Opioid-induced regulation of gene expression in PC12 cells stably transfected with mu-opioid receptor

Zarnegar, Parisa; Persson, Anders I.; Yu, Ming; Terenius, Lars

doi:10.1016/j.neulet.2005.11.040

Cited by 8 publications

(7 citation statements)

References 21 publications

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“…PC12 cells were transformed to express stably human MOP (hMOP) (43) or rMOP-EGFP (13) or EGFP alone (pEGFP-N1; Clontech, Mountain View, CA). Plasmids containing cDNAs for eukaryotic expression were encapsulated in cationic liposomes and delivered to the cells using Lipofectamine (Invitrogen).…”

Section: Plasmid Constructs and Stable Transfectionmentioning

confidence: 99%

µ‐Opioid receptor activation in live cells

Vukojević¹,

Yu²,

Hansen³

et al. 2008

FASEB j.

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Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80+/-10%) were characterized by a diffusion coefficient D(MOP,1) = (4+/-2) x 10(-11) m(2) s(-1), compared with the slowly moving fraction, D(MOP,2) = (4+/-2) x 10(-12) m(2) s(-1). On stimulation with selected agonists ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.

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Section: Plasmid Constructs and Stable Transfectionmentioning

confidence: 99%

µ‐Opioid receptor activation in live cells

Vukojević¹,

Yu²,

Hansen³

et al. 2008

FASEB j.

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“…In neuronal cell lines, expression of Rgs4 is reduced after treatment with nerve growth factor [13], cAMP [14] or camptothecin [15], whereas opioid receptor agonists lead to an increase in the expression levels of Rgs4 mRNA [16] and RGS4 protein [17]. Administration of corticosterone to adult rats decreases the level of Rgs4 mRNA in the paraventricular nucleus of the hypothalamus and increases the levels in locus coeruleus [18], but has no effect in the hippocampus [19], [20].…”

Section: Introductionmentioning

confidence: 99%

MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells

Zhang

Liu

et al. 2012

PLoS ONE

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Background Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1β is mediated by the activation of NFκB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells.Methodology/Principal FindingsCultured cells at first passage were treated with or without IL-1β (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1β stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1β. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1β treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1β increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter.Conclusion/SignificanceActivation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.

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“…However, the reports on the expression levels of Rgs4 under various neuropathologic conditions remain inconsistent (Guo et al, 2006; Li and He, 2006; Rizig et al, 2006; van Gemert et al, 2006a; Stuart Gibbons et al, 2008). In neuronal cell line, expression of Rgs4 is reduced after treatment with nerve growth factor (Krumins et al, 2004), cAMP (Pepperl et al, 1998) or camptothecin (Song and Jope, 2006), whereas opioid receptor agonists lead to an increase in Rgs4 mRNA expression (Zarnegar et al, 2006). Administration of corticosterone to adult rats decreased the level of Rgs4 mRNA in the paraventricular nucleus of the hypothalamus, and increased the levels in locus coeruleus (Ni et al, 1999), but had no effect in the hippocampus (van Gemert et al, 2006a; van Gemert et al, 2006b).…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

Murthy

Khalili

et al. 2010

Gene

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Regulator of G-protein signaling 4 (Rgs4) regulates the strength and duration of G-protein signaling, and plays an important role in cardiac development, smooth muscle contraction and psychiatric disorders. Rgs4 expression is regulated at both mRNA and protein levels. In order to examine the transcriptional mechanism of Rgs4 expression, we have cloned and characterized rabbit Rgs4 promoter. The coding sequence of rabbit Rgs4 was obtained by degenerative RT-PCR and used for Northern blot and 5′-RACE analysis. A single transcript was identified in rabbit colonic smooth muscle cells. The 5′-untranslated region (UTR) extended 120 bp nucleotides upstream of the Rgs4 start codon. A putative promoter sequence (1389 bp) showed a consensus TATA box and cis-acting binding sites for several potential transcriptional factors. Reporter gene assay identified strong promoter activity in various cell types. Further analysis by deletion mutagenesis suggested that the proximal region had a highest core promoter activity while the distal region is suppressive. IL-1β significantly increased the promoter activity. The in vitro and in vivo binding activities for NF-κB transcription factor were validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay respectively. Mutation of NF-κB site reduced the promoter activity. These data suggest that the cloned rabbit Rgs4 promoter is functionally active and NF-κB binding site possesses enhancer activity in regulating Rgs4 transcription. Our studies provide an important basis for further understanding of Rgs4 regulation and function in different diseases.

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Opioid-induced regulation of gene expression in PC12 cells stably transfected with mu-opioid receptor

Cited by 8 publications

References 21 publications

µ‐Opioid receptor activation in live cells

µ‐Opioid receptor activation in live cells

MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells

Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

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