Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24.
ABSTRACT: Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.
Project description:<h4>Introduction</h4>Neurotensin receptor 1 (NTR-1) is expressed and activated in prostate cancer cells. In this study, we explore the NTR expression in normal mouse tissues and study the positron emission tomography (PET) imaging of NTR in prostate cancer models.<h4>Materials and methods</h4>Three <sup>64</sup>Cu chelators (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid [DOTA], 1,4,7-triazacyclononane-N,N',N?-triacetic acid [NOTA], or AmBaSar) were conjugated to an NT analog. Neurotensin receptor binding affinity was evaluated using cell binding assay. The imaging profile of radiolabeled probes was compared in well-established NTR<sup>+</sup> HT-29 tumor model. Stability of the probes was tested. The selected agents were further evaluated in human prostate cancer PC3 xenografts.<h4>Results</h4>All 3 NT conjugates retained the majority of NTR binding affinity. In HT-29 tumor, all agents demonstrated prominent tumor uptake. Although comparable stability was observed, <sup>64</sup>Cu-NOTA-NT and <sup>64</sup>Cu-AmBaSar-NT demonstrated improved tumor to background contrast compared with <sup>64</sup>Cu-DOTA-NT. Positron emission tomography/computed tomography imaging of the NTR expression in PC-3 xenografts showed high tumor uptake of the probes, correlating with the in vitro Western blot results. Blocking experiments further confirmed receptor specificity.<h4>Conclusions</h4>Our results demonstrated that <sup>64</sup>Cu-labeled neurotensin analogs are promising imaging agents for NTR-positive tumors. These agents may help us identify NTR-positive lesions and predict which patients and individual tumors are likely to respond to novel interventions targeting NTR-1.
Project description:Pancreatic adenocarcinomas express neurotensin receptors in up to 90% of cases, however, their role in tumor biology and as a drug target is not clear. In the present study, a stable neurotensin (NT) analog induced intracellular calcium release and intracellular alkalinization in BxPC-3 and PANC-1 pancreatic cancer cells that was abolished by inhibitors of NT receptor (NTR) and sodium-proton exchanger 1 (NHE1), amiloride and SR 142948, respectively. Activation of NHE1 involved increased phosphorylation of dimethylfumarate-sensitive mitogen- and stress-activated kinase 1/2 (MSK1/2). NTR signaling appears to promote a metastatic phenotype in pancreatic cancer cells by induction of localized extracellular acidification in normoxic cells, preceeding acidosis induced by hypoxia and switch to glycolysis in addition to increased expression of interleukin-8 (IL-8).
Project description:Stress affects immunity, but the mechanism is not known. Neurotensin (NT) and corticotropin-releasing hormone (CRH) are secreted under stress in various tissues, and have immunomodulatory actions. We had previously shown that NT augments the ability of CRH to increase mast cell-dependent skin vascular permeability in rodents. Here we show that NT triggered human mast cell degranulation and significantly augmented CRH-induced vascular endothelial growth factor (VEGF) release. Investigation of various signaling molecules indicated that only NF-?B activation was involved. These effects were blocked by pretreatment with the NTR antagonist SR48692. NT induced expression of CRH receptor-1 (CRHR-1), as shown by Western blot and FACS analysis. Interestingly, CRH also induced NTR gene and protein expression. These results indicate unique interactions among NT, CRH, and mast cells that may contribute to auto-immune and inflammatory diseases that worsen with stress.
Project description:Functional maintenance of the mammalian main olfactory epithelium (MOE) is challenging because of its direct exposure to a wide spectrum of environmental chemicals. We previously reported that transient receptor potential channel M5-expressing microvillous cells (TRPM5-MCs) in the MOE play an important role in olfactory maintenance. To investigate the underpinning mechanisms, we exposed transcription factor Skn-1a knockout (Skn-1a-/-) mice lacking TRPM5-MCs, and TRPM5-GFP mice to either vehicle (water) or a mixture of odorous chemicals and chitin for two weeks and analyzed the expression of olfactory signaling proteins using immunolabeling and neurotrophin (NT) and NT receptor (NTR) gene transcripts using real-time quantitative PCR. The chemical exposure did not significantly attenuate the immunolabeling of olfactory signaling proteins. Vehicle-exposed Skn-1a-/- and TRPM5-GFP mice expressed similar levels of NT and NTR gene transcripts in the MOE and olfactory bulb. Chemical exposure significantly increased MOE expression of p75NTR in Skn-1a-/- mice, while p75NTR expression was reduced in TRPM5-GFP mice, as compared to vehicle-exposed mice. Additionally, our RNA in situ hybridization analysis and immunolabeling confirmed MOE expression of most NTs and NTRs. Together, these results indicate that TRPM5-MCs and chemical exposure influence expression of some NTs and NTRs in the MOE and olfactory bulb (OB).
Project description:Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives. The activity of NTRs in bacteria facilitates the metabolic activation and antibacterial activity of 5-nitroimidazoles. Therefore, NTR activity correlates with the drug susceptibility and resistance of pathogenic bacteria. As such, it is important to develop a rapid and visual assay for the real-time sensing of bacterial NTRs for the evaluation and development of antibiotics. Herein, an activatable near-infrared fluorescent probe (<b>HC-NO</b><sub><b>2</b></sub>) derived from a hemicyanine fluorophore was designed and developed based on two evaluation factors, including the calculated partition coefficient (Clog <i>P</i>) and fluorescence wavelength. Using <b>HC-NO</b><sub><b>2</b></sub> as the special substrate of NTRs, NTR activity can be assayed efficiently, and then, bacteria can be imaged based on the detection of NTRs. More importantly, a sensitive in-gel assay using <b>HC-NO</b><sub><b>2</b></sub> has been developed to selectively identify NTRs and sensitively determine NTR activity. Using the in-gel assay, NTRs from various bacterial species have been profiled visually from the "fluorescence fingerprints", which facilitates the rapid identification of NTRs from bacterial lysates. Thus, various homologous NTRs were identified from three metronidazole-susceptible bacterial species as well as seven unsusceptible species, which were confirmed by the whole-genome sequence. As such, the evaluation of NTRs from different bacterial species should help improve the rational usage of 5-nitroimidazole drugs as antibiotics.
Project description:Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with <i>in situ</i> hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.
Project description:This work describes the cloning and expression of the levocabastine-sensitive neurotensin (NT) receptor from mouse brain. The receptor protein comprises 417 amino acids and bears the characteristics of G-protein-coupled receptors. This new NT receptor (NTR) type is 39% homologous to, but pharmacologically distinct from, the only other NTR cloned to date from the rat brain and the human HT29 cell line. When the receptor is expressed in Xenopus laevis oocytes, the H1 antihistaminic drug levocabastine, like NT and neuromedin N, triggers an inward current. The pharmacological properties of this receptor correspond to those of the low-affinity, levocabastine-sensitive NT binding site described initially in membranes prepared from rat and mouse brain. It is expressed maximally in the cerebellum, hippocampus, piriform cortex, and neocortex of adult mouse brain.
Project description:Importin-(Imp)β family nucleocytoplasmic transport receptors (NTRs) are supposed to bind to their cargoes through interaction between a confined interface on an NTR and a nuclear localization or export signal (NLS/NES) on a cargo. Although consensus NLS/NES sequence motifs have been defined for cargoes of some NTRs, many experimentally identified cargoes of those NTRs lack those motifs, and consensus NLSs/NESs have been reported for only a few NTRs. Crystal structures of NTR-cargo complexes have exemplified 3D structure-dependent binding of cargoes lacking a consensus NLS/NES to different sites on an NTR. Since only a limited number of NTR-cargo interactions have been studied, whether most cargoes lacking a consensus NLS/NES bind to the same confined interface or to various sites on an NTR is still unclear. Addressing this issue, we generated four mutants of transportin-(Trn)SR, of which many cargoes lack a consensus NLS, and eight mutants of Imp13, where no consensus NLS has been defined, and we analyzed their binding to as many as 40 cargo candidates that we previously identified by a nuclear import reaction-based method. The cargoes bind differently to the NTR mutants, suggesting that positions on an NTR contribute differently to the binding of respective cargoes.
Project description:Hazara nairovirus (HAZV) is a member of the family <i>Nairoviridae</i> in the order <i>Bunyavirales</i> and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3' and 5' NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3' NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5' NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent <i>cis</i>-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed.<b>IMPORTANCE</b> Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3' and 5' ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3' and 5' NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.
Project description:<h4>Background</h4>Neo-tetraploid rice (NTR) is a useful new germplasm that developed from the descendants of the autotetraploid rice (ATR) hybrids. NTR showed improved fertility and yield potential, and produced high yield heterosis when crossed with indica ATR for commercial utilization. However, their classification, population structure and genomic feature remain elusive.<h4>Results</h4>Here, high-depth genome resequencing data of 15 NTRs and 18 ATRs, together with 38 publicly available data of diploid rice accessions, were analyzed to conduct classification, population structure and haplotype analyses. Five subpopulations were detected and NTRs were clustered into one independent group that was adjacent to japonica subspecies, which maybe the reason for high heterosis when NTRs crossed with indica ATRs. Haplotype patterns of 717 key genes that associated with yield and other agronomic traits were revealed in these NTRs. Moreover, a novel specific SNP variation was detected in the first exon of HSP101, a known heat-inducible gene, which was conserved in all NTRs but absent in ATRs, 3KRG and RiceVarMap2 databases. The novel allele was named as HSP101-1, which was confirmed to be a heat response factor by qRT-PCR, and knockout of HSP101-1 significantly decreased the thermotolerance capacity of NTR. Interestingly, HSP101-1 was also specifically expressed in the anthers of NTR at pre-meiotic and meiosis stages under optimal environment without heat stress, and its loss-of-function mutant showed significant decrease in fertility of NTR.<h4>Conclusion</h4>The construction of first genomic variation repository and the revelation of population structure provide invaluable information for optimizing the designs of tetraploid rice breeding. The detection of specific genomic variations offered useful genomic markers and new directions to resolve high fertility mechanism of NTR.