Analysis of the transcriptional and post-transcriptional regulation of human iNOS expression

诱导型一氧化氮合酶（iNOS）产生的大量NO具有有益的抗病毒、抗寄生虫和抗肿瘤作用。然而，异常的iNOS诱导参与了人类自身免疫性疾病（类风湿关节炎）、癌症和感染性休克的病理生理。因此，了解细胞调节inos相关NO产生的机制是很重要的。iNOS产生NO主要受iNOS表达调控。除了转录外，人类iNOS的表达还受到不同转录后机制的调控。在真正的iNOS编码序列（cds）上游的5- untranslation区（5-UTR）有一个小的上游开放阅读帧（µORF）。我们最近的数据表明，这种μ ORF似乎是可翻译的，因此应该阻止真正的iNOS cd的翻译。诱导细胞后可观察到iNOS蛋白的表达。我们的数据表明，poly-A结合蛋白（PABP）的一个结合位点和位于µORF后面的一个重叠的假定的内部核糖体进入位点（IRES）序列可能参与了人类iNOS mRNA的帽非依赖性翻译。在目前的应用中，我们想分析在μ ORF存在下iNOS cds翻译的机制。人类iNOS 5-UTR由外显子1和部分外显子2编码。µORF的终止密码子位于内含子1前部50bp处。这提示了无义介导的mRNA衰变（NMD）对iNOS表达的调节。我们的数据表明，sirna介导的Upf1 （NMD的中心介质）表达下调可增强人iNOS mRNA和蛋白的表达。在目前的应用中，我们想详细分析NMD在人类iNOS表达中的作用。Korneev等人描述了与真正的iNOS基因（as-iNOS-lncRNA）部分反义同源的长链非编码RNA （lncRNA）在人类细胞中的表达。我们的数据显示，as-iNOS-lncRNA的表达在细胞因子孵育后上调。此外，下调as-iNOS-lncRNA可显著降低细胞因子诱导的iNOS mRNA和蛋白的表达。这提示了as-iNOS-lncRNA对人类iNOS基因的调控作用。在目前的应用中，我们想分析这一规定的机制。在人C-28/I2软骨细胞中，iNOS的表达依赖于细胞分化。分化依赖性iNOS调控的重要机制尚不清楚。除了转录后机制的变化外，这可能是人类iNOS启动子诱导性的变化。我们的数据显示，与大多数细胞相比，人类iNOS启动子的诱导性与分化的C-28/I2细胞中iNOS mRNA表达的诱导性相似（约100倍）。在应用中，我们希望分析C-28/I2软骨细胞中调节人类iNOS表达的分化依赖机制。

The high amounts of NO produced by the inducible nitric oxide synthase (iNOS) have beneficial antiviral, antiparasital and antitumoral effects. However, aberrant iNOS induction is involved in the pathophysiology of human autoimmune diseases (rheumatoid arthritis), cancer and septic shock. Therefore it is important to understand the mechanisms by which cells regulate iNOS-related NO production. NO production by iNOS is mainly regulated by regulation of iNOS expression. Beside transcription human iNOS expression is regulated by different post-transcriptional mechanisms. In the 5-untranslated region (5-UTR) upstream of the bona fide iNOS coding sequence (cds) there is a small upstream open reading frame (µORF). Our recent data show that this µORF seems to be translatable and therefore should block the translation of the bona fide iNOS cds. However iNOS protein expression can be observed after induction of cells. Our data indicate that a binding site of the poly-A binding protein (PABP) and an overlapping putative internal ribosomal entry site (IRES) sequence located behind the µORF may be involved in a cap-independent translation of the human iNOS mRNA. In the present application we want to analyze the mechanisms enabling the translation of the iNOS cds in the presence of a µORF. The human iNOS 5-UTR is encoded by exon 1 and in part by exon 2. The stop codon of the µORF is located 50 bp in the front of the intron 1. This hints for a regulation of iNOS expression by the nonsense mediated mRNA decay (NMD). Our data show that siRNA-mediated downregulation of the expression of Upf1 (a central mediator of NMD) enhances human iNOS mRNA- and protein expression. In the present application we want to analyze the involvement of the NMD in human iNOS expression in detail. Korneev et al. describe the expression of a long non coding RNA (lncRNA) with a partial anti-sense homology to the bona fide iNOS gene (as-iNOS-lncRNA) in human cells. Our data show that the expression of the as-iNOS-lncRNA is upregulated by cytokine incubation. In addition down-regulation of as-iNOS-lncRNA significantly reduced cytokine-induced iNOS mRNA- and protein expression. This hints for an regulation of the human iNOS gene by the as-iNOS-lncRNA. In the present application we want to analyze the mechanisms of this regulation. In human C-28/I2 chondrocytes iNOS expression depends on cell differentiation. The mechanisms important for differentiation-dependent iNOS regulation are unknown. Beside changes in the post-transcriptional mechanisms this may be explained by changes in the inducibility of the human iNOS promoter. Our data show, that in contrast to most cells the inducibility of the human iNOS promoter resembles the inducibility of the iNOS mRNA expression (> 100 fold) in differentiated C-28/I2 cells. In the application we want to analyze the differentiation-dependent mechanisms regulating human iNOS expression in C-28/I2 chondrocytes.