SIGNALING PATHWAYS CONTROLLING NF-KB IN RHEUMATIOID ARTHRITIS

类风湿关节炎（RA）是一种典型的慢性炎症性疾病。我们在RA动物模型中的初步研究表明NF-kappaB在关节炎滑膜炎症、细胞凋亡和增殖的调节中起着至关重要的作用。因此，NF-kappaB成为风湿性关节炎和其他慢性炎症疾病治疗干预的非常有吸引力的靶点。抑制NF-kappaB激活的最合理的方法是调节控制NF-kappaB转录活性的信号级联反应。两个信号级联，NIK/IKK和p38 MAP激酶通路，在RA中NF-kappaB的调控中尤为重要。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不充分。本研究的主要目的是确定NIK/IKK和p38信号通路在RA中NF-kappaB激活中的生理作用，并评估这些通路在RA主要表现中的作用，如炎症、侵袭性滑膜的肿瘤样扩张、骨和软骨吸收，从而评估这些通路作为治疗干预的靶点。我们将采用基因转移技术来剖析NIK/IKK和p38 MAPK在治疗干预中的作用。我们将采用基因转移技术来剖析NIK/IKK和p38 MAPK通路在RA病理中NF-kappaB激活中的作用。使用显性阴性（DN）抑制剂的关节内（i.a）基因转移应该允许明确解释这些途径在RA中的作用，并将验证这些途径作为药物发现的靶点。在Aim 1中，我们将在体外滑膜成纤维细胞和单核细胞的细胞水平上研究NIK/IKK和p38通路在NF-kappaB激活、炎症和有丝分裂反应以及凋亡中的作用。接下来，我们将在体内评估NIK/IKK和p38通路在大鼠SCW关节炎中NF-kappaB激活、炎症、增生以及骨和软骨吸收中的作用（AIM 2）。这些数据与人类疾病的相关性将通过使用SCID小鼠/人类RA模型进行检验（目的3）。这些实验将确定NF-kappaB、NIK/IKK和p38通路在调节RA滑膜炎症、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，观察到滑膜中局部抑制NF-kappaB不仅在治疗中改善了疾病，而且在未治疗的对侧关节中也改善了疾病。这表明通过局部治疗减轻疾病全身性表现的可行性。目的4旨在探讨这一效应背后的两个假定机制。我们将研究局部抑制NF-kappaB对循环T细胞中促炎性和抗炎性TH1和Th2亚群平衡的影响，以及神经发生机制。

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.