Regulation Of Neuronal Nitric Oxide Synthase (nNOS) Gene Expression By Nuclear Factor (NF)-κB Acetylation

Regulation of Neuronal Nitric Oxide Synthase (nNOS) Gene Expression by Nuclear Factor (NF)-κB AcetylationIntroductionIn the nervous system, nitric oxide (NO) acts as a neurotransmitter, a neuromodulator or an intracellular signaling molecule that plays highly diversified biological roles. However deregulated excess generation of NO can initiate a neurotoxic cascade, which is involved in neuronal disorders. Regulation of NO production is therefore particularly important in maintaining the physiological function of the nervous system. NO is synthesized from L-arginine by three isoforms of NO synthase (NOS) (neuronal, nNOS; inducible, iNOS; and endothelial, eNOS), of which the nNOS is predominant in the neuronal system and participates in a number of physiological and pathological processes. The transcriptional regulation of nNOS gene is crucial for the nNOS-derived NO biosynthesis. Therefore, considerable efforts have been directed toward identifying the mechanisms that tightly regulate nNOS expression in response to physiological and pathophysiological stimuli. Recent findings support a complex pattern of transcriptional regulation of nNOS due to twelve alternative first exons (1a-11), leading to different transcripts. The expression of distinct exon 1 variants is facilitated by the strength of different promoters. The expression of exon If is relatively high in brain.Acetylation is now known to play a major role in governing transcription of a variety of genes. Two classes of enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in the regulation of the protein acetylation state and have opposite functions, p300 is a transcription coactivator that has HAT activity. Previous studies have demonstrated that trichostatin A (TSA) specifically inhibits HDAC activity and recruits HAT during transcription so as to augment acetylation of histones and certain transcription-related factors and results in changes of protein biological activities in regulating gene expression. Nuclear factor (NF)-κB is an important transcription factor family subject to acetylation that include five subunits such as p65 and p50.Up to date, there is no report concerning the regulation of nNOS gene expression by protein acetylation. In the present study, we used TSA as a treatment of acetylation and studied the effect of acetylation on nNOS expression in human neuroblastoma SK-N-SH cells. We identified that the influence of TSA-induced NF-κB acetylation on nNOS If promoter activity may be one of the mechanisms by which acetylation regulates nNOS expression. Furthermore, we probed the role of p300 in the regulation of NF-κB acetylation status by TSA.Materials and MethodsMaterials1. Human neuroblastoma cell line SK-N-SH2. Reagents for real-time RT-PCR and nuclear run-off assay3. Reagents for Western blot and NO-specific fluorescent dye DAF-2 DA4. Reagents for gene cloning and luciferase assay5. Reagents for electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (CHIP) and immunoprecipitation (IP)Methods1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to test the changes of total and exon 1f-specific nNOS mRNA level with TSA treatment. The effect of TSA on nNOS transcription rate was detected by nuclear run-off assay.2. Western blot was performed to test the influence of TSA on nNOS protein level and DAF-2 DA fluorescent dye was used to study the effect of TSA on nNOS-formed NO production.3. nNOS 1f promoter structure was analyzed by softwares. A series of truncated luciferase reporter vectors were constructed and Dual-Glo luciferase assay system was used to detect the activity of different parts of the promoter.4. EMSA was carried out to identify the NF-κB responsive element within nNOS 1f promoter and the effect of TSA on NF-κB binding under in vitro conditions. ChIP assay were used to further test the binding of NF-κB to this site and the effect of TSA on its binding in vivo.5. Immunoprecipitaion using specific antibodies of NF-κB p65 and p50 subunits combined with Western blot using monoclonal antibody against acetylated lysine as primary antibody was used to test the effect of TSA on p65 and p50 acetylation level.6. Luciferase assay was performed to detect the role of NF-κB element in the regulation of nNOS If promoter activity by TSA.7. Coimmunoprecipitation using p65, p50 or p300 anbitoby in immuoprecipitation and p300, p65 or p50 antibody in Western blot was carried out to analyze the interaction of p300 with NF-κB p65 and p50 subunits and the influence of TSA on their interations. ChIP assay with p300 antibody was performed to identify the binding of p300 to chromatin NF-κB responsive element region and the effect of TSA on the binding. Western blot was carried out to test the influence of TSA on p300 protein expression.8. After cells were transfected with wild-type or HAT domain-deleted mutant p300 expression vectors, immunoprecipitation was performed with p300 antibody followed by Western blot using specific antibody against acetylated lysine to detect the role of p300 in acetylating NF-κB and the effect of TSA on p300 function.Results1. Real-time RT-PCR results showed exon 1f-specific transcript was the major transcript of nNOS gene in SK-N-SH cells and TSA may upregulate total and exon 1f-specific nNOS mRNA in a time and concentration dependent manner. Meanwhile, TSA was demonstrated to increase nNOS transcription rate by nuclear run-off assay.2. Western blot showed TSA may increase nNOS protein expression and DAF-2 DA dye experiment indicated nNOS-derived NO output was increased by TSA in SK-N-SH cells.3. Software analysis suggested a number of putative responsive elements of transcription factors that are subject to acetylation within nNOS If promoter. Luciferase assay indicated two positive and two negative regulatory regions.4. EMSA demonstrated the position -893～-884 of the nNOS If promoter was an NF-κB responsive element and TSA may enhance the binding of NF-κB p65 and p50 subunits to this site in vitro. ChIP assay obtained the same results in vivo.5. Immunoprecipitation combined with Western blot showed acetylated NF-κB p65 and p50 subunits under basal state and demonstrated TSA may increase their acetylation level. 6. Results of luciferase assay showed the identified NF-κB responsive element acted as a powerful positive regulation element and took an important part in TSA-induced activation of nNOS 1f promoter.7. Coimmunoprecipitation demonstrated p300 may interact with NF-κB p65 and p50 subunits in nuclei and TSA enhanced their interactions. Results from ChIP assay suggested p300 may bind to the chromatin region of the NF-κB responsive element within the nNOS 1f promoter and TSA augmented the binding. Western blot analysis suggested TSA upregulated p300 protein expression.8. Results from immunoprecipitation and Western blot demonstrated p300 may directly acetylate NF-κB p65 and p50 subunits and TSA enhanced the function of p300.Conclusion1. The position -893～-884 of the nNOS 1f promoter was an NF-κB responsive element.2. TSA may increase the interaction of p300 with NF-κB p65 and p50 subunits within the identified NF-κB responsive element region so as to enhance the acetylation of p65 and p50 by p300. As a result, their DNA binding affinity was increased and the nNOS 1f promoter was activated. This is one of the mechanisms by which TSA increased nNOS mRNA and protein level and nNOS-derived NO production.3. Exon 1f-specific transcript was the major transcript of nNOS gene in SK-N-SH cells.