scholarly journals Protein Kinase A-Dependent Derepression of the Human Prodynorphin Gene via Differential Binding to an Intragenic Silencer Element

1998 ◽  
Vol 18 (12) ◽  
pp. 6921-6929 ◽  
Author(s):  
Angel M. Carrión ◽  
Britt Mellström ◽  
Jose R. Naranjo
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Time Course ◽  
Neuroblastoma Cells ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Human Neuroblastoma Cells ◽  
Basal Transcription ◽  
Human Neuroblastoma ◽  
Kinase A

ABSTRACT Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5′ deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5′ untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

scholarly journals DREAM-αCREM Interaction via Leucine-Charged Domains Derepresses Downstream Regulatory Element-Dependent Transcription

2000 ◽  
Vol 20 (24) ◽  
pp. 9120-9126 ◽  
Author(s):  
Fran Ledo ◽  
Angel M. Carrión ◽  
Wolfgang A. Link ◽  
Britt Mellström ◽  
José R. Naranjo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Regulatory Element ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cells ◽  
Binding Properties ◽  
Human Neuroblastoma ◽  
Protein Protein Interaction ◽  
Kinase A

ABSTRACT Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, αCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of αCREM is not required for the interaction, but phospho-αCREM shows higher affinity for DREAM. The interaction with αCREM is independent of the Ca2+-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both αCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca2+.

scholarly journals Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2295-2305 ◽  
Author(s):  
Yong Xu ◽  
Teresa L. Krukoff
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Protein Kinase A ◽  
Neuroblastoma Cells ◽  
No Synthase ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
No Release ◽  
Kinase A

Abstract We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pm to 100 nm) stimulated NO release from the cells, with a maximal response observed with 1 nm at 30 min. This response was blocked by 1 nm ADM22–52, an ADM receptor antagonist or 2 μm vinyl-l-NIO, a neuronal NO synthase inhibitor. In addition, 5 μm 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when l-type calcium channels were inhibited with 5 μm nifedipine or 10 μm nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 μm cyclopiazonic acid or 150 nm thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 μm ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mm dibutyryl cAMP, a cAMP analog, and were abrogated by 5 μm H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nm) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 μm) or H-89 (5 μm) and was reduced by ryanodine (10 μm). These results suggest that ADM stimulates calcium influx through l-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+]i cause activation of neuronal NO synthase and NO release.

scholarly journals Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A

2014 ◽  
Vol 307 (3) ◽  
pp. C266-C277 ◽  
Author(s):  
Shaima Salman ◽  
Alison C. Holloway ◽  
Colin A. Nurse
Keyword(s):  
Protein Kinase ◽  
Carbonic Anhydrase ◽  
Protein Kinase A ◽  
Chromaffin Cells ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Neonatal Rat ◽  
Pcr Analysis ◽  
Kinase A

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K+ (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ {[d-Arg2,Ly4]dermorphin-(1–4)-amide}- and δ ([d-Pen2,5,P-Cl-Phe4]enkephalin)-opioid agonists (2 μM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

Activation of DNA-Dependent Protein Kinase May Play a Role in Apoptosis of Human Neuroblastoma Cells

2008 ◽  
Vol 72 (3) ◽  
pp. 933-942 ◽  
Author(s):  
Balu R Chakravarthy ◽  
Teena Walker ◽  
Ingrid Rasquinha ◽  
Irene E Hill ◽  
John P MacManus
Keyword(s):  
Protein Kinase ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cells ◽  
Dependent Protein Kinase ◽  
Human Neuroblastoma ◽  
Dependent Protein
Sign in / Sign up

Export Citation Format

Share Document