidirectional selective breeding of mice for high (MAHDR) or low (MALDR) voluntary consumption of MA demonstrates a genetic influence on MA intake. Quantitative trait locus (QTL) mapping identified a QTL on mouse chromosome 10 that accounts for greater than 50% of the genetically-determined differences in MA intake in the MAHDR and MALDR lines. The trace amine-associated receptor 1 gene (Taar1) is within the confidence interval of the QTL and encodes a receptor (TAAR1) that modulates monoamine neurotransmission and at which MA serves as an agonist. We demonstrate the existence of a non-functional allele of Taar1 in the DBA/2J mouse strain, one of the founder strains of the selected lines, and show that this non-functional allele co-segregates with high MA drinking and with reduced sensitivity to MA-induced conditioned taste aversion (CTA) and hypothermia. The functional Taar1 allele, derived from the other founder strain, C57BL/6J, segregates with low MA drinking and heightened sensitivity to MA-induced CTA and hypothermia. A role for TAAR1 in these phenotypes is corroborated in Taar1 transgenic mice: Taar1 knockout mice consume more MA and exhibit insensitivity to MA-induced CTA and hypothermia, compared with Taar1 wild-type mice. These are the first data to show that voluntary MA consumption is, in part, regulated by TAAR1 function. Behavioral and physiological studies indicate that TAAR1 function increases sensitivity to aversive effects of MA, and may thereby protect against MA use. PMID:25740289

  17. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants.

    PubMed

    Heidenreich, Regina; Machein, Marcia; Nicolaus, Anke; Hilbig, Andreas; Wild, Carola; Clauss, Matthias; Plate, Karl H; Breier, Georg

    2004-09-01

    Vascular endothelial growth factor (VEGF) and the high-affinity VEGF receptor Flk-1/KDR (VEGFR-2) are key regulators of tumor angiogenesis. Strategies to block VEGF/VEGFR-2 signaling were successfully used to inhibit experimental tumor growth and indicated that VEGFR-2 is the main signaling VEGF receptor in proliferating tumor endothelium. Here, we investigated the role of the VEGF receptor-1 (VEGFR-1/Flt-1) in the vascularization of 2 different experimental tumors in vivo. VEGFR-1 mutants were generated that lack the intracellular tyrosine kinase domain. Retrovirus-mediated gene transfer of the VEGFR-1 mutants led to a strong reduction of tumor growth and angiogenesis in xenografted C6 glioma and in syngeneic BFS-1 fibrosarcoma. Histological analysis of the inhibited fibrosarcoma revealed reduced vascular density, decreased tumor cell proliferation as well as increased tumor cell apoptosis and the formation of necrosis. The retroviral gene transfer of the full length VEGFR-1 also caused a significant reduction of tumor growth in both models. The inhibitory effects of the VEGFR-1 mutants and the full length VEGFR-1 in BFS-1 fibrosarcoma were mediated through host tumor endothelial cells because the BFS-1 fibrosarcoma cells were not infected by the retrovirus. The formation of heterodimers between VEGFR-2 and full length or truncated VEGFR-1 was observed in vitro and might contribute to the growth inhibitory effect by modulating distinct signal transduction pathways. The results of our study underline the central role of the VEGF/VEGFR-1 signaling system in tumor angiogenesis and demonstrate that VEGFR-1 can serve as a target for anti-angiogenic gene therapy.

  18. Murine complement receptor 1 is required for germinal center B cell maintenance but not initiation.

    PubMed

    Donius, Luke R; Weis, Janis J; Weis, John H

    2014-06-01

    Germinal centers are the anatomic sites for the generation of high affinity immunoglobulin expressing plasma cells and memory B cells. The germinal center B cells that are precursors of these cells circulate between the light zone B cell population that interact with antigen laden follicular dendritic cells (FDC) and the proliferative dark zone B cell population. Antigen retention by follicular dendritic cells is dependent on Fc receptors and complement receptors, and complement receptor 1 (Cr1) is the predominant complement receptor expressed by FDC. The newly created Cr1KO mouse was used to test the effect of Cr1-deficiency on the kinetics of the germinal center reaction and the generation of IgM and switched memory B cell formation. Immunization of Cr1KO mice with a T cell-dependent antigen resulted in the normal initial expansion of B cells with a germinal center phenotype however these cells were preferentially lost in the Cr1KO animal over time (days). Bone marrow chimera animals documented the surprising finding that the loss of germinal center B cell maintenance was linked to the expression of Cr1 on B cells, not the FDC. Cr1-deficiency further resulted in antigen-specific IgM titer and IgM memory B cell reductions, but not antigen-specific IgG after 35-37 days. Investigations of nitrophenyl (NP)-specific IgG demonstrated that Cr1 is not necessary for affinity maturation during the response to particulate antigen. These data, along with those generated in our initial description of the Cr1KO animal describe unique functions of Cr1 on the surface of both B cells and FDC.

  19. Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells*

    PubMed Central

    Mizutani, Taketoshi; Ishizaka, Aya; Nihei, Coh-ichi

    2016-01-01

    As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS. PMID:26637351

  20. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer

    PubMed Central

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  1. Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction

    PubMed Central

    Fisch, Adam S.; Yerges-Armstrong, Laura M.; Backman, Joshua D.; Wang, Hong; Donnelly, Patrick; Ryan, Kathleen A.; Parihar, Ankita; Pavlovich, Mary A.; Mitchell, Braxton D.; O’Connell, Jeffrey R.; Herzog, William; Harman, Christopher R.; Wren, Jonathan D.; Lewis, Joshua P.

    2015-01-01

    Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease. PMID:26406321

  2. Complement receptor 1 is a sialic acid-independent erythrocyte receptor of Plasmodium falciparum.

    PubMed

    Spadafora, Carmenza; Awandare, Gordon A; Kopydlowski, Karen M; Czege, Jozsef; Moch, J Kathleen; Finberg, Robert W; Tsokos, George C; Stoute, José A

    2010-06-17

    Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.

  3. TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis.

    PubMed

    Duerrschmid, Clemens; Crawford, Jeffrey R; Reineke, Erin; Taffet, George E; Trial, Joann; Entman, Mark L; Haudek, Sandra B

    2013-04-01

    Angiotensin-II (Ang-II) is associated with many conditions involving heart failure and pathologic hypertrophy. Ang-II induces the synthesis of monocyte chemoattractant protein-1 that mediates the uptake of CD34(+)CD45(+) monocytic cells into the heart. These precursor cells differentiate into collagen-producing fibroblasts and are responsible for the Ang-II-induced development of non-adaptive cardiac fibrosis. In this study, we demonstrate that in vitro, using a human monocyte-to-fibroblast differentiation model, Ang-II required the presence of tumor necrosis factor-alpha (TNF) to induce fibroblast maturation from monocytes. In vivo, mice deficient in both TNF receptors did not develop cardiac fibrosis in response to 1week Ang-II infusion. We then subjected mice deficient in either TNF receptor 1 (TNFR1-KO) or TNF receptor 2 (TNFR2-KO) to continuous Ang-II infusion. Compared to wild-type, in TNFR1-KO, but not in TNFR2-KO hearts, collagen deposition was greatly attenuated, and markedly fewer CD34(+)CD45(+) cells were present. Quantitative RT-PCR demonstrated a striking reduction of key fibrosis-related, as well as inflammation-related mRNA expression in Ang-II-treated TNFR1-KO hearts. TNFR1-KO animals also developed less cardiac remodeling, cardiac hypertrophy, and hypertension compared to wild-type and TNFR2-KO in response to Ang-II. Our data suggest that TNF induced Ang-II-dependent cardiac fibrosis by signaling through TNFR1, which enhances the generation of monocytic fibroblast precursors in the heart.

  4. Synthesis of enantiomerically pure [(14) C]-labelled morpholine derivatives for a class of trace amine-associate receptor 1 agonists.

    PubMed

    Edelmann, Martin R; Hartung, Thomas; Trussardi, René; Iding, Hans; Galley, Guido; Pflieger, Philippe; Norcross, Roger D

    2016-12-01

    Various agonists of the trace amine-associate receptor 1, under consideration as potential clinical development candidates, were labelled with carbon-14 for use in preclinical in vitro and in vivo drug metabolism studies. Herein, the [(14) C]-radiosynthesis of 2-phenyl-substituted morpholines 1 is described. After evaluating and optimizing different synthetic routes, 4-iodonitrobenzene 3 was selected as starting material for the 14-step synthesis. Incorporation of carbon-14 into the acetyl moiety allowed a safe and efficient synthesis of [(14) C]-labelled 4-nitroacetophenone 2 in five steps and 38% yield. Further transformation of 2 to the target compounds 1 was achieved in a 9-step synthesis. In a representative example, [(14) C]-labelled 1 was obtained in an overall yield of 11% and was isolated in >99% radiochemical purity and a specific activity of 47 mCi/mmol.

  5. Association of 3'-UTR polymorphisms of the oxidised LDL receptor 1 (OLR1) gene with Alzheimer's disease.

    PubMed

    Lambert, J-C; Luedecking-Zimmer, E; Merrot, S; Hayes, A; Thaker, U; Desai, P; Houzet, A; Hermant, X; Cottel, D; Pritchard, A; Iwatsubo, T; Pasquier, F; Frigard, B; Conneally, P M; Chartier-Harlin, M-C; DeKosky, S T; Lendon, C; Mann, D; Kamboh, M I; Amouyel, P

    2003-06-01

    Although possession of the epsilon 4 allele of the apolipoprotein E gene appears to be an important biological marker for Alzheimer's disease (AD) susceptibility, strong evidence indicates that at least one additional risk gene exists on chromosome 12. Here, we describe an association of the 3'-UTR +1073 C/T polymorphism of the OLR1 (oxidised LDL receptor 1) on chromosome 12 with AD in French sporadic (589 cases and 663 controls) and American familial (230 affected sibs and 143 unaffected sibs) populations. The age and sex adjusted odds ratio between the CC+CT genotypes versus the TT genotypes was 1.56 (p=0.001) in the French sample and 1.92 (p=0.02) in the American sample. Furthermore, we have discovered a new T/A polymorphism two bases upstream of the +1073 C/T polymorphism. This +1071 T/A polymorphism was not associated with the disease, although it may weakly modulate the impact of the +1073 C/T polymorphism. Using 3'-UTR sequence probes, we have observed specific DNA protein binding with nuclear proteins from lymphocyte, astrocytoma, and neuroblastoma cell lines, but not from the microglia cell line. This binding was modified by both the +1071 T/A and +1073 C/T polymorphisms. In addition, a trend was observed between the presence or absence of the +1073 C allele and the level of astrocytic activation in the brain of AD cases. However, Abeta(40), Abeta(42), Abeta total, and Tau loads or the level of microglial cell activation were not modulated by the 3'-UTR OLR1 polymorphisms. Finally, we assessed the impact of these polymorphisms on the level of OLR1 expression in lymphocytes from AD cases compared with controls. The OLR1 expression was significantly lower in AD cases bearing the CC and CT genotypes compared with controls with the same genotypes. In conclusion, our data suggest that genetic variation in the OLR1 gene may modify the risk of AD.

  6. Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase

    PubMed Central

    Schmudde, Inken; Sun, Jing; Vollbrandt, Tillman; König, Peter; Köhl, Jörg

    2017-01-01

    C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma. PMID:28231307

  7. Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b.

    PubMed

    Bednarek, Maria A; MacNeil, Tanya; Tang, Rui; Fong, Tung M; Cabello, M Angeles; Maroto, Marta; Teran, Ana

    2008-06-01

    Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.

  8. Variants of the Adiponectin (ADIPOQ) and Adiponectin Receptor 1 (ADIPOR1) Genes and Colorectal Cancer Risk

    PubMed Central

    Kaklamani, Virginia G.; Wisinski, Kari B.; Sadim, Maureen; Gulden, Cassandra; Do, Albert; Offit, Kenneth; Baron, John A.; Ahsan, Habibul; Mantzoros, Christos; Pasche, Boris

    2008-01-01

    Context Current epidemiological evidence suggests an association between obesity, hyperinsulinemia, and colorectal cancer risk. Adiponectin is a hormone secreted by the adipose tissue, and serum levels are inversely correlated with obesity and hyperinsulinemia. While there is evidence of an association between circulating adiponectin levels and colorectal cancer risk, no association between genes of the adiponectin pathway and colorectal cancer have been reported to date. Objective To determine the association of 10 haplotype-tagging single-nucleotide polymorphisms (SNPs) of the adiponectin (ADIPOQ) and adiponectin receptor 1 (ADIPOR1) genes with colorectal cancer risk. Design, Setting, and Patients Two case-control studies including patients with a diagnosis of colorectal cancer and controls were recruited between 2000 and 2007. Case-control study 1 included a total of 441 patients with a diagnosis of colorectal cancer and 658 controls; both groups were of Ashkenazi Jewish ancestry and from New York, New York. Case-control study 2 included 199 patients with a diagnosis of colorectal cancer and 199 controls from Chicago, Illinois, matched 1:1 for sex, age, and ethnicity. Main Outcome Measures ADIPOQ and ADIPOR1 SNP frequency among cases and controls. Results In study 1, after adjustment for age, sex, and SNPs from the same gene, 3 ADIPOQ SNPs and 1 ADIPOR1 SNP were associated with colorectal cancer risk: rs266729 (adjusted odds ratio [AOR], 0.72; 95% confidence interval [CI], 0.55–0.95) and rs822396 (AOR, 0.37; 95% CI, 0.14–1.00) were associated with decreased risk whereas rs822395 (AOR, 1.76; 95% CI, 1.09–2.84) and rs1342387 (AOR, 1.79; 95% CI, 1.18–2.72) were associated with increased risk. In study 2, after adjustment for age, sex, race, and SNPs from the same gene, the ADIPOQ SNP rs266729 was associated with a decreased colorectal cancer risk of similar magnitude as in study 1 (AOR, 0.52; 95% CI, 0.34–0.78). Combined analysis of both studies shows an

  9. Human and Host Species Transferrin Receptor 1 Use by North American Arenaviruses

    PubMed Central

    Zong, Min; Fofana, Isabel

    2014-01-01

    ABSTRACT At least five New World (NW) arenaviruses cause hemorrhagic fevers in South America. These pathogenic clade B viruses, as well as nonpathogenic arenaviruses of the same clade, use transferrin receptor 1 (TfR1) of their host species to enter cells. Pathogenic viruses are distinguished from closely related nonpathogenic ones by their additional ability to utilize human TfR1 (hTfR1). Here, we investigate the receptor usage of North American arenaviruses, whose entry proteins share greatest similarity with those of the clade B viruses. We show that all six North American arenaviruses investigated utilize host species TfR1 orthologs and present evidence consistent with arenavirus-mediated selection pressure on the TfR1 of the North American arenavirus host species. Notably, one of these viruses, AV96010151, closely related to the prototype Whitewater Arroyo virus (WWAV), entered cells using hTfR1, consistent with a role for a WWAV-like virus in three fatal human infections whose causative agent has not been identified. In addition, modest changes were sufficient to convert hTfR1 into a functional receptor for most of these viruses, suggesting that a minor alteration in virus entry protein may allow these viruses to use hTfR1. Our data establish TfR1 as a cellular receptor for North American arenaviruses, highlight an “arms race” between these viruses and their host species, support the association of North American arenavirus with fatal human infections, and suggest that these viruses have a higher potential to emerge and cause human diseases than has previously been appreciated. IMPORTANCE hTfR1 use is a key determinant for a NW arenavirus to cause hemorrhagic fevers in humans. All known pathogenic NW arenaviruses are transmitted in South America by their host rodents. North American arenaviruses are generally considered nonpathogenic, but some of these viruses have been tentatively implicated in human fatalities. We show that these North American

  10. Dual Role of the Second Extracellular Loop of the Cannabinoid Receptor 1: Ligand Binding and Receptor Localization

    PubMed Central

    Ahn, Kwang H.; Bertalovitz, Alexander C.; Mierke, Dale F.

    2009-01-01

    The seven transmembrane α-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings

  11. Vitamin D-induced ectodomain shedding of TNF receptor 1 as a nongenomic action: D3 vs D2 derivatives.

    PubMed

    Yang, Won Seok; Yu, Hoon; Kim, Jin Ju; Lee, Mee Jeong; Park, Su-Kil

    2016-01-01

    As a nongenomic action, 1,25-dihydroxyvitamin D3 (1,25D3) induces L-type Ca(2+) channel-mediated extracellular Ca(2+) influx in human aortic smooth muscle cells (HASMCs), which activates a disintegrin and metalloprotease 10 (ADAM10) to cleave and shed the ectodomain of tumor necrosis factor receptor 1 (TNFR1). In this study, we examined the potencies of other vitamin D3 and D2 analogs to stimulate the ectodomain shedding of TNFR1 in HASMCs. 25-Hydroxyvitamin D3 (25D3), a precursor of 1,25D3, and elocalcitol, an analog of 1,25D3, caused ectodomain shedding of TNFR1 within 30 min, whereas 1,25-dihydroxyvitamin D2 (1,25D2) and paricalcitol, a derivative of 1,25D2, did not. Both 25D3 and elocalcitol rapidly induced extracellular Ca(2+) influx and markedly increased intracellular Ca(2+), while 1,25D2 and paricalcitol caused only small increases in intracellular Ca(2+). 25D3- and elocalcitol-induced TNFR1 ectodomain sheddings were abolished by verapamil and in Ca(2+)-free media. Both 25D3 and elocalcitol caused the translocation of ADAM10 to the cell surface, which was inhibited by verapamil, while 1,25D2 and paricalcitol did not cause ADAM10 translocation. When ADAM10 was depleted by ADAM10-siRNA, 25D3 and elocalcitol could not induce ectodomain shedding of TNFR1. The plasma membrane receptor, endoplasmic reticulum stress protein 57 (ERp57), but not the classic vitamin D receptor, mediated the nongenomic action of vitamin D to induce ectodomain shedding of TNFR1. In summary, like 1,25D3, 25D3 and elocalcitol caused ADAM10-mediated ectodomain shedding of TNFR1, whereas 1,25D2 and paricalcitol did not. The difference may depend on their affinities to ERp57 through which extracellular Ca(2+) influx is induced.

  12. Bioactive secondary metabolites of a marine Bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Lin, Chwan-Fwu; Chang, Wen-Yi; Kuo, Jimmy; Huang, Yin-Ting; Chung, Pei-Jen; Hwang, Tsong-Long

    2013-06-03

    It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils.

  13. Sphingosine-1-Phosphate Receptor-1 Selective Agonist Enhances Collateral Growth and Protects against Subsequent Stroke

    PubMed Central

    Ichijo, Masahiko; Ishibashi, Satoru; Li, Fuying; Yui, Daishi; Miki, Kazunori; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    Background and Purpose Collateral growth after acute occlusion of an intracranial artery is triggered by increasing shear stress in preexisting collateral pathways. Recently, sphingosine-1-phosphate receptor-1 (S1PR1) on endothelial cells was reported to be essential in sensing fluid shear stress. Here, we evaluated the expression of S1PR1 in the hypoperfused mouse brain and investigated the effect of a selective S1PR1 agonist on leptomeningeal collateral growth and subsequent ischemic damage after focal ischemia. Methods In C57Bl/6 mice (n = 133) subjected to unilateral common carotid occlusion (CCAO) and sham surgery. The first series examined the time course of collateral growth, cell proliferation, and S1PR1 expression in the leptomeningeal arteries after CCAO. The second series examined the relationship between pharmacological regulation of S1PR1 and collateral growth of leptomeningeal anastomoses. Animals were randomly assigned to one of the following groups: LtCCAO and daily intraperitoneal (ip) injection for 7 days of an S1PR1 selective agonist (SEW2871, 5 mg/kg/day); sham surgery and daily ip injection for 7 days of SEW2871 after surgery; LtCCAO and daily ip injection for 7 days of SEW2871 and an S1PR1 inverse agonist (VPC23019, 0.5 mg/kg); LtCCAO and daily ip injection of DMSO for 7 days after surgery; and sham surgery and daily ip injection of DMSO for 7 days. Leptomeningeal anastomoses were visualized 14 days after LtCCAO by latex perfusion method, and a set of animals underwent subsequent permanent middle cerebral artery occlusion (pMCAO) 7days after the treatment termination. Neurological functions 1hour, 1, 4, and 7days and infarction volume 7days after pMCAO were evaluated. Results In parallel with the increase in S1PR1 mRNA levels, S1PR1 expression colocalized with endothelial cell markers in the leptomeningeal arteries, increased markedly on the side of the CCAO, and peaked 7 days after CCAO. Mitotic cell numbers in the leptomeningeal arteries

  14. Design, synthesis and evaluation of novel potent angiotensin II receptor 1 antagonists.

    PubMed

    Bao, Xiaolu; Zhu, Weibo; Yuan, Weidong; Zhu, Xingbo; Yan, Yijia; Tang, Hesheng; Chen, Zhilong

    2016-11-10

    A series of new angiotensin II (Ang II) receptor 1 antagonists were designed, synthesized and evaluated. All compounds showed nanomolar affinities for the angiotensin II type 1 receptor in radioligand binding assays and could reduce blood pressure significantly in spontaneously hypertensive rats(SHRs). From which, compound 2b displayed higher affinity binding to angiotensin II type 1 receptor at the same order of magnitude to irbesartan with an IC50 value of 1.26 ± 0.08 nM in radioligand binding assays. 2b showed an efficient and long-lasting effect in reducing blood pressure, the maximal reducing responses were 40.62 ± 4.08 mmHg of MBP at 15 mg/kg and 28.39 ± 2.09 mmHg at 10 mg/kg in SHRs, 39.56 ± 4.83 mmHg at 15 mg/kg and 29.05 ± 2.20 mmHg at 10 mg/kg in RHRs, the significant antihypertensive effect lasted beyond 12 h both in SHRs and in RHRs. In the single-dose pharmacokinetic experiments, compound 2b could be absorbed efficiently and metabolized smoothly in Wistar rats after oral administration. The values of Cmax, Tmax, AUC0-72 and MRT0-72 were 885.61 ± 432.7 ng/mL, 5.67 ± 1.51 h, 6110.28 ± 7398.33 ng/mL h and 7.87 ± 2.30 h at 10 mg/kg, 2945.55 ± 1543.67 ng/mL, 4.33 ± 0.82 h, 26473.62 ± 12217.16 ng/mL h and 10.24 ± 6.94 h at 15 mg/kg, 5759.03 ± 1331.75 ng/mL, 5 ± 1.10 h, 89488.44 ± 18413.15 ng/mL·h and 12.89 ± 2.0 h at 30 mg/kg respectively. The T1/2 values of the three groups were similar, about 9-10 h. Compound 2b was distributed into tissues rapidly and extensively after oral administration. The level of it was the highest in the liver, followed by in spleen, kidney, and the lowest in brain. The acute toxicity assays of 2b proved its low acute toxicity with an LD50 value of 1551.71 mg/kg, and no toxicity reaction appeared at dose of 1200.00 mg/kg. These encouraging results make compound 2b an effective, long-lasting and safe anti-hypertensive drug candidate and worthy of

  15. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells.

    PubMed

    He, Kai-Li; Ting, Adrian T

    2002-09-01

    Tumor necrosis factor receptor 1 (TNFR1) can trigger distinct signaling pathways leading to either the activation of NF-kappaB transcription factors or apoptosis. NF-kappaB activation results in the expression of antiapoptotic genes that inhibit the apoptosis pathway that is activated in parallel. However, the molecular mechanism of this inhibition remains poorly characterized. We have isolated a Jurkat T-cell mutant that exhibits enhanced sensitivity to TNF-induced apoptosis as a result of a deficiency in I-kappaB kinase gamma (IKKgamma)/NEMO, an essential component of the IKK complex and NF-kappaB pathway. We show here that the zinc finger protein A20 is an NF-kappaB-inducible gene that can protect the IKKgamma-deficient cells from TNF-induced apoptosis by disrupting the recruitment of the death domain signaling molecules TRADD and RIP to the receptor signaling complex. Our study, together with reports on the role of other antiapoptotic proteins such as c-FLIP and c-IAP, suggests that, in order to ensure an effective shutdown of the apoptotic pathway, TNF induces multiple NF-kappaB-dependent genes that inhibit successive steps in the TNFR1 death signaling pathway.

  16. Cannabinoids modulate Olig2 and polysialylated neural cell adhesion molecule expression in the subventricular zone of post-natal rats through cannabinoid receptor 1 and cannabinoid receptor 2.

    PubMed

    Arévalo-Martín, Angel; García-Ovejero, Daniel; Rubio-Araiz, Ana; Gómez, Oscar; Molina-Holgado, Francisco; Molina-Holgado, Eduardo

    2007-09-01

    The subventricular zone (SVZ) is a source of post-natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the post-natal SVZ ependymocytes, radial glia and astrocyte-like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post-natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl-2-chloroethylamide and JWH-056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2-positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post-natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter.

  17. Integration of modeling with experimental and clinical findings synthesizes and refines the central role of inositol 1,4,5-trisphosphate receptor 1 in spinocerebellar ataxia

    PubMed Central

    Brown, Sherry-Ann; Loew, Leslie M.

    2015-01-01

    A suite of models was developed to study the role of inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in spinocerebellar ataxias (SCAs). Several SCAs are linked to reduced abundance of IP3R1 or to supranormal sensitivity of the receptor to activation by its ligand inositol 1,4,5-trisphosphate (IP3). Detailed multidimensional models have been created to simulate biochemical calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons. In these models, IP3R1-mediated calcium release is allowed to interact with ion channel response on the cell membrane. Experimental findings in mice and clinical observations in humans provide data input for the models. The SCA modeling suite helps interpret experimental results and provides suggestions to guide experiments. The models predict IP3R1 supersensitivity in SCA1 and compensatory mechanisms in SCA1, SCA2, and SCA3. Simulations explain the impact of calcium buffer proteins. Results show that IP3R1-mediated calcium release activates voltage-gated calcium-activated potassium channels in the plasma membrane. The SCA modeling suite unifies observations from experiments in a number of SCAs. The cadre of simulations demonstrates the central role of IP3R1. PMID:25653583

  18. Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b*

    PubMed Central

    Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A.; Hassan, Ali B.; Giles, Jennifer

    2015-01-01

    The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca2+-ATPase (PMCA) is essential for removal of cytoplasmic Ca2+ and for shaping the time courses of Ca2+-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca2+ extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca2+ signaling and GPER/GPR30-mediated activities. PMID:25847233

  19. Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

    PubMed

    Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

    2015-05-22

    The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities.

  20. Peroxisome proliferator-activated receptor gamma (PPARG) modulates free fatty acid receptor 1 (FFAR1) dependent insulin secretion in humans

    PubMed Central

    Wagner, Robert; Hieronimus, Anja; Lamprinou, Apostolia; Heni, Martin; Hatziagelaki, Erifili; Ullrich, Susanne; Stefan, Norbert; Staiger, Harald; Häring, Hans-Ulrich; Fritsche, Andreas

    2014-01-01

    Genetic variation in FFAR1 modulates insulin secretion dependent on non-esterified fatty acid (NEFA) concentrations. We previously demonstrated lower insulin secretion in minor allele carriers of PPARG Pro12Ala in high-NEFA environment, but the mode of action could not been revealed. We tested if this effect is mediated by FFAR1 in humans. Subjects with increased risk of diabetes who underwent oral glucose tolerance tests were genotyped for 7 tagging SNPs in FFAR1 and PPARG Pro12Ala. The FFAR1 SNPs rs12462800 and rs10422744 demonstrated interactions with PPARG on insulin secretion. FFAR1 rs12462800 (p = 0.0006) and rs10422744 (p = 0.001) were associated with reduced insulin secretion in participants concomitantly carrying the PPARG minor allele and having high fasting FFA. These results suggest that the minor allele of the PPARG SNP exposes its carriers to modulatory effects of FFAR1 on insulin secretion. This subphenotype may define altered responsiveness to FFAR1-agonists, and should be investigated in further studies. PMID:25161890

  1. Enkephalin levels and the number of neuropeptide Y-containing interneurons in the hippocampus are decreased in female cannabinoid-receptor 1 knock-out mice.

    PubMed

    Rogers, Sophie A; Kempen, Tracey A Van; Pickel, Virginia M; Milner, Teresa A

    2016-05-04

    Drug addiction requires learning and memory processes that are facilitated by activation of cannabinoid-1 (CB1) and opioid receptors in the hippocampus. This involves activity-dependent synaptic plasticity that is partially regulated by endogenous opioid (enkephalin and dynorphin) and non-opioid peptides, specifically cholecystokinin, parvalbumin and neuropeptide Y, the neuropeptides present in inhibitory interneurons that co-express CB1 or selective opioid receptors. We tested the hypothesis that CB1 receptor expression is a determinant of the availability of one or more of these peptide modulators in the hippocampus. This was achieved by quantitatively analyzing the immunoperoxidase labeling for each of these neuropeptide in the dorsal hippocampus of female wild-type (CB1+/+) and cannabinoid receptor 1 knockout (CB1-/-) C57/BL6 mice. The levels of Leu(5)-enkephalin-immunoreactivity were significantly reduced in the hilus of the dentate gyrus and in stratum lucidum of CA3 in CB1-/- mice. Moreover, the numbers of neuropeptide Y-immunoreactive interneurons in the dentate hilus were significantly lower in the CB1-/- compared to wild-type mice. However, CB1+/+ and CB1-/- mice did not significantly differ in expression levels of either dynorphin or cholecystokinin, and showed no differences in numbers of parvalbumin-containing interneurons. These findings suggest that the cannabinoid and opioid systems have a nuanced, regulatory relationship that could affect the balance of excitation and inhibition in the hippocampus and thus processes such as learning that rely on this balance.

  2. A synthetic peptide derived from A1 module in CRD4 of human TNF receptor-1 inhibits binding and proinflammatory effect of human TNF-alpha.

    PubMed

    Cao, Yingnan; Wang, Zhaohe; Bu, Xianzhang; Tang, Shu; Mei, Zhengrong; Liu, Peiqing

    2009-06-01

    Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Proinflammatory effect of TNF-alpha is activated mainly through human TNF receptor-1 (TNF-R1). However, the role of the fourth cystein-rich domain (CRD4) of TNF-R1 extracellular portion in the interaction of TNF-alpha with TNF-R1 is still unclear. In the present study, binding activity of TNF-alpha to TNF-R1 and protein levels of IkappaB-alpha and nuclear transcription factor kappa B (NF-kappaB) p65 subunit in HeLa cells were measured using enzyme-linked immunosorbent assay (ELISA) and western-blot analysis. Pep 3 (LRENECVS) which was derived from the hydrophilic region of A1 module in CRD4 remarkably inhibited the binding of TNF-alpha to TNF-R1, and also reversed TNF-alpha-induced degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit in HeLa cells. Our results confirmed that the hydrophilic region of A1 module in CRD4 participated in the interaction of TNF-alpha with TNF-R1, and demonstrated the potential of small-molecule TNF-alpha extracellular inhibitors targeting at A1 module in CRD4 of TNF-R1 in suppressing proinflammatory effect of TNF-alpha.

  3. A new strategy to improve the predictive ability of the local lazy regression and its application to the QSAR study of melanin-concentrating hormone receptor 1 antagonists.

    PubMed

    Li, Jiazhong; Li, Shuyan; Lei, Beilei; Liu, Huanxiang; Yao, Xiaojun; Liu, Mancang; Gramatica, Paola

    2010-04-15

    In the quantitative structure-activity relationship (QSAR) study, local lazy regression (LLR) can predict the activity of a query molecule by using the information of its local neighborhood without need to produce QSAR models a priori. When a prediction is required for a query compound, a set of local models including different number of nearest neighbors are identified. The leave-one-out cross-validation (LOO-CV) procedure is usually used to assess the prediction ability of each model, and the model giving the lowest LOO-CV error or highest LOO-CV correlation coefficient is chosen as the best model. However, it has been proved that the good statistical value from LOO cross-validation appears to be the necessary, but not the sufficient condition for the model to have a high predictive power. In this work, a new strategy is proposed to improve the predictive ability of LLR models and to access the accuracy of a query prediction. The bandwidth of k neighbor value for LLR is optimized by considering the predictive ability of local models using an external validation set. This approach was applied to the QSAR study of a series of thienopyrimidinone antagonists of melanin-concentrating hormone receptor 1. The obtained results from the new strategy shows evident improvement compared with the commonly used LOO-CV LLR methods and the traditional global linear model.

  4. Ovarian cancer G protein-coupled receptor 1 is involved in acid-induced apoptosis of endplate chondrocytes in intervertebral discs.

    PubMed

    Yuan, Feng-Lai; Wang, Hui-Ren; Zhao, Ming-Dong; Yuan, Wei; Cao, Lu; Duan, Ping-Guo; Jiang, Yun-Qi; Li, Xi-Lei; Dong, Jian

    2014-01-01

    Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a receptor for protons. We investigated the role of proton-sensing G protein-coupled receptors in the apoptosis of endplate chondrocytes induced by extracellular acid. The expression of proton-sensing G protein-coupled receptors was examined in rat lumbar endplate chondrocytes. Knockdown of OGR1 was achieved by transfecting chondrocytes with specific short hairpin RNA (shRNA) for OGR1. Apoptotic changes were evaluated by DNA fragmentation ELISA, electron microscopy, and flow cytometry. Intracellular calcium ([Ca(2+) ]i) was analyzed with laser scanning confocal microscopy. The mechanism of OGR1 in acid-induced apoptosis of endplate chondrocytes was also investigated. We found that OGR1 was predominantly expressed in rat endplate chondrocytes, and its expression was highly upregulated in response to acidosis. Knocking down OGR1 with shRNAs effectively attenuated acid-induced apoptosis of endplate chondrocytes and increased [Ca(2+) ]i. Blocking OGR1-mediated [Ca(2+) ]i elevation inhibited acid-induced calcium-sensitive proteases such as calpain and calcineurin, and also inhibited the activation of Bid, Bad, and Caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). OGR1-mediated [Ca(2+) ]i elevation has a crucial role in apoptosis of endplate chondrocytes by regulating activation of calcium-sensitive proteases and their downstream signaling.

  5. Administration of orexin receptor 1 antagonist into the rostral ventromedial medulla increased swim stress-induced antinociception in rat

    PubMed Central

    Soliemani, Neda; Moslem, Alireza; Shamsizadeh, Ali; Azhdari-Zarmehri, Hassan

    2016-01-01

    Objective(s): Intracerebroventricular injection of orexin-A (hypocretin-1) antagonist has been shown to inhibit stress-induced analgesia. However the locations of central sites that may mediate these effects have not been totally demonstrated. This study was performed to investigate the role of rostral ventromedial medulla (RVM) orexin receptor 1 in stress-induced analgesia (SIA). Materials and Methods: Forced swim stress in water was employed to adult male rats (200-250 g). Nociceptive responses were measured by formalin test (50 µl injection of formalin 2% subcutaneously into hind paw) and, pain related behaviors were monitored for 90 min following intra-microinjection of SB-334867 (orexin receptor 1 antagonist) into RVM. Results: Exposure to swimming stress test after administration of SB-334867 into RVM significantly reduces the formalin-induced nociceptive behaviors in phase1, interphase, and phase 2 in rats. Conclusion: The result demonstrated the involvement of OXR1 in antinociceptive behaviors induced by swim stress in RVM. PMID:27403261

  6. Human fear acquisition deficits in relation to genetic variants of the corticotropin releasing hormone receptor 1 and the serotonin transporter.

    PubMed

    Heitland, Ivo; Groenink, Lucianne; Bijlsma, Elisabeth Y; Oosting, Ronald S; Baas, Johanna M P

    2013-01-01

    The ability to identify predictors of aversive events allows organisms to appropriately respond to these events, and failure to acquire these fear contingencies can lead to maladaptive contextual anxiety. Recently, preclinical studies demonstrated that the corticotropin-releasing factor and serotonin systems are interactively involved in adaptive fear acquisition. Here, 150 healthy medication-free human subjects completed a cue and context fear conditioning procedure in a virtual reality environment. Fear potentiation of the eyeblink startle reflex (FPS) was measured to assess both uninstructed fear acquisition and instructed fear expression. All participants were genotyped for polymorphisms located within regulatory regions of the corticotropin releasing hormone receptor 1 (CRHR1 - rs878886) and the serotonin transporter (5HTTLPR). These polymorphisms have previously been linked to panic disorder and anxious symptomology and personality, respectively. G-allele carriers of CRHR1 (rs878886) showed no acquisition of fear conditioned responses (FPS) to the threat cue in the uninstructed phase, whereas fear acquisition was present in C/C homozygotes. Moreover, carrying the risk alleles of both rs878886 (G-allele) and 5HTTLPR (short allele) was associated with increased FPS to the threat context during this phase. After explicit instructions regarding the threat contingency were given, the cue FPS and context FPS normalized in all genotype groups. The present results indicate that genetic variability in the corticotropin-releasing hormone receptor 1, especially in interaction with the 5HTTLPR, is involved in the acquisition of fear in humans. This translates prior animal findings to the human realm.

  7. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    SciTech Connect

    Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

    2013-03-29

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

  8. Tumor necrosis factor (TNF)-receptor 1 and 2 mediate homeostatic synaptic plasticity of denervated mouse dentate granule cells

    PubMed Central

    Becker, Denise; Deller, Thomas; Vlachos, Andreas

    2015-01-01

    Neurological diseases are often accompanied by neuronal cell death and subsequent deafferentation of connected brain regions. To study functional changes after denervation we generated entorhino-hippocampal slice cultures, transected the entorhinal pathway, and denervated dentate granule cells in vitro. Our previous work revealed that partially denervated neurons respond to the loss of input with a compensatory, i.e., homeostatic, increase in their excitatory synaptic strength. TNFα maintains this denervation-induced homeostatic strengthening of excitatory synapses. Here, we used pharmacological approaches and mouse genetics to assess the role of TNF-receptor 1 and 2 in lesion-induced excitatory synaptic strengthening. Our experiments disclose that both TNF-receptors are involved in the regulation of denervation-induced synaptic plasticity. In line with this result TNF-receptor 1 and 2 mRNA-levels were upregulated after deafferentation in vitro. These findings implicate TNF-receptor signaling cascades in the regulation of homeostatic plasticity of denervated networks and suggest an important role for TNFα-signaling in the course of neurological diseases accompanied by deafferentation. PMID:26246237

  9. APP, APOE, complement receptor 1, clusterin and PICALM and their involvement in the herpes simplex life cycle.

    PubMed

    Carter, C J

    2010-10-11

    The major Alzheimer's disease susceptibility genes (APOE, clusterin, complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein, PICALM) can be implicated directly (APOE, CR1) or indirectly (clusterin and PICALM) in the herpes simplex life cycle. The virus binds to proteoliposomes containing APOE or APOA1 and also to CR1, and both clusterin and PICALM are related to a mannose-6-phosphate receptor used by the virus for cellular entry and intracellular transport. PICALM also binds to a nuclear exportin used by the virus for nuclear egress. Clusterin and complement receptor 1 are both related to the complement pathways and play a general role in pathogen defence. In addition, the amyloid precursor protein APP is involved in herpes viral transport and gamma-secretase cleaves a number of receptors used by the virus for cellular entry. APOE, APOA1 and clusterin, or alpha 2-macroglobulin, insulysin and caspase 3, which also bind to the virus, are involved in beta-amyloid clearance or degradation, as are the viral binding complement components, C3 and CR1. There are multiple ways in which the products of key susceptibility genes might be able to modify the viral life cycle and in turn the virus interacts with key proteins involved in APP and beta-amyloid processing. These interactions support a role for the herpes simplex virus in Alzheimer's disease pathology and suggest that antiviral agents or vaccination might be considered as viable therapeutic strategies in Alzheimer's disease.

  10. Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)*

    PubMed Central

    Diepenhorst, Natalie A.; Petrie, Emma J.; Chen, Catherine Z.; Wang, Amy; Hossain, Mohammed Akhter; Bathgate, Ross A. D.; Gooley, Paul R.

    2014-01-01

    Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists. PMID:25352603

  11. Trace Amine-Associated Receptor 1 – Family Archetype or Iconoclast?

    PubMed Central

    Grandy, David K.

    2009-01-01

    Interest has recently been rekindled in receptors that are activated by low molecular weight, non-catecholic, biogenic amines that are typically found as trace constituents of various vertebrate and invertebrate tissues and fluids. The timing of this resurgent focus on receptors activated by the ‘trace amines’ (TAs) β-phenylethylamine (PEA), tyramine (TYR), octopamine (OCT), synephrine (SYN), and tryptamine (TRYP) is the direct result of two publications that appeared in 2001 describing the cloning of a novel G protein-coupled receptor (GPCR) referred to by their discoverers as TA1 (Borowsky et al., 2001) and TAR1 (Bunzow et al., 2001). When heterologously expressed in Xenopus laevis oocytes and various eukaryotic cell lines recombinant rodent and human TA receptors dose-dependently couple to the stimulation of cAMP production. Structure-activity profiling based on this functional response has revealed that in addition to the TAs, other biologically active compounds containing a 2 carbon aliphatic side chain linking an amino group to at least one benzene ring are potent and efficacious TA receptor agonists with amphetamine, methamphetamine, 3-iodothyronamine, thyronamine, and dopamine among the most notable. Almost 100 years after the search for TA receptors began numerous TA1/TAR1-related sequences, now called Trace Amine-Associated Receptors (TAARs), have been identified in the genome of every species of vertebrate examined to date. Consequently, even though heterologously expressed TAAR1 fits the pharmacological criteria established for a bona fide TA receptor a major challenge for those working in the field is to discern the in vivo pharmacology and physiology of each purported member of this extended family of GPCRs. Only then will it be possible to establish whether TAAR1 is the family archetype or an iconoclast. PMID:17888514

  12. Effect of soluble complement receptor-1 on neutrophil accumulation after traumatic brain injury in rats.

    PubMed

    Kaczorowski, S L; Schiding, J K; Toth, C A; Kochanek, P M

    1995-09-01

    As part of the acute inflammatory response, neutrophils accumulate in the central nervous system after injury. Recently, a soluble human recombinant complement receptor (sCR1; BRL 55730; T Cell Sciences, Inc., Cambridge, MA, U.S.A.) has been developed that inhibits the activation of both the classical and the alternative pathways of complement. sCR1 attenuates the effects of the acute inflammatory response in several models of injury outside the central nervous system. The role of complement in traumatic brain injury, however, remains undefined. We hypothesized that treatment with sCR1 would attenuate neutrophil accumulation in the brain after cerebral trauma. Using a randomized, blinded protocol, 18 anesthetized Sprague-Dawley rats were pre-treated with sCR1 or saline (control) at both 2 h and 2 min before trauma (weight drop) to the exposed right parietal cortex. A third dose of sCR1 (or saline) was given 6 h after trauma. Coronal brain sections centered on the site of trauma were obtained at 24 h after trauma and analyzed for myeloperoxidase (MPO) activity as a marker of neutrophil accumulation. Complete blood counts with differential were obtained before treatment with sCR1 and at 24 h after trauma. At 24 h after trauma, brain MPO activity was reduced by 41% in sCR1-treated rats compared with control rats [0.1599 +/- 0.102 versus 0.2712 +/- 0.178 U/g (mean +/- SD); p = 0.02]. The neutrophil count in peripheral blood increased approximately twofold in both groups. Neutrophil accumulation occurring in the brain after trauma is inhibited by sCR1 treatment. This suggests that complement activation is involved in the local inflammatory response to traumatic brain injury and plays an important role in neutrophil accumulation in the injured brain.

  13. Potent, selective, and orally efficacious antagonists of melanin-concentrating hormone receptor 1.

    PubMed

    Tavares, Francis X; Al-Barazanji, Kamal A; Bigham, Eric C; Bishop, Michael J; Britt, Christy S; Carlton, David L; Feldman, Paul L; Goetz, Aaron S; Grizzle, Mary K; Guo, Yu C; Handlon, Anthony L; Hertzog, Donald L; Ignar, Diane M; Lang, Daniel G; Ott, Ronda J; Peat, Andrew J; Zhou, Hui-Qiang

    2006-11-30

    The high expression of MCH in the hypothalamus with the lean hypophagic phenotype coupled with increased resting metabolic rate and resistance to high fat diet-induced obesity of MCH KO mice has spurred considerable efforts to develop small molecule MCHR1 antagonists. Starting from a lead thienopyrimidinone series, structure-activity studies at the 3- and 6-positions of the thienopyrimidinone core afforded potent and selective MCHR1 antagonists with representative examples having suitable pharmacokinetic properties. Based on structure-activity relationships, a structural model for MCHR1 was constructed to explain the binding mode of these antagonists. In general, a good correlation was observed between pKas and activity in the right-hand side of the template, with Asp123 playing an important role in the enhancement of binding affinity. A representative example when evaluated chronically in diet-induced obese mice resulted in good weight loss effects. These antagonists provide a viable lead series in the discovery of new therapies for the treatment of obesity.

  14. Soluble Tumor Necrosis Factor Receptor 1 Released by Skin-Derived Mesenchymal Stem Cells Is Critical for Inhibiting Th17 Cell Differentiation

    PubMed Central

    Ke, Fang; Zhang, Lingyun; Liu, Zhaoyuan; Yan, Sha; Xu, Zhenyao; Bai, Jing; Zhu, Huiyuan; Lou, Fangzhou; Cai, Wei; Sun, Yang; Gao, Yuanyuan; Wang, Hong

    2016-01-01

    T helper 17 (Th17) cells play an important role in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Th17 cell differentiation from naïve T cells can be induced in vitro by the cytokines transforming growth factor β1 and interleukin-6. However, it remains unclear whether other regulatory factors control the differentiation of Th17 cells. Mesenchymal stem cells (MSCs) have emerged as a promising candidate for inhibiting Th17 cell differentiation and autoimmune diseases. Despite the fact that several molecules have been linked to the immunomodulatory function of MSCs, many other key MSC-secreted regulators that are involved in inhibiting Th17 cell polarization are ill-defined. In this study, we demonstrated that the intraperitoneal administration of skin-derived MSCs (S-MSCs) substantially ameliorated the development of EAE in mice. We found that the proinflammatory cytokine tumor necrosis factor (TNF)-α, a key mediator in the pathophysiology of MS and EAE, was capable of promoting Th17 cell differentiation. Moreover, under inflammatory conditions, we demonstrated that S-MSCs produced high amounts of soluble TNF receptor 1 (sTNFR1), which binds TNF-α and antagonizes its function. Knockdown of sTNFR1 in S-MSCs decreased their inhibitory effect on Th17 cell differentiation ex vivo and in vivo. Thus, our data identified sTNFR1 and its target TNF-α as critical regulators for Th17 cell differentiation, suggesting a previously unrecognized mechanism for MSC therapy in Th17-mediated autoimmune diseases. Significance This study showed that administration of skin-derived mesenchymal stem cells (S-MSCs) was able to alleviate the clinical score of experimental autoimmune encephalomyelitis by inhibiting the differentiation of T helper 17 (Th17) cells. Tumor necrosis factor (TNF)-α is a critical cytokine for promoting Th17 cell differentiation. It was discovered that activated S-MSCs produced high amount of soluble TNF receptor 1

  15. Intestinal CX3C chemokine receptor 1high (CX3CR1high) myeloid cells prevent T-cell-dependent colitis

    PubMed Central

    Kayama, Hisako; Ueda, Yoshiyasu; Sawa, Yukihisa; Jeon, Seong Gyu; Ma, Ji Su; Okumura, Ryu; Kubo, Atsuko; Ishii, Masaru; Okazaki, Taku; Murakami, Masaaki; Yamamoto, Masahiro; Yagita, Hideo; Takeda, Kiyoshi

    2012-01-01

    Adequate activation of CD4+ T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4+ T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX3C chemokine receptor 1high (CX3CR1high) CD11b+ CD11c+ cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4+ T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX3CR1high CD11b+ CD11c+ cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX3CR1high CD11b+ CD11c+ cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis. PMID:22403066

  16. Intestinal CX3C chemokine receptor 1(high) (CX3CR1(high)) myeloid cells prevent T-cell-dependent colitis.

    PubMed

    Kayama, Hisako; Ueda, Yoshiyasu; Sawa, Yukihisa; Jeon, Seong Gyu; Ma, Ji Su; Okumura, Ryu; Kubo, Atsuko; Ishii, Masaru; Okazaki, Taku; Murakami, Masaaki; Yamamoto, Masahiro; Yagita, Hideo; Takeda, Kiyoshi

    2012-03-27

    Adequate activation of CD4(+) T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4(+) T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX(3)C chemokine receptor 1(high) (CX(3)CR1(high)) CD11b(+) CD11c(+) cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4(+) T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX(3)CR1(high) CD11b(+) CD11c(+) cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX(3)CR1(high) CD11b(+) CD11c(+) cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis.

  17. Artesunate Protected Blood-Brain Barrier via Sphingosine 1 Phosphate Receptor 1/Phosphatidylinositol 3 Kinase Pathway After Subarachnoid Hemorrhage in Rats.

    PubMed

    Zuo, Shilun; Ge, Hongfei; Li, Qiang; Zhang, Xuan; Hu, Rong; Hu, Shengli; Liu, Xin; Zhang, John H; Chen, Yujie; Feng, Hua

    2017-03-01

    Blood-brain barrier preservation plays an important role in attenuating vasogenic brain edema after subarachnoid hemorrhage (SAH). This study was designed to investigate the protective effect and mechanism of artesunate, a traditional anti-malaria drug, on blood-brain barrier after SAH. Three hundred and seventy-seven (377) male Sprague-Dawley rats were subjected to endovascular perforation model for SAH. The rats received artesunate alone or in combination with Sphingosine-1-phosphate receptor-1 (S1P1) small interfering RNA (siRNA), antagonist VPC23019, or phosphatidylinositol 3-kinase inhibitor wortmannin after SAH. Modified Garcia score, SAH grades, brain water content, Evans blue leakage, transmission electron microscope, immunohistochemistry staining, Western blot, and cultured endothelial cells were used to investigate the optimum concentration and the therapeutic mechanism of artesunate. We found that artesunate (200 mg/kg) could do better in raising modified Garcia score, reducing brain water content and Evans blue leakage than other groups after SAH. Moreover, artesunate elevated S1P1 expression, enhanced phosphatidylinositol 3-kinase activation, lowered GSK-3β activation, stabilized β-catenin, and improved the expression of Claudin-3 and Claudin-5 after SAH in rats. These effects were eliminated by S1P1 siRNA, VPC23019, and wortmannin. This study revealed that artesunate could preserve blood-brain barrier integrity and improve neurological outcome after SAH, possibly through activating S1P1, enhancing phosphatidylinositol 3-kinase activation, stabilizing β-catenin via GSK-3β inhibition, and then effectively raising the expression of Claudin-3 and Claudin-5. Therefore, artesunate may be favorable for the blood-brain barrier (BBB) protection after SAH and become a potential candidate for the treatment of SAH patients.

  18. Radiolabeling and evaluation of 64Cu-DOTA-F56 peptide targeting vascular endothelial growth factor receptor 1 in the molecular imaging of gastric cancer

    PubMed Central

    Zhu, Hua; Zhao, Chuanke; Liu, Fei; Wang, Lixin; Feng, Junnan; Zhou, Zheng; Qu, Like; Shou, Chengchao; Yang, Zhi

    2015-01-01

    Noninvasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in early diagnosis of gastric cancer. Here we reported the synthesis, radiolabeling, and evaluation of a novel 64Cu-radiolabeled peptide for noninvasive positron emission tomography (PET) imaging of VEGFR1 positive gastric cancer. The binding of modified peptide WHSDMEWWYLLG (termed as F56) to VEGER-1 expressed in gastric cancer cell BCG823 has been confirmed by immune-fluorescence overlap. DOTA-F56 was designed and prepared by solid-phase synthesis and folded in vitro. 64Cu-DOTA-F56 was synthesized in high radiochemical yield and high specific activity (S.A. up to 255.6 GBq/mmol). It has excellent in vitro stability. Micro-PET imaging of 64Cu-DOTA-F56 identifies tumor in BCG823 tumor-bearing mice, while that of 18F-FDG does not. Immunohistochemical analysis of excised BCG823 xenograft showed colocalization between the PET images and the staining of VEGFR1. These results demonstrated that 64Cu-DOTA-F56 peptide has potential as a noninvasive imaging agent in VEGFR1 positive tumors. PMID:26807312

  19. Fcγ Receptor-induced Soluble Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Production Inhibits Angiogenesis and Enhances Efficacy of Anti-tumor Antibodies*

    PubMed Central

    Justiniano, Steven E.; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M.; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D.; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P.; Byrd, John C.; Tridandapani, Susheela

    2013-01-01

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. PMID:23902770

  20. Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

    PubMed

    Justiniano, Steven E; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P; Byrd, John C; Tridandapani, Susheela

    2013-09-13

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

  1. Comprehensive Behavioral Analysis of Male Ox1r (-/-) Mice Showed Implication of Orexin Receptor-1 in Mood, Anxiety, and Social Behavior.

    PubMed

    Abbas, Md G; Shoji, Hirotaka; Soya, Shingo; Hondo, Mari; Miyakawa, Tsuyoshi; Sakurai, Takeshi

    2015-01-01

    Neuropeptides orexin A and orexin B, which are exclusively produced by neurons in the lateral hypothalamic area, play an important role in the regulation of a wide range of behaviors and homeostatic processes, including regulation of sleep/wakefulness states and energy homeostasis. The orexin system has close anatomical and functional relationships with systems that regulate the autonomic nervous system, emotion, mood, the reward system, and sleep/wakefulness states. Recent pharmacological studies using selective antagonists have suggested that orexin receptor-1 (OX1R) is involved in physiological processes that regulate emotion, the reward system, and autonomic nervous system. Here, we examined Ox1r (-/-) mice with a comprehensive behavioral test battery to screen additional OX1R functions. Ox1r (-/-) mice showed increased anxiety-like behavior, altered depression-like behavior, slightly decreased spontaneous locomotor activity, reduced social interaction, increased startle response, and decreased prepulse inhibition. These results suggest that OX1R plays roles in social behavior and sensory motor gating in addition to roles in mood and anxiety.

  2. Plasma FGF21 concentrations, adipose fibroblast growth factor receptor-1 and β-klotho expression decrease with fasting in northern elephant seals.

    PubMed

    Suzuki, Miwa; Lee, Andrew Y; Vázquez-Medina, José Pablo; Viscarra, Jose A; Crocker, Daniel E; Ortiz, Rudy M

    2015-05-15

    Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting.

  3. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

    PubMed Central

    Daniels-Wells, Tracy R.; Helguera, Gustavo; Rodríguez, José A.; Leoh, Lai Sum; Erb, Michael A.; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L.

    2012-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin. PMID:23085102

  4. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

    PubMed

    Daniels-Wells, Tracy R; Helguera, Gustavo; Rodríguez, José A; Leoh, Lai Sum; Erb, Michael A; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L

    2013-02-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

  5. Overexpression of cannabinoid receptor 1 in esophageal squamous cell carcinoma is correlated with metastasis to lymph nodes and distant organs, and poor prognosis.

    PubMed

    Hijiya, Naoki; Shibata, Tomotaka; Daa, Tsutomu; Hamanaka, Ryoji; Uchida, Tomohisa; Matsuura, Keiko; Tsukamoto, Yoshiyuki; Nakada, Chisato; Iha, Hidekatsu; Inomata, Masafumi; Moriyama, Masatsugu

    2017-02-01

    In patients with esophageal squamous cell carcinoma (ESCC), the status of metastasis to lymph nodes is strongly associated with prognosis. Consequently, development of a biomarker to detect the presence of metastasis would be clinically valuable. In this study, we found that overexpression of cannabinoid receptor 1 (CB1R) was applicable as a marker for prediction of metastasis in ESCC. CB1R overexpression was detected immunohistochemically in 54 of 88 cases (61.4%). The intensity of CB1R expression was uniform in both intraepithelial and invasive regions in each case, and was significantly correlated with the status of metastasis to lymph nodes (P = 0.046) and distant organs (P = 0.047). Furthermore, multivariate analysis revealed that CB1R overexpression was independently associated with poor prognosis (P = 0.019). Biological analysis of CB1R overexpression using ESCC cell lines revealed that CB1R activation appeared to promote cell proliferation and invasion. On the basis of these findings, we propose that evaluation of CB1R expression status in biopsy specimens of ESCC using immunohistochemistry might be clinically useful for prediction of metastasis to lymph nodes and distant organs.

  6. Azapeptide analogues of the growth hormone releasing peptide 6 as cluster of differentiation 36 receptor ligands with reduced affinity for the growth hormone secretagogue receptor 1a.

    PubMed

    Proulx, Caroline; Picard, Émilie; Boeglin, Damien; Pohankova, Petra; Chemtob, Sylvain; Ong, Huy; Lubell, William D

    2012-07-26

    The synthetic hexapeptide growth hormone releasing peptide-6 (GHRP-6) exhibits dual affinity for the growth hormone secretagogue receptor 1a (GHS-R1a) and the cluster of differentiation 36 (CD36) receptor. Azapeptide GHRP-6 analogues have been synthesized, exhibiting micromolar affinity to the CD36 receptor with reduced affinity toward the GHS-R1a. A combinatorial split-and-mix approach furnished aza-GHRP-6 leads, which were further examined by alanine scanning. Incorporation of an aza-amino acid residue respectively at the D-Trp(2), Ala(3), or Trp(4) position gave aza-GHRP-6 analogues with reduced affinity toward the GHS-R1a by at least a factor of 100 and in certain cases retained affinity for the CD36 receptor. In the latter cases, the D-Trp(2) residue proved important for CD36 receptor affinity; however, His(1) could be replaced by Ala(1) without considerable loss of binding. In a microvascular sprouting assay using a choroid explant, [azaTyr(4)]-GHRP-6 (15), [Ala(1), azaPhe(2)]-GHRP-6 (16), and [azaLeu(3), Ala(6)]-GHRP-6 (33) all exhibited antiangiogenic activity.

  7. Distinct role of interleukin-6 and tumor necrosis factor receptor-1 in oval cell- mediated liver regeneration and inflammation-associated hepatocarcinogenesis

    PubMed Central

    Zhao, Jie; Li, Xi; Lin, Hui; Cai, Xiujun; Cang, Yong

    2016-01-01

    Interleukin 6 (IL6), tumor necrosis factor α (TNFα) and TNF receptor-1(TNFR1) have been shown to involve in oval cell proliferation and hepatocellular carcinoma (HCC) development. However, their role in these processes is still unclear. In the present study, by using hepatocytes-specific DDB1 deletion mouse models, we explored the role and mechanism of IL6, TNFα and TNFR1 in oval cell proliferation and HCC development in the context of inflammation, which is the common features of HCC pathogenesis in humans. Our results showed that IL6 promotes oval cell proliferation and liver regeneration, while TNFα/TNFR1 does not affect this process. Deletion of IL6 accelerates HCC development and increases tumor burden. The number of natural killer(NK) cells is significantly decreased in tumors without IL6, implying that IL6 suppresses HCC by NK cells. In contrast to IL6, TNFR1-mediated signaling pathway promotes HCC development, and deletion of TNFR1 reduced tumor incidence. Increased apoptosis, compensatory proliferation and activation of MAPK/MEK/ERK cascade contribute to the oncogenic function of TNFR1-mediated signaling pathway. Intriguingly, deletion of TNFα accelerates tumor development, which shows divergent roles of TNFα and TNFR1 in hepatocarcinogenesis. PMID:27556180

  8. Neurotensin-induced Proinflammatory Signaling in Human Colonocytes Is Regulated by β-Arrestins and Endothelin-converting Enzyme-1-dependent Endocytosis and Resensitization of Neurotensin Receptor 1*

    PubMed Central

    Law, Ivy Ka Man; Murphy, Jane E.; Bakirtzi, Kyriaki; Bunnett, Nigel W.; Pothoulakis, Charalabos

    2012-01-01

    The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT. PMID:22416137

  9. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

    PubMed

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2015-01-01

    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

  10. The effect of soluble complement receptor 1 (sCR1) and human thyroid antibodies on the course of experimental autoimmune thyroiditis in rats.

    PubMed

    Metcalfe, R A; McIntosh, R S; Morgan, B P; Levin, J L; Weetman, A P

    1996-01-01

    Experimental autoimmune thyroiditis (EAT), induced by immunisation of rats with thyroid extract and complete Freund's adjuvant, has been used as a model to study the effects of complement inhibition mediated by soluble complement receptor 1 (sCR1) administration during the initial phase of the disease. There was no effect of sCR1 on the severity of thyroiditis at day 28 after immunisation or on the levels of thyroid antibodies, whether sCR1 was given during the first or second week after immunisation. Human IgG containing high levels of thyroid peroxidase antibodies given to rats at the time of immunisation caused significant worsening of thyroiditis severity (P < 0.01 compared to animals receiving normal IgG) but sCR1 again had no effect in this variant of the EAT model. The results indicate that complement does not play a major role in the initial phase of tissue injury in EAT and complement inhibition does not impair the generation of an autoimmune response against the thyroid, although it remains possible that complement activation is important during the chronic phase of disease maintenance in human autoimmune thyroid disease.

  11. Vanilloid Receptor 1 Agonists, Capsaicin and Resiniferatoxin, Enhance MHC Class I-restricted Viral Antigen Presentation in Virus-infected Dendritic Cells

    PubMed Central

    Lee, Young-Hee; Im, Sun-A; Kim, Ji-Wan

    2016-01-01

    DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that the VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), enhance antiviral CTL responses by increasing MHC class I-restricted viral antigen presentation in dendritic cells (DCs). Bone marrow-derived DCs (BM-DCs) were infected with a recombinant vaccinia virus (VV) expressing OVA (VV-OVA), and then treated with CP or RTX. Both CP and RTX increased MHC class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Oral administration of CP or RTX significantly increased MHC class I-restricted OVA presentation by splenic and lymph node DCs in VV-OVA-infected mice, as assessed by directly measuring OVA peptide SIINFEKL-Kb complexes on the cell surface and by performing functional assays using OVA-specific CD8 T cells. Accordingly, oral administration of CP or RTX elicited potent OVA-specific CTL activity in VV-OVA-infected mice. The results from this study demonstrate that VR1 agonists enhance anti-viral CTL responses, as well as a neuro-immune connection in anti-viral immune responses. PMID:27574502

  12. Reversible kallmann syndrome, delayed puberty, and isolated anosmia occurring in a single family with a mutation in the fibroblast growth factor receptor 1 gene.

    PubMed

    Pitteloud, Nelly; Acierno, James S; Meysing, Astrid U; Dwyer, Andrew A; Hayes, Frances J; Crowley, William F

    2005-03-01

    Kallmann syndrome (KS) is a clinically and genetically heterogeneous disorder. Recently, loss-of-function mutations in the fibroblast growth factor receptor 1 (FGFR1) gene have been shown to cause autosomal dominant KS. To date, the detailed reproductive phenotype of KS associated with mutations in the FGFR1 has yet to be described. We report a kindred comprising a male proband with KS and spontaneous reversibility, whose mother had delayed puberty and whose maternal grandfather isolated anosmia. The proband presented at age 18 yr with KS and was subsequently treated with testosterone (T) therapy. Upon discontinuation of T therapy, he recovered from his hypogonadotropic hypogonadism, as evidenced by a normal LH secretion pattern, sustained normal serum T levels, and active spermatogenesis. The three members of this single family harbor the same FGFR1 mutation (Arg(622)X) in the tyrosine kinase domain. This report demonstrates 1) the first genetic cause of the rare variant of reversible KS, 2) the reversal of hypogonadotropic hypogonadism in a proband carrying an FGFR1 mutation suggests a role of FGFR1 beyond embryonic GnRH neuron migration, and 3) a loss of function mutation in the FGFR1 gene causing delayed puberty.

  13. Identification of small molecule agonists of human relaxin family receptor 1 (RXFP1) by utilizing a homogenous cell-based cAMP assay

    PubMed Central

    Chen, Catherine Z.; Southall, Noel; Xiao, Jingbo; Marugan, Juan J.; Ferrer, Marc; Hu, Xin; Jones, Raisa E.; Feng, Shu; Agoulnik, Irina U.

    2016-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the receptor could have a wide range of pharmacological utility, up to date, there are no reported small molecule agonists for relaxin receptors. Here, we report the development of quantitative high-throughput platform for RXFP1 agonist screen based on homogenous cell-based HTRF cAMP assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365,677 compounds. Neither compound showed activity in a counter screen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  14. Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

    PubMed

    Sasmal, Sanjita; Balaji, Gade; Kanna Reddy, Hariprasada R; Balasubrahmanyam, D; Srinivas, Gujjary; Kyasa, Shivakumar; Sasmal, Pradip K; Khanna, Ish; Talwar, Rashmi; Suresh, J; Jadhav, Vikram P; Muzeeb, Syed; Shashikumar, Dhanya; Harinder Reddy, K; Sebastian, V J; Frimurer, Thomas M; Rist, Øystein; Elster, Lisbeth; Högberg, Thomas

    2012-05-01

    Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays a role in metabolic and CNS disorders. The modeling-supported design, synthesis and multi-parameter optimization (biological activity, solubility, metabolic stability, hERG) of novel quinazoline derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified. Clusters of refined hMCHR1 homology models derived from the X-ray structure of the β2-adrenergic receptor, including extracellular loops, were developed and used to guide the design.

  15. Mapping the dopamine receptor. 1. Features derived from modifications in ring E of the neuroleptic butaclamol.

    PubMed

    Humber, L G; Bruderlein, F T; Philipp, A H; Götz, M

    1979-07-01

    Several analogues of the neuroleptic agent butaclamol, having modifications in the ring E region of the molecule, have been synthesized. Pharmacological evaluation identified two of the analogues as being equipotent to butaclamol, namely, anhydrobutaclamol (8) and deoxybutaclamol (9a). The molecular structures of both the active and inactive analogues were analyzed and the results have been used for mapping the central dopamine receptor. The existence of a previously proposed lipophilic accessory binding site on the receptor macromolecule has been confirmed. Its minimum dimensions, as well as its locus with respect to the primary binding sites, have been defined. A receptor model incorporating the above features is proposed.

  16. Beta-Adrenergic Receptor 1 Selective Antagonism Inhibits Norepinephrine-Mediated TNF-Alpha Downregulation in Experimental Liver Cirrhosis

    PubMed Central

    Zapater, Pedro; Gómez-Hurtado, Isabel; Peiró, Gloria; González-Navajas, José Manuel; García, Irma; Giménez, Paula; Moratalla, Alba; Such, José; Francés, Rubén

    2012-01-01

    Background Bacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA. Animals and Methods Forty-six mice were included in a 16-week protocol of CCl4-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl4 was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received E. coli-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured. Results Bacterial-DNA translocation was more frequent in CCl4-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with vs without bacterial-DNA (319.7±120.6 vs 120.7±68.6 pg/g for norepinephrine, 38.4±6.1 vs 29.7±4.2 pg/g for TNF-alpha, 41.8±7.4 vs 28.7±4.3 pg/g for IL-6). Only beta-adrenergic receptor-1 was significantly increased in treated vs control animals (34.6±7.3 vs 12.5±5.3, p = 0.01) and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist) treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist) didn’t inhibit liver norepinephrine modulation of pro-inflammatory cytokines. Conclusions Beta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl4-treated mice with bacterial-DNA. PMID:22916250

  17. Regulation of adiponectin receptor 1 in human hepatocytes by agonists of nuclear receptors

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas; Kirchner, Stefan; Laberer, Sabine; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2005-09-02

    The adiponectin receptors AdipoR1 and AdipoR2 have been identified to mediate the insulin-sensitizing effects of adiponectin. Although AdipoR2 was suggested to be the main receptor for this adipokine in hepatocytes, AdipoR1 protein is highly abundant in primary human hepatocytes and hepatocytic cell lines. Nuclear receptors are main regulators of lipid metabolism and activation of peroxisome proliferator-activated receptor {alpha} and {gamma}, retinoid X receptor (RXR), and liver X receptor (LXR) by specific ligands may influence AdipoR1 abundance. AdipoR1 protein is neither altered by RXR or LXR agonists nor by pioglitazone. In contrast, fenofibric acid reduces AdipoR1 whereas hepatotoxic troglitazone upregulates AdipoR1 protein in HepG2 cells. Taken together this work shows for the first time that AdipoR1 protein is expressed in human hepatocytes but that it is not a direct target gene of nuclear receptors. Elevated AdipoR1 induced by hepatotoxic troglitazone may indicate a role of this receptor in adiponectin-mediated beneficial effects in liver damage.

  18. Soluble forms of VEGF receptor-1 and -2 promote vascular maturation via mural cell recruitment.

    PubMed

    Lorquet, Sophie; Berndt, Sarah; Blacher, Silvia; Gengoux, Emily; Peulen, Olivier; Maquoi, Erik; Noël, Agnès; Foidart, Jean-Michel; Munaut, Carine; Péqueux, Christel

    2010-10-01

    Two soluble forms of vascular endothelial growth factor (VEGF) receptors, sVEGFR-1 and sVEGFR-2, are physiologically released and overproduced in some pathologies. They are known to act as anti-VEGF agents. Here we report that these soluble receptors contribute to vessel maturation by mediating a dialogue between endothelial cells (ECs) and mural cells that leads to blood vessel stabilization. Through a multidisciplinary approach, we provide evidence that these soluble VEGF receptors promote mural cell migration through a paracrine mechanism involving interplay in ECs between VEGF/VEGFR-2 and sphingosine-1-phosphate type-1 (S1P)/S1P1 pathways that leads to endothelial nitric oxyde synthase (eNOS) activation. This new paradigm is supported by the finding that sVEGFR-1 and -2 perform the following actions: 1) induce an eNOS-dependent outgrowth of a mural cell network in an ex vivo model of angiogenesis, 2) increase the mural cell coverage of neovessels in vitro and in vivo, 3) promote mural cell migration toward ECs, and 4) stimulate endothelial S1P1 overproduction and eNOS activation that promote the migration and the recruitment of neighboring mural cells. These findings provide new insights into mechanisms regulating physiological and pathological angiogenesis and vessel stabilization.

  19. Nogo receptor 1 is expressed in both primary cultured glial cells and neurons

    PubMed Central

    Ukai, Junichi; Imagama, Shiro; Ohgomori, Tomohiro; Ito, Zenya; Ando, Kei; Ishiguro, Naoki; Kadomatsu, Kenji

    2016-01-01

    ABSTRACT Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR’s roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells. PMID:27578914

  20. Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity

    PubMed Central

    Miao, Xinyan; Zhang, Wei; Huang, Zhangsen

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities. PMID:27612088

  1. Molecular Cloning and Functional Characterization of a Zebrafish Nuclear Progesterone Receptor1

    PubMed Central

    Chen, Shi X.; Bogerd, Jan; García-López, Ángel; de Jonge, Hugo; de Waal, Paul P.; Hong, Wan S.; Schulz, Rüdiger W.

    2009-01-01

    Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially—via Sertoli cells—also to germ cell differentiation in zebrafish testis. PMID:19741208

  2. Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

    PubMed

    Savoia, Carlo P; Liu, Qing-Hua; Zheng, Yun-Min; Yadav, Vishal; Zhang, Zhen; Wu, Ling-Gang; Wang, Yong-Xiao

    2014-11-15

    Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

  3. Sphingosine 1-Phosphate Receptor-1 Enhances Mitochondrial Function and Reduces Cisplatin-Induced Tubule Injury

    PubMed Central

    Rosin, Diane L.; Chroscicki, Piotr; Lee, Sangju; Dondeti, Krishna; Ye, Hong; Kinsey, Gilbert R.; Stevens, Brian K.; Jobin, Katarzyna; Kenwood, Brandon M.; Hoehn, Kyle L.; Lynch, Kevin R.; Okusa, Mark D.

    2015-01-01

    Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein– coupled receptors (S1P1–S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan-S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null (PepckCreS1pr1fl/fl) and control (PepckCreS1pr1w/wt) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-α, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI. PMID:25145931

  4. Montelukast targeting the cysteinyl leukotriene receptor 1 ameliorates Aβ1-42-induced memory impairment and neuroinflammatory and apoptotic responses in mice.

    PubMed

    Lai, Jin'e; Hu, Meng; Wang, Hao; Hu, Mei; Long, Yan; Miao, Ming-xing; Li, Jia-chang; Wang, Xiao-bing; Kong, Ling-yi; Hong, Hao

    2014-04-01

    Montelukast, known as a cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, is currently used for treatment of inflammatory diseases such as asthma. Here, we investigated effects of montelukast on neuroinflammatory, apoptotic responses, and memory performance following intracerebral infusions of amyloid-β (Aβ). The data demonstrated that intracerebroventrical infusions of aggregated Aβ1-42 (410 pmol/mouse) produced deficits in learning ability and memory, as evidenced by increase in escape latency during acquisition trials and decreases in exploratory activities in the probe trial in Morris water maze (MWM) task, and by decrease in the number of correct choices and increase in latency to enter the shock-free compartment in Y-maze test, and caused significant increases in pro-inflammatory cytokines such as NF-κB p65, TNF-α and IL-1β as well as pro-apoptotic molecule caspase-3 activation and anti-apoptotic protein Bcl-2 downregulation in hippocampus and cortex. Interestingly, this treatment resulted in upregulation of protein or mRNA of CysLT1R in both hippocampus and cortex. Blockade of CysLT1R by repeated treatment with montelukast (1 or 2 mg/kg, ig, 4 weeks) reduced Aβ1-42-induced CysLT1R expression and also suppressed Aβ1-42-induced increments of NF-κB p65, TNF-α, IL-1β and caspase-3 activation, and Bcl-2 downregulation in the hippocampus and cortex. Correspondingly, montelukast treatment significantly improved Aβ1-42-induced memory impairment in mice, but had little effect on normal mice. Our results show that montelukast may ameliorate Aβ1-42-induced memory impairment via inhibiting neuroinflammation and apoptosis mediated by CysLT1R signaling, suggesting that CysLT1R antagonism represents a novel treatment strategy for Alzheimer's disease.

  5. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

    PubMed Central

    Deniger, Drew C.; Yu, Jianqiang; Huls, M. Helen; Figliola, Matthew J.; Mi, Tiejuan; Maiti, Sourindra N.; Widhopf, George F.; Hurton, Lenka V.; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E.; Wierda, William G.; Kipps, Thomas J.; Cooper, Laurence J. N.

    2015-01-01

    T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire. PMID:26030772

  6. Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during renal allograft rejection in the rat.

    PubMed

    Pratt, J R; Hibbs, M J; Laver, A J; Smith, R A; Sacks, S H

    1996-12-01

    Complement is both an effector of the humoral immune response and a stimulator of leukocyte activation. To examine the influence of complement on the allograft response, we inhibited complement using recombinant human soluble complement receptor-1 (sCR1; TP10), in an unsensitized model of rat renal allograft rejection. Lewis to DA renal transplant recipients were treated daily with 25 mg/kg sCR1 or saline and sacrificed on days 1 to 5 after transplant. Transplanted organs were examined histologically and immunohistochemically for leukocyte subset markers and for the third component of complement, C3, and membrane attack complex deposition. A second set of recipients was followed from day 5 to day 9 to assess graft survival. sCR1-treated recipients displayed > 90% inhibition of plasma complement activity and a marked reduction in tissue C3 and membrane attack complex deposition. Inactivation of complement reduced the vascular injury such that there was almost complete sparing of vascular damage in day 5 sCR1-treated rats. There was a significant reduction in infiltrating leukocytes by day 5 after transplant, and complement inhibition delayed the time to reach a histologically defined end point of graft survival from 5 days in controls to 9 days in the sCR1-treated group. These results imply that the vascular and cell-mediated injury arises, in part, from complement activation. The partial inhibition of these injuries by sCR1 may have functional implications for strategies to inhibit allograft rejection.

  7. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

    PubMed

    Chen, Cheng; Li, Kesang; Jiang, Hua; Song, Fei; Gao, Huiping; Pan, Xiaorong; Shi, Bizhi; Bi, Yanyu; Wang, Huamao; Wang, Hongyang; Li, Zonghai

    2017-04-01

    Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1(+)GPC3(-) tumor cells, but they exhibited a similar cytotoxicity against GPC3(+)ASGR1(-) and GPC3(+)ASGR1(+) HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3(+)ASGR1(+) HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3(+)ASGR1(+) HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3(+)ASGR1(+) HCC.

  8. Disseminated Mycobacterium avium complex infection in a child with partial dominant interferon gamma receptor 1 deficiency in India.

    PubMed

    Sharma, Varun K; Pai, Gautham; Deswarte, Caroline; Lodha, Rakesh; Singh, Sarman; Kang, Liew Woei; Yin, Chong Chia; Casanova, Jean-Laurent; Bustamante, Jacinta; Kabra, Sushil K

    2015-07-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare condition characterized by clinical disease caused by weakly virulent mycobacteria. All genes mutated in MSMD patients are involved in IFN-γ immunity. Autosomal partial dominant (PD) interferon-γ receptor 1 (IFN-γR1) deficiency is the most frequent abnormality affecting the group of MSMD patients leading to impaired response of IFN-γ. We describe here a patient from India with disseminated infection due to Mycobacterium avium intracellulare (MAC) including multifocal osteomyelitis and BCG disease. A heterozygous mutation in exon 6 of IFNGR1 gene was identified, conferring an autosomal PD IFN-γR1 deficiency. Patient had recurrence of mycobacterial disease during antibiotic therapy for which subcutaneous IFN-γ was added as a modality of treatment for resistant MAC infection.

  9. Identification of cardiac hemo-vascular precursors and their requirement of sphingosine-1-phosphate receptor 1 for heart development

    PubMed Central

    Hu, Yan; Belyea, Brian C.; Li, Minghong; Göthert, Joachim R.; Gomez, R. Ariel; Sequeira-Lopez, Maria Luisa S.

    2017-01-01

    The cardiac endothelium plays a crucial role in the development of a functional heart. However, the precise identification of the endocardial precursors and the mechanisms they require for their role in heart morphogenesis are not well understood. Using in vivo and in vitro cell fate tracing concomitant with specific cell ablation and embryonic heart transplantation studies, we identified a unique set of precursors which possess hemogenic functions and express the stem cell leukemia (SCL) gene driven by its 5′ enhancer. These hemo-vascular precursors give rise to the endocardium, atrioventricular cushions and coronary vascular endothelium. Furthermore, deletion of the sphingosine-1-phosphate receptor 1 (S1P1) in these precursors leads to ventricular non-compaction cardiomyopathy, a poorly understood condition leading to heart failure and early mortality. Thus, we identified a distinctive population of hemo-vascular precursors which require S1P1 to exert their functions and are essential for cardiac morphogenesis. PMID:28338096

  10. Plasmodium falciparum field isolates use complement receptor 1 (CR1) as a receptor for invasion of erythrocytes.

    PubMed

    Awandare, Gordon A; Spadafora, Carmenza; Moch, J Kathleen; Dutta, Sheetij; Haynes, J David; Stoute, José A

    2011-05-01

    A majority of Plasmodium falciparum strains invade erythrocytes through interactions with sialic acid (SA) on glycophorins. However, we recently reported that complement receptor 1 (CR1) is a SA-independent invasion receptor of many laboratory strains of P. falciparum. To determine the role of CR1 in erythrocyte invasion among P. falciparum field isolates, we tested eight isolates obtained from children in Kenya. All the parasites examined were capable of invading in a SA-independent manner, and invasion of neuraminidase-treated erythrocytes was nearly completely blocked by anti-CR1 and soluble CR1 (sCR1). In addition, anti-CR1 and sCR1 partially inhibited invasion of intact erythrocytes in a majority of isolates tested. Sequencing of the hypervariable region of P. falciparum AMA-1 showed considerable diversity among all the isolates. These data demonstrate that CR1 mediates SA-independent erythrocyte invasion in P. falciparum field isolates.

  11. Abluminal stimulation of sphingosine 1-phosphate receptors 1 and 3 promotes and stabilizes endothelial sprout formation.

    PubMed

    Das, Anusuya; Lenz, Steven M; Awojoodu, Anthony O; Botchwey, Edward A

    2015-01-01

    Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications.

  12. Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1.

    PubMed

    Ingram, R J; Harris, A; Ascough, S; Metan, G; Doganay, M; Ballie, L; Williamson, E D; Dyson, H; Robinson, J H; Sriskandan, S; Altmann, D M

    2013-07-01

    Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

  13. Functional characterization of lysophosphatidic acid receptor 1 mutants identified in rat cancer tissues.

    PubMed

    Ishii, Shoichi; Tsujiuchi, Toshifumi; Fukushima, Nobuyuki

    2017-05-06

    Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts various cellular effects through activation of LPA receptors, LPA1-LPA6, in many types of cells including cancer cells. We recently found several missense mutations of Lpar1 in rat cancer tissues. One of these mutations is located at the extracellular tip of the seventh transmembrane domain of LPA1, and another three mutations are found within the NPXXY motif in the seventh transmembrane domain. These mutants are designated F295S LPA1 and P308S, I310T, and Y311H LPA1, respectively. Here, we examined the functions of these LPA1 mutants. Compared with wild-type (WT) LPA1, F295S, P308S, and I310T LPA1 showed decreased maximal responses in inhibition of cAMP formation, Ca(2+) mobilization, and cytoskeletal changes. Y311H LPA1 failed to show LPA-induced cellular responses. However, these LPA1 mutants were internalized in response to LPA exposure. Finally, while WT and F295S LPA1 showed a similar, broad distribution throughout the cell, P308S, I310T, and Y311H LPA1 displayed a restricted cellular distribution and co-localized with the endoplasmic reticulum. These data suggest that the LPA1 mutants perturb LPA signaling in cancer tissues.

  14. Surfactant prevents quartz induced down-regulation of complement receptor 1 in human granulocytes.

    PubMed

    Zetterberg, G; Lundahl, J; Curstedt, T; Eklund, A

    1997-02-01

    Quartz is known to induce an inflammatory response in the alveolar space by recruitment of different effector cells. We investigated the interaction between granulocytes and quartz with respect to expression of complement receptor type 1 (CR1) and CR3, with and without the presence of surfactant. Granulocytes from hemolyzed blood were stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), which mobilize the intracellular pool of CR1 to the surface, and the mean fluorescence intensity (MFI) measured by cytofluorometry was 47.4 (46-63.6) (median; interquartile range). Quartz exposure reduced the CR1 expression to 23.2 (22.8-30.6) MFI units (P < 0.01), a porcine surfactant preparation added during quartz exposure abolished the down-regulation completely, 47.7 (43.2-62.3) MFI units (P < 0.001). Similar results were obtained after preincubation of the cells with surfactant followed by quartz exposure. No significant influence on CR1 expression was found by a synthetic lipid mixture, nor was the CR3 expression affected. In conclusion, this study demonstrates that the presence of surfactant inhibits quartz induced down-regulation of CR1 on activated granulocytes.

  15. Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

    SciTech Connect

    P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

    2011-12-31

    NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

  16. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

    PubMed

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-02-13

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

  17. The Neurotensin Receptor-1 Pathway Contributes to Human Ductal Breast Cancer Progression

    PubMed Central

    Dupouy, Sandra; Viardot-Foucault, Véronique; Alifano, Marco; Souazé, Frédérique; Plu-Bureau, Geneviève; Chaouat, Marc; Lavaur, Anne; Hugol, Danielle; Gespach, Christian

    2009-01-01

    Background The neurotensin (NTS) and its specific high affinity G protein coupled receptor, the NT1 receptor (NTSR1), are considered to be a good candidate for one of the factors implicated in neoplastic progression. In breast cancer cells, functionally expressed NT1 receptor coordinates a series of transforming functions including cellular migration and invasion. Methods and Results we investigated the expression of NTS and NTSR1 in normal human breast tissue and in invasive ductal breast carcinomas (IDCs) by immunohistochemistry and RT-PCR. NTS is expressed and up-regulated by estrogen in normal epithelial breast cells. NTS is also found expressed in the ductal and invasive components of IDCs. The high expression of NTSR1 is associated with the SBR grade, the size of the tumor, and the number of metastatic lymph nodes. Furthermore, the NTSR1 high expression is an independent factor of prognosis associated with the death of patients. Conclusion these data support the activation of neurotensinergic deleterious pathways in breast cancer progression. PMID:19156213

  18. Abluminal Stimulation of Sphingosine 1-Phosphate Receptors 1 and 3 Promotes and Stabilizes Endothelial Sprout Formation

    PubMed Central

    Lenz, Steven M.; Awojoodu, Anthony O.

    2015-01-01

    Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications. PMID:25315888

  19. Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by G(α)i/o /RhoA/ROCK signaling pathway.

    PubMed

    Mai, Ping; Tian, Lei; Yang, Le; Wang, Lin; Yang, Lin; Li, Liying

    2015-07-01

    Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence-staining, real time RT-PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence-activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA-enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP-bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G((α)i/o) protein inhibitor) decreased GTP-bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho-associated kinase ROCK inhibitor) attenuated CB1-mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G((α)i/o)/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up-regulation of CB1 expression by real time RT-PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up-regulation of CB1 was RhoA-independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G((α)i/o)/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process.

  20. Orexin receptor 1 signaling contributes to ethanol binge-like drinking: Pharmacological and molecular evidence.

    PubMed

    Carvajal, Francisca; Alcaraz-Iborra, Manuel; Lerma-Cabrera, Jose Manuel; Valor, Luis Miguel; de la Fuente, Leticia; Sanchez-Amate, Maria Del Carmen; Cubero, Inmaculada

    2015-01-01

    Orexins (OX) have been recently implicated in ethanol seeking and self-administration. A few recent studies have provided additional evidence that OX receptor antagonists effectively reduce voluntary ethanol consumption in subjects spontaneously showing high levels of ethanol intake. The present study further evaluates the contribution of OXR1 to excessive binge-like drinking of ethanol in ad libitum-fed C57BL/6J mice from a pharmacological and molecular approach. The main findings in the study are: (1) Icv administration of SB-334867 (3 μg/μl) blunted ethanol (20% v/v), but not saccharin (0.15% w/v) binge-like drinking in a drinking in the dark procedure, without any alteration of chow consumption or total calories ingested; (2) Icv administration of SB-334867 (3 μg/μl) increased the latency to recover the righting reflex after a sedative dose of ethanol without any significant alteration in ethanol peripheral metabolism; (3) four repetitive, 2-h daily episodes of saccharin, but not ethanol binge-like drinking blunted OXR1 mRNA expression in the lateral hypothalamus. Present findings extend the current knowledge pointing to a role for OX signaling in ethanol sedation, which might partially explain the inhibitory effect of OXR1 antagonists on ethanol consumption. Combined pharmacological and molecular data suggesting the contribution of OXR1 in ethanol binge-drinking leading us to propose the idea that targeting OXR1 could represent a novel pharmacological approach to control binge-consumption episodes of ethanol in vulnerable organisms failing to spontaneously reduce OX activity.

  1. The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1.

    PubMed

    Schmid, Gerald; Guba, Markus; Ischenko, Ivan; Papyan, Armine; Joka, Mareile; Schrepfer, Sabine; Bruns, Christiane J; Jauch, Karl-Walter; Heeschen, Christopher; Graeb, Christian

    2007-05-01

    FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.

  2. Sulfonylurea receptor 1 contributes to the astrocyte swelling and brain edema in acute liver failure.

    PubMed

    Jayakumar, A R; Valdes, V; Tong, X Y; Shamaladevi, N; Gonzalez, W; Norenberg, M D

    2014-02-01

    Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation, and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, nonselective cation (NCCa-ATP) channel. We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a threefold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by cotreatment of cells with BAY 11-7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.

  3. The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

    PubMed Central

    Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

    2015-01-01

    The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267

  4. Cooperative Signaling between Homodimers of Metabotropic Glutamate Receptors 1 and 5

    PubMed Central

    Sevastyanova, Tatyana N.

    2014-01-01

    Metabotropic glutamate receptors (mGluRs) function as dimers. Recent work suggests that mGluR1 and mGluR5 may physically interact, but the nature and functional consequences of this relationship have not been addressed. In this study, the functional and pharmacological consequences of this interaction were investigated. Using heterologous expression of mGluR cDNA in rat sympathetic neurons from the superior cervical ganglion and inhibition of the native calcium currents as an assay for receptor activation, a functional interdependence between mGluR1 and mGluR5 was demonstrated. In neurons coexpressing these receptors, combining a selective mGluR1 competitive antagonist with either an mGluR1- or mGluR5-selective negative allosteric modulator (NAM) BAY36-7620 [(3aS,6aS)-hexahydro-5-methylene-6a-(2-naphthalenylmethyl)-1H-cyclopenta[c]furan-1-one] or MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride], respectively, strongly occluded signaling by both receptors to an approximately equal degree. By contrast, in cells coexpressing mGluR1 and mGluR2, combining the same mGluR1 competitive inhibitor with an mGluR1 or mGluR2 NAM yielded partial and full inhibition of the response, respectively, as expected for independently acting receptors. In neurons expressing mGluR1 and mGluR5, the selective NAMs each strongly inhibited the response to glutamate, suggesting that these receptors do not interact as heterodimers, which would not be inhibited by selective NAMs. Finally, evidence for a similar mGluR1/mGluR5 functional dependence is shown in medium spiny striatal neurons. Together, these data demonstrate cooperative signaling between mGluR1 and mGluR5 in a manner inconsistent with heterodimerization, and thus suggest an interaction between homodimers. PMID:25113912

  5. Allosteric antagonist binding sites in class B GPCRs: corticotropin receptor 1

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Supriyo; Subramanian, Govindan; Hall, Spencer; Lin, Jianping; Laoui, Abdelazize; Vaidehi, Nagarajan

    2010-08-01

    ,000 compounds seeded with 350 CRF1-R specific active antagonists performed on the NBI-27914 stabilized conformation of CRF1-R yielded a 44% increase in enrichment compared to the initially modeled receptor conformation at a 10% cutoff. The NBI-27914 stabilized conformation also shows a high enrichment for high affinity antagonists compared to the weaker ones. Thus, the conformational changes induced by NBI-27914 improved the ligand screening efficiency of the CRF1-R model and demonstrate a generalized application of the method in drug discovery.

  6. Tryptophan hydroxylase and serotonin receptor 1A expression in the retina of the sea lamprey.

    PubMed

    Cornide-Petronio, María Eugenia; Anadón, Ramón; Barreiro-Iglesias, Antón; Rodicio, María Celina

    2015-06-01

    The dual development of the retina of lampreys is exceptional among vertebrates and offers an interesting EvoDevo (evolutionary developmental biology) model for understanding the origin and evolution of the vertebrate retina. Only a single type of photoreceptor, ganglion cell and bipolar cell are present in the early-differentiated central retina of lamprey prolarvae. A lateral retina appears later in medium-sized larvae (about 3 years after hatching in the sea lamprey), growing and remaining largely neuroblastic until metamorphosis. In this lateral retina, only ganglion cells and optic fibers differentiate in larvae, whereas differentiation of amacrine, horizontal, photoreceptor and bipolar cells mainly takes place during metamorphosis, which gives rise to the adult retina. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter found in the retina of vertebrates whose synthesis is mediated by the rate-limiting enzyme tryptophan hydroxylase (TPH). TPH is also the first enzyme in the biosynthetic pathways of melatonin in photoreceptor cells. The serotonin 1A receptor (5-HT1A) is a major determinant of the activity of both serotonergic cells and their targets due to its pre- and post-synaptic location. Here, we report the developmental pattern of expression of tph and 5-ht1a transcripts in the sea lamprey retina by means of in situ hybridization. In larvae, strong tph mRNA signal was observed in photoreceptors and putative ganglion cells of the central retina, and in some neuroblasts of the lateral retina. In adults, strong tph expression was observed in bipolar, amacrine and ganglion cells and in photoreceptors. In the prolarval (central) retina, all the differentiated retinal cells expressed 5-ht1a transcripts, which were not observed in undifferentiated cells. In larvae, photoreceptors, bipolar cells and ganglion cells in the central retina, and neuroblasts in the lateral retina, showed 5-ht1a expression. In the adult retina, expression of 5-ht1a transcript

  7. Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis

    PubMed Central

    Wurfel, Mark M.; Gordon, Anthony C.; Holden, Tarah D.; Radella, Frank; Strout, Jeanna; Kajikawa, Osamu; Ruzinski, John T.; Rona, Gail; Black, R. Anthony; Stratton, Seth; Jarvik, Gail P.; Hajjar, Adeline M.; Nickerson, Deborah A.; Rieder, Mark; Sevransky, Jonathan; Maloney, James P.; Moss, Marc; Martin, Greg; Shanholtz, Carl; Garcia, Joe G. N.; Gao, Li; Brower, Roy; Barnes, Kathleen C.; Walley, Keith R.; Russell, James A.; Martin, Thomas R.

    2008-01-01

    Rationale: Polymorphisms affecting Toll-like receptor (TLR)–mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. Objectives: To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. Methods: We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case–control study in a cohort of intensive care unit patients with sepsis, and a case–control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. Measurements and Main Results: The SNP demonstrating the most hypermorphic effect was the G allele of TLR1−7202A/G (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 × 10−20). TLR1−7202G marked a coding SNP that causes higher TLR1-induced NF-κB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1−7202G predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07–3.09). In the case-control study TLR1−7202G was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59–7.27). TLR1−7202G also associated with a higher prevalence of gram-positive cultures in both clinical studies. Conclusions: Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis. PMID

  8. Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body.

    PubMed

    Xiaopeng, Bai; Tanaka, Yoshimasa; Ihara, Eikichi; Hirano, Katsuya; Nakano, Kayoko; Hirano, Mayumi; Oda, Yoshinao; Nakamura, Kazuhiko

    2017-02-15

    Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK1) and NK2 antagonists, but not by an NK3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK1, NK2 and NK3) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK1 and NK2 antagonists, but not by the NK3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK1/2. Gap junctions were indispensable in this tachykinin-induced response.

  9. V101L of human formyl peptide receptor 1 (FPR1) increases receptor affinity and augments the antagonism mediated by cyclosporins

    PubMed Central

    Zhou, Caihong; Zhou, Yan; Wang, Jia; Feng, Yang; Wang, Haonan; Xue, Jinglun; Chen, Yani; Ye, Richard D.; Wang, Ming-Wei

    2013-01-01

    Genetic variation plays a major role in drug response variability. CsA (cyclosporin A), a widely used immunosuppressive agent, is a specific antagonist for FPR1 (formyl peptide receptor 1), which is an important G-protein-coupled chemoattractant receptor in the innate immune system. In order to study the variable responses of cyclosporins to different FPR1 mutants, we investigated the distribution of human FPR1 haplotypes among 209 healthy Han Chinese subjects. The haplotype pattern in Han Chinese were characterized on the basis of five SNPs (single nucleotide polymorphisms), including rs5030878 (p.T11I), rs2070745 (p.V101L), rs5030880 (p.R190W), rs1042229 (p.N192K) and rs867228 (p.A346E). Receptor binding affinity of cyclosporins to FPR1 haplotypes was assessed using N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys–FITC in CHO-Gα16 cells stably transfected with cDNAs encoding the top 12 FPR1 haplotypes in the Han Chinese. Variants of FPR1 carrying a single amino acid substitution of leucine for valine at position 101 (p.Leu101) displayed significantly higher pKi values for CsA and CsH (cyclosporin H), indicative of an improved receptor affinity. The polymorphism of FPR1 p.Leu101 also enhanced the inhibitory effects of cyclosporins on fMLF (N-formyl-methionyl-leucyl-phenylalanine)-induced activities, including calcium mobilization, cell chemotaxis and MAPK (mitogen-activated protein kinase) phosphorylation. These results point to a possible complication for clinical use of CsA in patients carrying the p.Leu101 allele of FPR1. PMID:23373827

  10. Beta-phenylethylamine alters monoamine transporter function via trace amine-associated receptor 1: implication for modulatory roles of trace amines in brain.

    PubMed

    Xie, Zhihua; Miller, Gregory M

    2008-05-01

    Brain monoamines include common biogenic amines (dopamine, norepinephrine, and serotonin) and trace amines [beta-phenylethylamine (beta-PEA), tyramine, tryptamine, and octopamine]. Common biogenic amines are well established as neurotransmitters, but the roles and functional importance of trace amines remain elusive. Here, we re-evaluated the interaction of trace amines with trace amine-associated receptor 1 (TAAR1) and investigated effects of beta-PEA on monoamine transporter function and influence of monoamine autoreceptors on TAAR1 signaling. We confirmed that TAAR1 was activated by trace amines and demonstrated that TAAR1 activation by beta-PEA significantly inhibited uptake and induced efflux of [3H]dopamine, [3H]norepinephrine, and [3H]serotonin in transfected cells. In brain synaptosomes, beta-PEA significantly inhibited uptake and induced efflux of [3H]dopamine and [3H]serotonin in striatal and [3H]norepinephrine in thalamic synaptosomes of rhesus monkeys and wild-type mice, but it lacked the same effects in synaptosomes of TAAR1 knockout mice. The effect of beta-PEA on efflux was blocked by transporter inhibitors in either the transfected cells or wild-type mouse synaptosomes. We also demonstrated that TAAR1 signaling was not affected by monoamine autoreceptors at exposure to trace amines that we show to have poor binding affinity for the autoreceptors relative to common biogenic amines. These results reveal that beta-PEA alters monoamine transporter function via interacting with TAAR1 but not monoamine autoreceptors. The functional profile of beta-PEA may reveal a common mechanism by which trace amines exert modulatory effects on monoamine transporters in brain.

  11. A novel gene silencer, pyrrole-imidazole polyamide targeting human lectin-like oxidized low-density lipoprotein receptor-1 gene improves endothelial cell function.

    PubMed

    Ueno, Takahiro; Fukuda, Noboru; Tsunemi, Akiko; Yao, En-Hui; Matsuda, Hiroyuki; Tahira, Kazunobu; Matsumoto, Taro; Matsumoto, Koichi; Matsumoto, Yoshiaki; Nagase, Hiroki; Sugiyama, Hiroshi; Sawamura, Tatsuya

    2009-03-01

    Pyrrole-imidazole polyamide can be combined in antiparallel side-by-side dimeric complexes along the minor groove of DNA in a sequence-specific manner. Pyrrole-imidazole polyamides are effective inhibitors of transcription factors as well as viral repressors and transactivators. Recently, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was reported to be a major factor contributing to the pathogenesis of coronary atherosclerosis. In this study, we designed a pyrrole-imidazole polyamide specific for the LOX-1 gene and evaluated its effect on LOX-1 gene transcription. A pyrrole-imidazole polyamide was designed to target the AP-1 binding site of the LOX-1 gene and synthesized by solid phase methods. This pyrrole-imidazole polyamide significantly inhibited LOX-1 promoter activity in HEK293 cells, determined by the luciferase assay. LOX-1 mRNA expression was also inhibited by the pyrrole-imidazole polyamide at a concentration of 10-9 mol/l in human umbilical vein endothelial cells (HUVEC), determined by the real-time PCR method. HUVEC were treated by pyrrole-imidazole polyamide targeting the LOX-1 gene, and apoptosis was assessed using Hoechst stain, terminal deoxy nucleotidyl transferase-mediated UTP end labeling method, and dye-uptake bioassay. Treatment of HUVEC for 72 h with LOX-1 targeted pyrrole-imidazole polyamide decreased apoptosis induced by angiotensin II and oxidized low-density lipoprotein (ox-LDL) loading in all assays. This novel therapeutic agent, pyrrole-imidazole polyamide, could specifically inhibit LOX-1 gene expression by reducing the promoter activity of the gene. Pyrrole-imidazole polyamide seems to be a powerful promising new agent that can be used to explore therapies based on inhibition of transcription. Molecular recognition of DNA by small molecules could provide insight into the development of new human medicines.

  12. A Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) – Discoidin Domain Receptor 1 Axis Regulates Collagen-Induced Apoptosis in Breast Cancer Cells

    PubMed Central

    Assent, Delphine; Bourgot, Isabelle; Hennuy, Benoît; Geurts, Pierre; Noël, Agnès; Foidart, Jean-Michel; Maquoi, Erik

    2015-01-01

    During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and pro-apoptotic properties. To develop metastatic capabilities, tumour cells must acquire the capacity to cope with this novel microenvironment. How cells interact with and respond to their microenvironment during cancer dissemination remains poorly understood. To address the impact of type I collagen on the fate of tumour cells, human breast carcinoma MCF-7 cells were cultured within three-dimensional type I collagen gels (3D COL1). Using this experimental model, we have previously demonstrated that membrane type-1 matrix metalloproteinase (MT1-MMP), a proteinase overexpressed in many aggressive tumours, promotes tumour progression by circumventing the collagen-induced up-regulation of BIK, a pro-apoptotic tumour suppressor, and hence apoptosis. Here we performed a transcriptomic analysis to decipher the molecular mechanisms regulating 3D COL1-induced apoptosis in human breast cancer cells. Control and MT1-MMP expressing MCF-7 cells were cultured on two-dimensional plastic plates or within 3D COL1 and a global transcriptional time-course analysis was performed. Shifting the cells from plastic plates to 3D COL1 activated a complex reprogramming of genes implicated in various biological processes. Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling. By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis. Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a

  13. V101L of human formyl peptide receptor 1 (FPR1) increases receptor affinity and augments the antagonism mediated by cyclosporins.

    PubMed

    Zhou, Caihong; Zhou, Yan; Wang, Jia; Feng, Yang; Wang, Haonan; Xue, Jinglun; Chen, Yani; Ye, Richard D; Wang, Ming-Wei

    2013-04-15

    Genetic variation plays a major role in drug response variability. CsA (cyclosporin A), a widely used immunosuppressive agent, is a specific antagonist for FPR1 (formyl peptide receptor 1), which is an important G-protein-coupled chemoattractant receptor in the innate immune system. In order to study the variable responses of cyclosporins to different FPR1 mutants, we investigated the distribution of human FPR1 haplotypes among 209 healthy Han Chinese subjects. The haplotype pattern in Han Chinese were characterized on the basis of five SNPs (single nucleotide polymorphisms), including rs5030878 (p.T11I), rs2070745 (p.V101L), rs5030880 (p.R190W), rs1042229 (p.N192K) and rs867228 (p.A346E). Receptor binding affinity of cyclosporins to FPR1 haplotypes was assessed using N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC in CHO-G(α16) cells stably transfected with cDNAs encoding the top 12 FPR1 haplotypes in the Han Chinese. Variants of FPR1 carrying a single amino acid substitution of leucine for valine at position 101 (p.Leu(101)) displayed significantly higher pK(i) values for CsA and CsH (cyclosporin H), indicative of an improved receptor affinity. The polymorphism of FPR1 p.Leu(101) also enhanced the inhibitory effects of cyclosporins on fMLF (N-formyl-methionyl-leucyl-phenylalanine)-induced activities, including calcium mobilization, cell chemotaxis and MAPK (mitogen-activated protein kinase) phosphorylation. These results point to a possible complication for clinical use of CsA in patients carrying the p.Leu(101) allele of FPR1.

  14. Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

    PubMed Central

    Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

    2010-01-01

    Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

  15. Divergent responses to thermogenic stimuli in BAT and subcutaneous adipose tissue from interleukin 18 and interleukin 18 receptor 1-deficient mice

    PubMed Central

    Pazos, Patricia; Lima, Luis; Tovar, Sulay; González-Touceda, David; Diéguez, Carlos; García, María C.

    2015-01-01

    Brown and beige adipocytes recruitment in brown (BAT) or white adipose tissue, mainly in the inguinal fat pad (iWAT), meet the need for temperature adaptation in cold-exposure conditions and protect against obesity in face of hypercaloric diets. Using interleukin18 (Il18) and Il18 receptor 1- knockout (Il18r1-KO) mice, this study aimed to investigate the role of IL18 signaling in BAT and iWAT activation and thermogenesis under both stimuli. Il18-KO, extremely dietary obesity-prone as previously described, failed to develop diet-induced thermogenesis as assessed by BAT and iWAT Ucp1 mRNA levels. Overweight when fed standard chow but not HFD, HFD-fed Il18r1-KO mice exhibited increased iWAT Ucp1 gene expression. Energy expenditure was reduced in pre-obese Il18r1-KO mice and restored upon HFD-challenge. Cold exposure lead to similar results; Il18r1-KO mice were protected against acute body temperature drop, displaying a more brown-like structure, alternative macrophage activation and thermogenic gene expression in iWAT than WT controls. Opposite effects were observed in Il18-KO mice. Thus, Il18 and Il18r1 genetic ablation disparate effects on energy homeostasis are likely mediated by divergent BAT responses to thermogenic stimuli as well as iWAT browning. These results suggest that a more complex receptor-signaling system mediates the IL18 adipose-tissue specific effects in energy expenditure. PMID:26656097

  16. Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors.

    PubMed

    Yonamine, Camila M; Kondo, Marcia Y; Nering, Marcela B; Gouvêa, Iuri E; Okamoto, Débora; Andrade, Douglas; da Silva, José Alberto A; Prieto da Silva, Alvaro R B; Yamane, Tetsuo; Juliano, Maria A; Juliano, Luiz; Lapa, Antônio J; Hayashi, Mirian A F; Lima-Landman, Maria Teresa R

    2014-03-01

    Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or

  17. Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

    PubMed Central

    Wu, Jing-xiang; Yuan, Xiao-min; Wang, Qiong; Wei, Wang

    2016-01-01

    Background Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain. Results In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak. Conclusions Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer. PMID:27094551

  18. Cardiac specific deletion of N-methyl-d-aspartate receptor 1 ameliorates mtMMP-9 mediated autophagy/mitophagy in hyperhomocysteinemia.

    PubMed

    Tyagi, Neetu; Vacek, Jonathan C; Givvimani, Srikanth; Sen, Utpal; Tyagi, Suresh C

    2010-04-01

    Autophagy is an important process in the pathogenesis of cardiovascular diseases; however, the proximal triggers for mitochondrial autophagy were unknown. The N-methyl-d-aspartate receptor 1 (NMDA-R1) is a receptor for homocysteine (Hcy) and plays a key role in cardiac dysfunction. Cardiac-specific deletion of NMDA-R1 has been shown to ameliorate Hcy-induced myocyte contractility. Hcy activates mitochondrial matrix metalloproteinase-9 (mtMMP-9) and induces translocation of connexin-43 (Cxn-43) to the mitochondria (mtCxn-43). We sought to show cardiac-specific deletion of NMDA-R1 mitigates Hcy-induced mtCxn-43 translocation, mtMMP-9-mediated mtCxn-43 degradation, leading to mitophagy, in part, by decreasing mitochondrial permeability (MPT). Cardiac-specific knockout (KO) of NAMDA-R1 was generated using the cre/lox approach. The myocyte mitochondria were isolated from wild type (WT), WT + Hcy (1.8 g of DL-Hcy/L in the drinking water for 6 weeks), NMDA-R1 KO + Hcy, and NR1(fl/fl)/Cre (NR1(fl/fl)) genetic control mice. Mitochondrial respiratory capacity and MPT were measured by fluorescence-dye methods. The mitochondrial superoxide and peroxinitrite levels were detected by confocal microscopy using Mito-SOX and dihydrorhodamine-123. The mtMMP-9 activity and expression were detected by zymography and RT-PCR analyses. The mtCxn-43 translocation was detected by confocal microscopy. The degradation of mtCxn-43 and LC3-I/II (a marker of autophagy) were detected by Western blot. These results suggested that Hcy enhanced intramitochondrial nitrosative stress in myocytes. There was a robust increase in mtMMP-9 activity. An increase in translocation and degradation of mtCxn-43 was also noted. These increases led to mitophagy. The effects were ameliorated by cardiac-specific deletion of NMDA-R1. We concluded that HHcy increased mitochondrial nitrosative stress, thereby activating mtMMP-9 and inciting the degradation of mtCxn-43. This led to mitophagy, in part, by activating NMDA

  19. Efficacy and safety of DALI-LDL-apheresis in two patients treated with the angiotensin II-receptor 1 antagonist losartan.

    PubMed

    Bosch, Thomas; Wendler, Thorsten

    2004-08-01

    Direct adsorption of lipids (DALI) is the first low-density lipoprotein (LDL)-apheresis technique capable of adsorbing LDL and lipoprotein (a) directly from whole blood. The adsorber consists of negatively charged polyacrylate ligands linked to a Eupergit matrix. Negatively charged ligands give rise to activation of bradykinin, which is rapidly degraded by the angiotensin converting enzyme (ACE). Thus, angiotensin converting enzyme inhibitors are contraindicated in DALI-LDL-apheresis. This is the first paper to describe the efficacy and safety of DALI-LDL-apheresis in patients treated with 50 mg of the angiotensin II-receptor 1 antagonist (ARA) losartan. Two hypercholesterolemic patients were treated for 79 patient months with weekly or biweekly DALI sessions (N = 221 sessions). Approximately 1.4 patient blood volumes were treated per session. Acute reductions of LDL-cholesterol (63%) and lipoprotein (a) (62%) exceeded 60% and laboratory safety parameters remained in the apheresis typical range. Mean bradykinin plasma levels peaked in the efferent line post-adsorber at 1000 mL of treated blood volume; 467 fmol/mL (N = 6 sessions) in the ARA-treated patients and 671 fmol/mL (N = 9 sessions) in a control group of three DALI patients without ARA medication (P = 0.69, n.s.). Clinically, the DALI sessions for the ARA-treated patients were completely uneventful and blood pressure was not significantly different in the two groups. In summary, according to this retrospective pilot study, DALI-LDL-apheresis was shown for the first time to be safe and effective in patients on ARA medication.

  20. Early-life stress-induced anxiety-related behavior in adult mice partially requires forebrain corticotropin-releasing hormone receptor 1.

    PubMed

    Wang, Xiao-Dong; Labermaier, Christiana; Holsboer, Florian; Wurst, Wolfgang; Deussing, Jan M; Müller, Marianne B; Schmidt, Mathias V

    2012-08-01

    Early-life stress may lead to persistent changes in central corticotropin-releasing hormone (CRH) and the CRH receptor 1 (CRHR1) system that modulates anxiety-related behavior. However, it remains unknown whether CRH-CRHR1 signaling is involved in early-life stress-induced anxiety-related behavior in adult animals. In the present study, we used conditional forebrain CRHR1 knockout (CRHR1-CKO) mice and examined the potential role of forebrain CRHR1 in the anxiogenic effects of early-life stress. As adults, wild-type mice that received unstable maternal care during the first postnatal week showed reduced body weight gain and increased anxiety levels in the open field test, which were prevented in stressed CRHR1-CKO mice. In the light-dark box test, control CRHR1-CKO mice were less anxious, but early-life stress increased anxiety levels in both wild-type and CRHR1-CKO mice. In the elevated plus maze test, early-life stress had only subtle effects on anxiety-related behavior. Moreover, early-life stress did not alter the basal home cage activity and gene expression levels of key hypothalamic-pituitary-adrenal axis regulators in adult wild-type and CRHR1-CKO mice, but enhanced neuroendocrine reactivity to acute immobilization stress in CRHR1-CKO mice. Our findings highlight the importance of forebrain CRHR1 in modulating some of the anxiogenic effects of early-life stress, and suggest that other neural circuits are also involved in the programming effects of early-life stress on anxiety-related behavior.

  1. Influences of melatonin treatment, melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) gene polymorphisms on first conception in Sarda ewe lambs.

    PubMed

    Luridiana, S; Mura, M C; Daga, C; Cosso, G; Bodano, S; Farci, F; Zidda, F; Carcangiu, V

    2016-04-01

    In order to investigate if the melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) genes influence the reproductive response to melatonin treatment, 510 Sarda ewe lambs were divided into groups C (control) and M; Group M received one melatonin implant (18mg). After 35 days rams were introduced for 40 days and subsequent lambing dates and number of newborns were recorded. The MTNR1A gene Exon II and KiSS-1 gene Exon I were amplified and genotyped by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism analysis. Two single nucleotide polymorphisms (SNPs; C606T and G612A) in MTNR1A and one (G1035A) in KiSS-1 were found. The most frequent genotypes were G/G (63%) and C/C (53%) for MTNR1A and G/G (92%) for KiSS-1. Treated animals showed a higher lambing rate (P<0.05) and an advanced lambing date (P<0.05) compared with controls. The three SNPs did not influence the onset of reproductive activity. The majority of the G/G animals of Group M lambed before 190 days after ram introduction (P<0.05), while in Group C a higher number of G/G animals lambed after this date. Data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that the G/G genotype of the MTNR1A gene is able to influence the reproductive response to melatonin treatment.

  2. Adult Lysophosphatidic Acid Receptor 1-Deficient Rats with Hyperoxia-Induced Neonatal Chronic Lung Disease Are Protected against Lipopolysaccharide-Induced Acute Lung Injury

    PubMed Central

    Chen, Xueyu; Walther, Frans J.; Laghmani, El H.; Hoogeboom, Annemarie M.; Hogen-Esch, Anne C. B.; van Ark, Ingrid; Folkerts, Gert; Wagenaar, Gerry T. M.

    2017-01-01

    Aim: Survivors of neonatal chronic lung disease or bronchopulmonary dysplasia (BPD) suffer from compromised lung function and are at high risk for developing lung injury by multiple insults later in life. Because neonatal lysophosphatidic acid receptor-1 (LPAR1)-deficient rats are protected against hyperoxia-induced lung injury, we hypothesize that LPAR1-deficiency may protect adult survivors of BPD from a second hit response against lipopolysaccharides (LPS)-induced lung injury. Methods: Directly after birth, Wistar control and LPAR1-deficient rat pups were exposed to hyperoxia (90%) for 8 days followed by recovery in room air. After 7 weeks, male rats received either LPS (2 mg kg−1) or 0.9% NaCl by intraperitoneal injection. Alveolar development and lung inflammation were investigated by morphometric analysis, IL-6 production, and mRNA expression of cytokines, chemokines, coagulation factors, and an indicator of oxidative stress. Results: LPAR1-deficient and control rats developed hyperoxia-induced neonatal emphysema, which persisted into adulthood, as demonstrated by alveolar enlargement and decreased vessel density. LPAR1-deficiency protected against LPS-induced lung injury. Adult controls with BPD exhibited an exacerbated response toward LPS with an increased expression of pro-inflammatory mRNAs, whereas LPAR1-deficient rats with BPD were less sensitive to this “second hit” with a decreased pulmonary influx of macrophages and neutrophils, interleukin-6 (IL-6) production, and mRNA expression of IL-6, monocyte chemoattractant protein-1, cytokine-induced neutrophil chemoattractant 1, plasminogen activator inhibitor-1, and tissue factor. Conclusion: LPAR1-deficient rats have increased hyperoxia-induced BPD survival rates and, despite the presence of neonatal emphysema, are less sensitive to an aggravated “second hit” than Wistar controls with BPD. Intervening in LPA-LPAR1-dependent signaling may not only have therapeutic potential for neonatal chronic

  3. Inhibition of formyl peptide receptor 1 reduces the efficacy of anticancer chemotherapy against carcinogen-induced breast cancer

    PubMed Central

    Baracco, Elisa E.; Pietrocola, Federico; Buqué, Aitziber; Bloy, Norma; Senovilla, Laura; Zitvogel, Laurence; Vacchelli, Erika; Kroemer, Guido

    2016-01-01

    ABSTRACT The loss-of-function mutation of formyl peptide receptor 1 (FPR1) has a negative impact on the progression-free and overall survival of breast cancer patients treated with anthracycline-based adjuvant chemotherapy. This effect may be attributed to the fact that chemotherapy-induced antitumor immunity requires FPR1 and that such anticancer immune responses are responsible for the long-term effects of chemotherapy. Here, we investigated the possible contribution of FPR1 to the efficacy of a combination of mitoxantrone (MTX) and cyclophosphamide (CTX) for the treatment of hormone-induced breast cancer. Breast cancer induced by a combination of medroxyprogesterone acetate (MPA) and 7,12-Dimethylbenz[a]anthracene (DMBA) could be successfully treated with MTX plus CTX in thus far that tumor growth was retarded and overall survival was extended (as compared to vehicle-only treated controls). However, the therapeutic efficacy of the combination therapy was completely abolished when FPR1 receptors were blocked by means of cyclosporin H (CsH). Future genetic studies on neoadjuvant chemotherapy-treated breast cancers are warranted to validate these findings at the clinical level. PMID:27471610

  4. Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis

    PubMed Central

    Bai, Qifeng; Yao, Xiaojun

    2016-01-01

    Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1. PMID:26887338

  5. Association of estrogen receptor 1 (ESR1) haplotypes with risk for systemic lupus erythematosus among South Indians.

    PubMed

    Rupasree, Yedluri; Naushad, Shaik Mohammad; Rajasekhar, Liza; Uma, Addepally; Kutala, Vijay Kumar

    2015-11-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disorder involving genetic, epigenetic and environmental factors and has higher incidence in women. In this study, we explored the association of estrogen receptor 1 (ESR1) rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms with susceptibility to SLE. PCR-RFLP and ELISA were used for genetic analysis and determination of specific autoantibodies, respectively. The univariate analysis showed no independent association of rs2234693 (OR: 1.14, 95% CI: 0.87 - 1.49, p = 0.36) and rs9340799 (OR: 0.87, 95% CI: 0.66-1.14, p = 0.34). The haplotype analysis using SHEsis platform revealed strong linkage disequilibrium between these two polymorphisms (D': 0.81, r2: 0.55). Among the four haplotype groups, the C-A haplotype (rs2234693-rs9340799) was strongly associated with the risk for SLE (OR: 2.10, 95% CI: 1.32 - 3.34, p = 0.001). The homozygous variant genotype of rs2234693 exhibited elevated TNF-α and depleted IFN-α, while the effects of rs9340799 were contradictory. The wild genotype of rs2234693 exhibited lower levels of IL-12 with number of rs9340799 variant alleles pronouncing this effect. From this study, it is concluded that the ESR1 haplotypes may influence the Th2 cytokine profile and susceptibility to SLE among the South Indians.

  6. Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells.

    PubMed

    Rodríguez, José A; Luria-Pérez, Rosendo; López-Valdés, Héctor E; Casero, David; Daniels, Tracy R; Patel, Shabnum; Avila, David; Leuchter, Richard; So, Sokuntheavy; Ortiz-Sánchez, Elizabeth; Bonavida, Benjamin; Martínez-Maza, Otoniel; Charles, Andrew C; Pellegrini, Matteo; Helguera, Gustavo; Penichet, Manuel L

    2011-11-01

    A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.

  7. Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells

    PubMed Central

    Rodríguez, JoséA.; Luria-Pérez, Rosendo; López-Valdés, Héctor E.; Casero, David; Daniels, Tracy R.; Patel, Shabnum; Avila, David; Leuchter, Richard; So, Sokuntheavy; ánchez, Elizabeth Ortiz-S; Bonavida, Benjamin; Martínez-Maza, Otoniel; Charles, Andrew .C; Pellegrini, Matteo; Helguera, Gustavo; Penichet, Manuel L.

    2013-01-01

    A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies. PMID:21870996

  8. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    SciTech Connect

    Tsujiuchi, Toshifumi . E-mail: ttujiuch@life.kindai.ac.jp; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-10-27

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.

  9. Reevaluation of Fatty Acid Receptor 1 as a Drug Target for the Stimulation of Insulin Secretion in Humans

    PubMed Central

    Wagner, Robert; Kaiser, Gabriele; Gerst, Felicia; Christiansen, Elisabeth; Due-Hansen, Maria E.; Grundmann, Manuel; Machicao, Fausto; Peter, Andreas; Kostenis, Evi; Ulven, Trond; Fritsche, Andreas; Häring, Hans-Ulrich; Ullrich, Susanne

    2013-01-01

    The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists. PMID:23378609

  10. Sulfonylurea receptor -1 (SUR1): genetic and metabolic evidences for a role in the susceptibility to type 2 diabetes mellitus.

    PubMed

    Reis, A F; Velho, G

    2002-02-01

    The pancreatic B-cell ATP-sensitive potassium channel (K(ATP)) is composed of two distinct subunits, an inwardly rectifying ion channel forming the pore (Kir6.2), and a regulatory subunit, namely the sulfonylurea receptor-1 (SUR1), which binds this widely used class of insulin-secreting drugs. Mutations in the genes encoding Kir6.2 and SUR1 may result in familial persistent hyperinsulinemic hypoglycaemia of infancy, demonstrating their role in the regulation of insulin secretion. Studies in various populations with different ethnic background provided evidence that various alleles of single nucleotide polymorphisms (SNPs) in the SUR1 gene, and to a less extent in the Kir6.2 gene, confer a significantly increased risk for the development of type 2 diabetes mellitus (T2DM). Allelic variations of these SNPs were shown to modulate insulin secretion and insulin sensitivity in vivo, thus providing a pathophysiological background to explain their contribution to the genetic susceptibility to T2DM. The aim of this review is to summarise and discuss the significant results of recent literature on the implication of K(ATP), and particularly of SUR1, in the genetic and pathopysiological mechanisms of T2DM.

  11. Functional Genetic Variation of the Cannabinoid Receptor 1 and Cannabis Use Interact on Prefrontal Connectivity and Related Working Memory Behavior

    PubMed Central

    Colizzi, Marco; Fazio, Leonardo; Ferranti, Laura; Porcelli, Annamaria; Masellis, Rita; Marvulli, Daniela; Bonvino, Aurora; Ursini, Gianluca; Blasi, Giuseppe; Bertolino, Alessandro

    2015-01-01

    Cannabinoid signaling is involved in different brain functions and it is mediated by the cannabinoid receptor 1 (CNR1), which is encoded by the CNR1 gene. Previous evidence suggests an association between cognition and cannabis use. The logical interaction between genetically determined cannabinoid signaling and cannabis use has not been determined. Therefore, we investigated whether CNR1 variation predicts CNR1 prefrontal mRNA expression in postmortem prefrontal human tissue. Then, we studied whether functional variation in CNR1 and cannabis exposure interact in modulating prefrontal function and related behavior during working memory processing. Thus, 208 healthy subjects (113 males) were genotyped for the relevant functional SNP and were evaluated for cannabis use by the Cannabis Experience Questionnaire. All individuals performed the 2-back working memory task during functional magnetic resonance imaging. CNR1 rs1406977 was associated with prefrontal mRNA and individuals carrying a G allele had reduced CNR1 prefrontal mRNA levels compared with AA subjects. Moreover, functional connectivity MRI demonstrated that G carriers who were also cannabis users had greater functional connectivity in the left ventrolateral prefrontal cortex and reduced working memory behavioral accuracy during the 2-back task compared with the other groups. Overall, our results indicate that the deleterious effects of cannabis use are more evident on a specific genetic background related to its receptor expression. PMID:25139064

  12. Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

    SciTech Connect

    Lee, Dong Hoon; Jeon, Byeong Tak; Jeong, Eun Ae; Kim, Joon Soo; Cho, Yong Woon; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung; Roh, Gu Seob

    2010-03-12

    Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P{sub 1}) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P{sub 1} in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Flu