Dual role of cAMP in iNOS expression in glial cells and macrophages is mediated by differential regulation of p38-MAPK/ATF-2 activation and iNOS stability.
We reported previously that cAMP analogues or cAMP synthesis activator (forskolin; FSK) inhibit lipopolysaccharide (LPS)-induced inducible nitric-oxide systase (iNOS) gene expression in astrocytes, while they enhance that in macrophages. Here, we report that the FSK-mediated inhibition of iNOS expression in C6 glial cells is due to its reduced transcriptional activity, while the FSK-mediated enhancement of iNOS expression in RAW264.7 macrophages is a result of increased stability of iNOS protein without transcriptional enhancement. The LPS/interferon-gamma (IFN)-induced iNOS transcription was inhibited by FSK via inhibition of p38-MAPK/ATF-2 activity in glial cells while it was not affected in macrophages. In both cell types, proteasome activities were required for the spontaneous degradation of iNOS protein, and the inhibition of proteasome activity by MG132 after maximum increase of iNOS protein levels further enhanced iNOS protein induction by LPS/IFN, suggesting the involvement of proteasome in iNOS degradation. More importantly, the iNOS protein levels were equalized by the MG132 posttreatment in macrophages treated with LPS/IFN alone and along with FSK, and ubiquitinated iNOS protein levels were reduced by FSK posttreatment, suggesting that the FSK-mediated inhibition of ubiquitination of iNOS protein and the following increased stability of iNOS protein are one of the mechanisms of cAMP-pathway-mediated enhancement of iNOS gene expression in macrophages. To our knowledge, this is the first evidence that cAMP regulates iNOS expression at the posttranslational level in macrophages.
Won
Je-Seong
JS
Developmental Neurogenetics, Department of Pediatrics; Department of Pathology, Medical University of South Carolina, 316 CSB, Charleston, SC 29425, USA.
Im
Yeong-Bin
YB
Singh
Avtar K
AK
Singh
Inderjit
I
D051698:Activating Transcription Factor 2; D000818:Animals; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects; D007976:Leupeptins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D007976:Leupeptins; D008070:Lipopolysaccharides; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D025801:Ubiquitin; D005576:Colforsin; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D048051:p38 Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde