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具有良好的抗病毒、抗副反应和抗肿瘤作用。然而，诱导型一氧化氮合酶的异常诱导参与了人类自身免疫性疾病(类风湿性关节炎)、癌症和感染性休克的病理生理过程。因此，了解细胞调节iNOS相关NO产生的机制是很重要的。INOS产生的NO主要受iNOS表达的调节。除了转录外，人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-非翻译区(5-UTR)有一个小的上游开放阅读框(UORF)。我们最近的数据显示，这一µORF似乎是可翻译的，因此应该会阻止真正的iNOS CD的翻译。诱导后可观察到iNOS蛋白的表达。我们的数据表明，PolyA结合蛋白(PABP)的结合位点和位于µORF后面的重叠的内部核糖体进入位点(IRES)序列可能参与了人iNOS mRNA的帽非依赖性翻译。在本申请中，我们要分析在µORF存在的情况下实现iNOS CD翻译的机制。人诱导型一氧化氮合酶5-非编码区由外显子1编码，部分由外显子2编码。uORF的终止密码子位于内含子1前面50个碱基对。这提示无意义介导的信使核糖核酸衰变(NMD)对iNOS的表达有调节作用。我们的数据表明，siRNA介导的UPF1(NMD的中心介质)表达下调增强了人iNOSmRNA和蛋白的表达。在本应用中，我们想要详细分析NMD在人iNOS表达中的参与。Kornev等人。描述与真正的iNOS基因部分反义同源的长非编码RNA(LncRNA)在人类细胞中的表达(AS-iNOS-lncRNA)。我们的数据表明，细胞因子孵育上调了AS-iNOS-lncRNA的表达。此外，下调AS-iNOS-lncRNA显着减少细胞因子诱导的iNOSmRNA和蛋白表达。这暗示了AS-iNOS-lncRNA对人类iNOS基因的调节。在目前的应用中，我们想要分析这一规则的机制。在人C-28/I2软骨细胞中，iNOS的表达依赖于细胞分化。依赖于分化的iNOS调控的重要机制尚不清楚。除了转录后机制的变化外，这可能是因为人类iNOS启动子的诱导性发生了变化。我们的数据显示，与大多数细胞相反，在分化的C-28/I2细胞中，人iNOS启动子的诱导性类似于iNOS mRNA表达的诱导性(&gt；100倍)。在应用中，我们想要分析人iNOS在C-28/I2软骨细胞中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.