We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.
Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up-or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is downregulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.
Arnt2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1) that was completely established. Whereas Arnt2 gene expression, established by Northern blotting and in situ hybridization histochemistry, occurred selectively in brain and kidney, that of Arnt1 was ubiquitous, suggesting that the two proteins play distinct roles, presumably via dimerization and DNA binding with different partners.Arnt, the Ah receptor nuclear translocator, is a member of the basic helix-loop-helix (bHLH) superfamily of DNA binding proteins, originally found to form a heterodimer with the aryl hydrocarbon receptor (Ahr) in response to environmental pollutants such as dioxins. The Ahr/Arnt heteromer translocates to the nucleus and binds specific recognition sequences localized upstream of various dioxin-inducible genes such as cytochromes P450 [reviewed in refs. 1-3].Arnt sequence contains a conserved region termed the PAS domain and shared by other transcription factors [4]. Recent observations suggest that Arnt might have regulatory roles via heterodimerization not only with Ahr but also with other bHLH-containing PAS partners such as the single-minded protein (Sim), involved in central nervous system development [5,6] and the hypoxia-inducible factor 1a (HIF-1a) involved in adaptive responses of various cells and tissues to hypoxia [7].Following RTPCR amplification of cerebral mRNAs using degenerate amplimers of heptahelical receptors, we unexpectedly cloned a cDNA of unknown sequence displaying however distinct homology with that of human or mouse Arnt (called Arnt1 here) [4,8]. Here we report the full length sequence of this novel protein (designated Arnt2) as well as that of rat Arnt1, of which only a fraction was previously reported [9]. The latter was established for purpose of comparison and in order to design selective hybridization probes. Northern blot and in situ hybridization studies indicate that the two homologous gene transcripts are differently distributed among rat tissues. Recently, while this manuscript was in preparation, the sequence and properties of a mouse Arnt2 were disclosed [10].
We described previously the cDNA cloning of three functional rat histamine H 3 receptor (rH 3 R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH 3A , rH 3B , and rH 3C receptor isoforms (Mol Pharmacol 59:1-8). In the current report, we describe the cDNA cloning, mRNA localization in the rat central nervous system, and pharmacological characterization of three additional rH 3 R splice variants (rH 3D , rH 3E , and rH 3F ) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H 3 R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH 3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H 3 R ligands could not be detected for the 6TM-rH 3 isoforms. In contrast to the rH 3A receptor (rH 3A R), detection of the rH 3D isoform using hemagglutinin antibodies revealed that the rH 3D isoform remains mainly intracellular. The expression of the rH 3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H 3 R isoforms. Coexpression of the rH 3A R and the rH 3D isoforms resulted in the intracellular retention of the rH 3A R and reduced rH 3A R functionality. Finally, we show that in rat brain, the H 3 R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH 3 R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.
1. The pharmacological properties and location of H3 receptors modulating acetylcholine release have been investigated in non-superfused slices and synaptosomes of rat entorhinal cortex preloaded with [3H]-choline. 2. (R)alpha-methylhistamine, an H3-receptor agonist, potently inhibited the K(+)-evoked tritium release from slices, an effect antagonized by thioperamide, an H3-receptor antagonist, with nanomolar potency. 3. The K(+)-evoked tritium release from synaptosomes remained unaltered in the presence of the potent and selective H3-receptor agonists, imetit and (R)alpha-methylhistamine, suggesting that H3 receptors modulating acetylcholine release are not presynaptically located on cholinergic nerve terminals. 4. Phenylbutanoylhistamine and phenylpropylhistamine, two H3-receptor antagonists of moderate potency, failed to antagonize the inhibitory effects of (R)alpha-methylhistamine observed in slices. Unexpectedly, both compounds when used alone, inhibited tritium release from slices and synaptosomes with micromolar potency and to the same extent (by approximately 50% when added at a final concentration of 200 microM). This inhibitory effect did not involve H1, H2 or H3 receptors and was not mediated by an unknown histamine receptor site, since histamine used at a high concentration neither reproduced nor antagonized the effect of phenylbutanoylhistamine. It remained unaltered in the presence of scopolamine and was neither mimicked nor antagonized by vasoactive intestinal peptide, previously shown to be colocalized with acetylcholine in some neurones. 5. It is concluded that acetylcholine release in rat entorhinal cortex is modulated by H3 receptors presumably not located on cholinergic axon terminals. It remains to be established whether these H3 receptors belong to a receptor subtype different from those previously described since the potency ofphenylbutanoylhistamine and phenylpropylhistamine as H3-receptor antagonists was probably greatly underestimated by additional properties of both drugs.
The two isoforms of p190 RhoGAP (p190A and p190B) are important regulators of RhoGTPase activity in mammalian cells. Both proteins are ubiquitously expressed, are involved in the same signalling pathways and interact with the same identified binding partners. In search of isoform functional specificity, we knocked down the expression of each p190 protein using siRNA and examined the resulting phenotypic changes in human umbilical vein endothelial cells (HUVECs). We provide evidence that p190B plays a crucial role in the regulation of MT1-MMP expression and cell-surface presentation, as well as subsequent MMP2 activation. p190B is involved in both local extracellular matrix degradation at podosomes and endothelial cell assembly into tube-like structures in Matrigel. In addition, whereas p190B knockdown does not affect podosome formation, p190A knockdown increases the number of cells showing podosome structures in HUVECs. We conclude that the two p190 RhoGAP isoforms play distinct roles in endothelial cells. In addition, our data reveal an unsuspected role for p190B in the expression of the two collaborative proteases MT1-MMP and MMP2, thereby affecting matrix remodelling and angiogenesis.
The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix-loop-helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival.
RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.
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