Cyclic AMP-mediated upregulation of the expression of neuronal NO synthase in human A673 neuroepithelioma cells results in a decrease in the level of bioactive NO production: analysis of the signaling mechanisms that are involved.
The expression level of neuronal nitric oxide synthase (nNOS) can vary depending on the (patho)physiological conditions. Here we document a marked induction of nNOS mRNA, protein, and total NO production in response to dibutyryl cyclic AMP (db-cAMP) in human A673 neuroepithelial cells. However, the upregulation of nNOS was associated with a decreased level of production of bioactive NO and by an increase in the level of generation of reactive oxygen species (ROS). ROS production could be prevented by the NOS inhibitor L-NAME, suggesting nNOS itself is involved in ROS generation. Sepiapterin supplementation of db-cAMP-treated A673 cells could restore full bioactive NO production, most likely by preventing the uncoupling of nNOS. nNOS was upregulated by other stable analogues of cAMP, by the activator of adenylyl cyclase forskolin, by isoproterenol or by dopamine through activation of D1 receptors, and by inhibitors of phosphodiesterase. cAMP did not change the half-life of the nNOS mRNA. Inhibitors of protein kinase A (PKA), H-89 and R(p)-cAMPS, produced a partial inhibition of basal and cAMP-induced nNOS expression. cAMP response element binding and modulator transcription factors (CREB and CREM), typical target proteins of PKA, were expressed in A673 cells, as was the coactivator CREB binding protein (CBP). cAMP-stimulated induction of nNOS was significantly enhanced in A673 cells stably transfected with wild-type CREB and almost abolished in cells transfected with KCREB (containing a mutation of the DNA binding domain). In A673 cells transfected with CREB(133) (containing a mutation of the phosphorylatable serine 133), the overall level of nNOS expression was reduced, but the expressional stimulation by cAMP remained. This suggests that CREB bypasses, in part, the classical requirement for phosphorylation and association with CBP. Three members of the recently described four-and-a-half-LIM-domain proteins (FHL1-FHL3) were found to be expressed in A673 cells; FHL-1 and FHL-3 were upregulated by cAMP. These proteins can provide direct activation function to both CREB and CREM, and may be responsible for the PKA-independent component of CREB and CREM activity.
Boissel
Jean-Paul
JP
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. boissel@mail.uni-mainz.de
Bros
Matthias
M
Schröck
Andreas
A
Gödtel-Armbrust
Ute
U
Förstermann
Ulrich
U
D000818:Animals; D016375:Antisense Elements (Genetics); D001798:Blood Proteins / Q000378:metabolism; D003994:Bucladesine; D003180:Complement C3b Inactivator Proteins; D017242:Complement Factor H; D000242:Cyclic AMP / Q000494:pharmacology*; D051786:Cyclic AMP Response Element Modulator; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006136:GTP Cyclohydrolase / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D018241:Neuroectodermal Tumors, Primitive, Peripheral / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D015854:Up-Regulation
D016375:Antisense Elements (Genetics); D001798:Blood Proteins; C517621:CFHR1 protein, human; D003180:Complement C3b Inactivator Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C117563:FHL3 protein, human; C501160:Fhl3 protein, mouse; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D011622:Pterins; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D051786:Cyclic AMP Response Element Modulator; D009569:Nitric Oxide; D003994:Bucladesine; D017242:Complement Factor H; C016727:sepiapterin; D000242:Cyclic AMP; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D006136:GTP Cyclohydrolase; D019331:NG-Nitroarginine Methyl Ester