Abstract
Inducible nitric oxide synthase, the critical enzyme responsible for the enhanced synthesis of nitric oxide in inflammatory states, is widely expressed in mammalian cells. To evaluate potential regulatory roles of the 5'-untranslated region (UTR) in the human inducible nitric oxide synthase gene, the transcription initiation sites and structure of the 5'-UTR of human inducible nitric oxide synthase were examined. Freshly isolated human alveolar macrophages, bronchial epithelial cells, and several types of cultured cells were evaluated following stimulation with cytokines (i.e. interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6). The mRNA was analyzed by reverse transcription-polymerase chain reaction. Northern analysis, and 5'-rapid amplification of cDNA ends. Despite the presence of a TATA box in the promoter region, multiple transcription initiation sites were observed, some extending several hundred base pairs upstream from the main TATA-directed initiation site. Alternative splicing in the 5'-UTR of human inducible nitric oxide synthase mRNA resulted in further diversity. The TATA-independent inducible nitric oxide synthase mRNA transcripts were up-regulated by cytokines. The long and complex 5'-UTRs contain eight partially overlapping open reading frames upstream of the putative inducible nitric oxide synthase ATG, which may have an important role in translational regulation of human inducible nitric oxide synthase mRNA.
Highlights
Nitric oxide (NO)l appears to be critical in the physiology or pathophysiology of every organ system [1,2,3,4]
Human iNOS mRNA Transcripts with Deletion ofPreviously Reported Exon i-Evaluation ofiNOS mRNA transcripts in the region encompassing the 5'-flanking region and exons 1-5 by reverse transcriptionpolymerase chain reaction (RT-PCR) (Fig. 1, top panel) demonstrated two different transcripts in cytokine-stimulated A549 cells, DLD-1 cells, and human bronchial epithelial primary culture (HBEC) cells (Fig. lA, lanes 2, 4, and 6), resting DLD-1 and T84 cells (Fig. lA, lanes 3 and 7), and freshly isolated NHBE cells and alveolar macrophages (Fig. lA, lanes 8 and 9). iNOS transcripts were not observed in unstimulated A549 or HBEC cells (Fig. lA, lanes 1 and 5)
We describe here the complexity of the 5'-untranslated regions (UTRs) of human iNOS mRNA
Summary
Nitric oxide (NO)l appears to be critical in the physiology or pathophysiology of every organ system [1,2,3,4]. In 425 bp of its 5'-flanking region are found a TATA box and consensus sequences for three interferon-y response elements, an NF-KB site, an A activator-binding site, and a degenerate (but palindromic) tumor necrosis factor response element These findings are consistent with the observation that the iNOS gene is inducible by cytokines. Due to the diversity and multiplicity of transcription element consensus sequences in the promoter region of the iNOS gene, previously unidentified transcription initiation sites could exist in the 5'-UTR. To evaluate this hypothesis, the 5'-UTR of human iNOS mRNA was characterized by using reverse transcriptionpolymerase chain reaction (RT-PCR), Northern analysis, and 5' -rapid amplification of cDNA ends (RACE). The study demonstrates that, due to multiple transcription initiation sites and alternative splicing, there exist multiple forms of exon 1 in iNOS mRNA transcripts from human epithelial cells and alveolar macrophages, with production of these iNOS mRNAs enhanced by cytokines
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