SIGNALING PATHWAYS CONTROLLING NF-KB IN RHEUMATIOID ARTHRITIS

类风湿关节炎(RA)是慢性炎症性疾病的原型。我们对类风湿关节炎动物模型的初步研究表明，核因子-kappaB在调节关节炎滑膜的炎症、细胞凋亡和增殖中起着至关重要的作用。因此，对于RA和其他慢性炎症性疾病的治疗干预，核因子-kappaB成为非常有吸引力的靶点。抑制核因子-kappaB活性最合理的方法是调节控制核因子-kappaB转录活性的信号级联反应。NIKK/IKK和p38MAPK这两个信号通路在调节类风湿关节炎中的核因子-kappaB方面尤为重要。我们对这些通路的生理功能的了解非常有限，主要是由于实验方法的不足。本研究的主要目的是确定Nik/IKK和p38信号通路在RA核因子-kappaB活化中的生理作用，并评估这些信号通路在RA的主要表现中的作用，即炎症、侵袭性滑膜肿瘤样扩张以及骨和软骨吸收，从而评估这些信号通路作为治疗干预的靶点。我们将利用基因转移技术来剖析Nik/IKK和p38 MAPK的作用，以进行治疗干预。我们将利用基因转移技术来分析Nik/IKK和p38 MAPK通路在类风湿关节炎病理过程中对核因子-kappaB活化的作用。使用关节内(i.a.)显性负性(DN)抑制剂的基因转移应该能够清楚地解释这些途径在RA中的作用，并将验证这些途径作为药物发现的靶点。在目标1中，我们将在细胞水平上研究Nik/ikk和p38信号通路在滑膜成纤维细胞和单核细胞的核因子-kappaB活化、炎症和有丝分裂反应以及细胞凋亡中的作用。接下来，我们将评估体内Nik/IKK和p38通路在大鼠SCW关节炎中核因子-kappaB的激活、炎症、增殖以及骨和软骨吸收中的作用(目的2)。这些数据与人类疾病的相关性将通过使用类风湿性关节炎的SCID小鼠/人类模型(目标3)来检验。这些实验将确定核因子-kappaB以及Nik/IKK和p38通路在调节人类RA滑膜的炎症、细胞凋亡和软骨破坏中的作用。我们对动物关节炎的初步研究的一个意想不到的结果是观察到，局部抑制滑膜中的核因子-kappaB不仅可以改善已治疗的疾病，而且还可以改善未治疗的对侧关节。这表明通过局部治疗来缓解该病的全身症状是可行的。目标4旨在探索这种效应背后的两种假定机制。我们将研究局部抑制核因子-kappaB对循环T细胞中促炎症和抗炎的TH1和Th2亚群平衡的影响，以及在神经发生机制中的作用。

Rheumatoid arthritis (RA) is a prototype of chronic inflammatory disease. Our preliminary studies in animal models of RA demonstrated crucial involvement of NF-kappaB in regulation of inflammation, apoptosis, and proliferation in the arthritic synovium. Thus, NF-kappaB emerges as very attractive target for therapeutic intervention in RA and other chronic inflammatory conditions. The most logical way to inhibit NF-kappaB activation is to modulate the signaling cascades which controls transcriptional activity of NF-kappaB. Two signaling cascades, the NIK/IKK and p38 MAP kinase pathways, are particularly important in regulation of NF-kappaB in RA. Our knowledge of the physiological function of these pathways is very limited, mainly due to inadequate experimental approaches. The major purpose of this proposal is to determine the physiological role of the NIK/IKK and p38 signaling pathways in activation of NF-kappaB in RA, and to assess the contribution of these pathways in major manifestations of RA, i.e. inflammation, tumor-like expansion of invasive synovium, and bone and cartilage resorption, and thus evaluate these pathways as targets for therapeutic intervention. We will employ gene transfer technology for dissecting the role of the NIK/IKK and p38 MAPK for therapeutic intervention. We will employ gene transfer technology for dissecting the role of the NIK/IKK and p38 MAPK pathways in NF-kappaB activation in the RA pathology. Using intraarticular (i.a.) gene transfer of dominant negative (DN) inhibitors should allow for the clear-cut interpretation of the role of these pathways in RA and will validate these pathways as targets for drug discovery. In Aim 1, we will examine the role of the NIK/IKK and p38 pathways in NF-kappaB activation, inflammatory and mitogenic responses, and apoptosis on the cellular level in synovial fibroblasts and monocytic cells in vitro. Next, we will assess the role of the NIK/IKK and p38 pathways in vivo in NF-kappaB activation, inflammation, hyperplasia, and bone and cartilage resorption in SCW arthritis in rats (AIM 2). The relevance of these data to human disease will be examined by using a SCID mice/human models of RA (Aim 3). These experiments will determine the role of NF-kappaB, and the NIK/IKK and p38 pathways in regulation of inflammation, apoptosis, and cartilage destruction in human RA synovium. One unexpected result of our preliminary studies in animal arthritis was observation that local suppression of NF-kappaB in the synovium ameliorated disease not only in treated, but also in untreated, contralateral joints. This indicates the feasibility of alleviating systemic manifestations of the disease through local treatment. Aim 4 serves to explore two putative mechanisms underlying this effect. We will examine the influence of local suppression of NF-kappaB on the balance of pro- and anti-inflammatory TH1 and Th2 subsets in circulating T cells, and in neurogenic mechanisms.