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）的产生改变多种功能，包括心肌收缩力、血管紧张素、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表征，但对下调iNOS的相应途径知之甚少。利用在体内和体外小鼠巨噬细胞和大鼠肝细胞模型的LPS刺激，我们已经证明，NO反馈抑制其自身的合成，通过增加基因转录和骨桥蛋白（OPN），一个有效的反式抑制iNOS表达的启动子激活。 这种NO依赖性OPN基因转录和蛋白质合成的负反馈途径以前没有描述过。 我们假设，骨桥蛋白，一个有效的反式抑制剂的iNOS表达，转录是NO依赖于LPS刺激的小鼠巨噬细胞。 在永生化ANA-1小鼠巨噬细胞系中，我们建议在LPS刺激NO产生的背景下功能性地定位OPN启动子。 具体目标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-报告基因构建体进行缺失分析和定点突变。 具体目标2。为了明确NO依赖的反式调节作用，我们将分别利用生物素-链霉亲和素亲和法和以DNA识别位点为探针筛选cDNA表达文库来分离NO依赖的转录因子及其cDNA克隆。 具体目标3。为了确定我们的转录因子的S-亚硝基化状态及其对DNA结合的影响，我们将使用CuCl-半胱氨酸偶联化学发光。具体目标4。为了证实在LPS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，或者通过瞬时转染过表达转录因子。具体目标5.为了证实体内相关性，我们将利用内毒素血症的鼠OPN敲除模型来证明在不存在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，但尚未充分表征的NO依赖性转录途径，其在内毒素血症的情况下抑制iNOS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.