Regulation of human neuronal Nitric Oxide Synthase gene transcription containing a splicing form exon 1f by Creb and NF-kB-like AP-2 transcriptional factors

초록

Mammalian neuronal NO synthase(nNOS) gene is one of the most complicated genes. It is regulated differentially by several transcriptional factors and alternative spliced exons distributed in tissue-specific manner. Particularly in human nNOS gene, nine alternatively spliced first exons( exon 1a to li) in its mRNA with different sixed 5’-UTRs and altered translational efficiency have been reported. These include also the tissue-specific transcripts like the major nNOS isoforms. However, little is known about the transcriptional regulation of nNOS gene both in CNS and PNS. In the present study, we report that transcription of human nNOS exon 1f gene transcripts distributed predominantly in brain and gastrointestinal tract is controlled by dinucleotide repeated sequences (suppressive function of Z-DNA structure) and by two major transcriptional factors, cyclic-AMP response element binding protein (CREB) and nuclear factor kappa B-like activating protein-2 (NF-Κb-ke AP-2,) In various neuronal cells (SH-SY5Y, CATH.a, and PC12) and one muscle cells (NOR-1) cells trnsfected transiently with luciferase construct (pGL-2 basic vector) and containing CRE and NF-Κb-like AP-2 binding sites modified by site-directed mutagenesis, the luciferase activity was decreased partially and completely ,respectively . Furthermore, in the cells containing the modified mutant forms, the complexicity of the major binding proteins decreased markedly. Based on the results, we suggest that transcription nNOS gene with exon 1f spliced form can be regulated by the proteins that bind to CRE and NF-Κb-like AP-2,This may be involved in the differential release of NO in the neuronal systems contained in brain and gastrointestinal tract.