NO induces osteopontin,a potent trans-repressor of iNOS

在内毒素(LPS)介导的脓毒症中，诱导型一氧化氮合酶(INOS)的表达和一氧化氮(NO)的产生改变了多种功能，包括心脏收缩、血管紧张性、肠上皮通透性和白细胞募集。虽然内毒素血症中上调iNOS的分子途径已被广泛研究，但下调iNOS的相应途径却知之甚少。利用小鼠体内和体外小鼠巨噬细胞和大鼠肝细胞的内毒素刺激模型，我们证明了NO反馈通过增加骨桥蛋白(OPN)的基因转录和启动子激活来抑制自身的合成，骨桥蛋白是一种强有力的iNOS表达的反式抑制因子。这种依赖NO的OPN基因转录和蛋白质合成的负反馈途径以前从未被描述过。我们假设，在脂多糖刺激的小鼠巨噬细胞中，OPN的转录是NO依赖的，它是iNOS表达的有力反式抑制因子。在永生化的ANA-1小鼠巨噬细胞系中，我们建议在脂多糖刺激NO产生的背景下对OPN启动子进行功能定位。具体目的1.为了确定NO依赖的顺式作用转录控制区，我们将利用OPN启动子-报告结构进行缺失分析和定点突变。具体目的2.为了明确NO依赖的反式作用调节，我们分别采用生物素-链霉亲和素亲和方法分离了NO依赖的转录因子及其cDNA克隆，并以DNA识别位点为探针筛选了表达文库。具体目的3.利用CuCl-半胱氨酸偶联化学发光法测定转录因子的S亚硝化状态及其对DNA结合的影响。具体目的4.为了证实在内毒素刺激的巨噬细胞中的相关性，我们将用反义技术抑制转录因子mRNA的翻译，或者通过瞬时转染法过表达转录因子。具体目的5.为了证实体内相关性，我们将利用内毒素血症的小鼠OPN基因敲除模型来证明在没有OPN的情况下缺乏iNOS抑制。我们的研究将OPN的产生定义为一种独特的、目前尚不明确的、依赖于NO的转录途径，它在内毒素血症的背景下抑制iNOS的表达。

In endotoxin (LPS)-mediated sepsis, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production alter multiple functions, including cardiac contractility, vasomotor tone, intestinal epithelial permeability, and leukocyte recruitment. While the molecular pathways which upregulate iNOS in endotoxemia have been extensively characterized, little is known of the corresponding pathways which downregulate iNOS. Utilizing both in vivo murine and in vitro murine macrophage and rat hepatocyte models of LPS stimulation, we have demonstrated that NO feedback inhibits its own synthesis by increasing gene transcription and promoter activation of osteopontin (OPN), a potent trans-repressor of iNOS expression. This negative feedback pathway of NO-dependent OPN gene transcription and protein synthesis has not been previously described. We hypothesize that transcription of OPN, a potent trans-repressor of iNOS expression, is NO-dependent in LPS-stimulated murine macrophages. In the immortalized ANA-1 murine macrophage cell line, we propose to functionally map the OPN promoter in the context of LPS stimulated NO production. Specific Aim 1. To define NO-dependent cis-acting transcriptional control regions, we will utilize OPN promoter-reporter constructs with deletion analysis and site-directed mutagenesis. Specific Aim 2. To define NO-dependent trans-acting regulation, we will isolate the NO-dependent transcription factor and its cDNA clone by using the biotin-strepavidin affinity method and screening a cDNA expression library, respectively, using the DNA recognition site as a probe. Specific Aim 3. To determine the S-nitrosylation status of our transcription factor and its effect upon DNA binding, we will use CuCl-cysteine coupled chemiluminescence. Specific Aim 4. To confirm relevancy in the LPS-stimulated macrophage, we will inhibit translation of the transcription factor mRNA with antisense techniques and alternatively, over- express the transcription factor by transient transfection. Specific Aim 5. To confirm in vivo relevancy, we will utilize a murine OPN-knockout model of endotoxemia to demonstrate lack of iNOS inhibition in the absence of OPN. Our studies will define OPN production as a unique and as yet, poorly characterized, NO- dependent transcriptional pathway which inhibits iNOS expression in the setting of endotoxemia.