Role of BH3-Domain Proteins in the Effector Phase of RA

描述（由申请人提供）：风湿性关节炎（RA）是一种自身免疫性疾病，其特征为滑膜衬里增生、炎症以及软骨和骨的破坏。在RA中，Bcl-2家族的抗凋亡和促凋亡成员之间的平衡可能会向生存转移。虽然我们证明了抗凋亡蛋白Bcl-2和Mcl-1与对照相比在RA滑膜组织中增加，但没有研究检查拮抗Bcl-2抗凋亡成员在关节炎中的治疗潜力。研究Bcl-2家族抗凋亡成员的缺陷是复杂的胚胎致死或出生后早期死亡。相反，缺乏促凋亡Bcl-2成员如巴克、Bax和Bim的小鼠存活并达到成年。由于Bim通过其对Bcl-2/Mcl- 1的隔离和/或其对巴克和Bax的激活而成为细胞凋亡的关键激活剂，因此其是治疗RA的潜在靶点。我们证明，与对照组相比，RA滑膜组织中凋亡启动子Bim的表达显著降低。此外，缺乏Bim但不缺乏下游效应物巴克或Bax的小鼠发展成更严重形式的炎性关节炎。Bim-/-小鼠中的这种恶化的疾病与细胞凋亡减少、促炎分子表达增加和血管翳中更多的巨噬细胞相关。Bim-/-巨噬细胞响应于LPS刺激显示升高的IL-6、TNF 1和IL-12水平，增强的CD 40和CD 69表达，以及增加和维持的活性p38水平。基于这些数据，我们假设Bim与Bcl-2和/或Mcl-1的比率充当分子变阻器，其决定关节中巨噬细胞增生和活化的程度。我们将使用药理学方法、整体动物方法和细胞特异性方法来确定Bim缺乏如何加剧炎症性关节炎。这些研究将可能导致新的治疗方法，类风湿关节炎。A.1.检查改变Bcl-2家族成员的关联在炎症性关节炎发展中的影响。A.2.确定巴克、Bax或Bcl-2的功能丧失对Bim-/-小鼠中炎性关节炎发展的影响。A.3.确定单核细胞和巨噬细胞中Bim的靶向缺失对炎症性关节炎发展的影响。A.4.确定Bim介导的抑制巨噬细胞活化的机制。细胞死亡和生长的调节对于维持人体的平衡至关重要。然而，在自身免疫性疾病类风湿性关节炎（RA）的开始和/或进展期间，这种平衡被破坏。在RA中，存在细胞生长的增加和伴随的细胞死亡的减少，导致附着于软骨/骨连接部（滑膜衬里）的组织的异常增加。在RA期间，滑膜衬里侵入并破坏邻近的软骨和骨。对RA患者关节组织的分析显示，巨噬细胞的数量与预后不良相关。我们证明了由促死亡蛋白Bim介导的死亡信号级联在RA患者的巨噬细胞中是功能失调的。此外，我们已经证明，在所有细胞类型中缺乏Bim的小鼠都会患上更严重的关节炎，并且这些小鼠的巨噬细胞高度活化，这意味着它们会产生大量加剧炎症的有害因素。我们已经开发了一种潜在的治疗分子，它由Bim的一部分组成，称为BH 3结构域。我们现在已经证明，将BH 3肽全身递送至小鼠可预防炎性关节炎的发展，而对小鼠没有毒性。这些研究首次显示了BH 3肽作为治疗RA的潜在分子的功效。我们的近期目标是开发BH 3肽疗法作为RA和其他风湿性疾病的一线治疗。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. In RA, the balance between anti- and pro-apoptotic members of the Bcl-2 family may be shifted towards survival. While we demonstrated that the anti-apoptotic proteins, Bcl-2 and Mcl-1 are increased in RA synovial tissue as compared to controls, no study has examined the therapeutic potential of antagonizing Bcl-2 anti-apoptotic members in arthritis. The study of deficiencies in anti-apoptotic members of the Bcl-2 family is complicated by embryonic lethality or early post-natal death. In contrast, mice deficient in pro-apoptotic Bcl-2 members such as Bak, Bax, and Bim survive and reach adulthood. Since Bim is a critical activator of apoptosis by virtue of its sequestration of Bcl-2/Mcl- 1 and/or its activation of Bak and Bax, it is a potential target for treatment of RA. We demonstrated that expression of the apoptotic initiator Bim is markedly reduced in synovial tissue from RA compared to controls. Further, mice lacking Bim but not the downstream effectors Bak or Bax develop a more severe form of inflammatory arthritis. This exacerbated disease in Bim-/- mice is associated with decreased apoptosis, increased expression of pro-inflammatory molecules, and more macrophages in pannus. Bim-/- macrophages display elevated levels of IL-6, TNF1, and IL-12, enhanced expression of CD40 and CD69, and increased and sustained level of active p38 in response to stimulation with LPS. Based on these data, we hypothesize that the ratio of Bim to Bcl-2 and/or Mcl-1 serves as molecular rheostat that determines the extent of hyperplasia and activation of macrophages in the joint. We will use a pharmacological approach, a whole animal approach, and a cell-specific approach to identify how deficiency in Bim exacerbates inflammatory arthritis. These studies will potentially lead to novel therapeutic approaches to RA. A.1. Examine the effect of altering the association of Bcl-2 family members in the development of inflammatory arthritis. A.2. Determine the effect of loss of function in Bak, Bax, or Bcl-2 on the development of inflammatory arthritis in Bim-/- mice. A.3. Determine the effect of targeted deletion of Bim in monocytes and macrophages on the development of inflammatory arthritis. A.4. Determine the mechanism of Bim-mediated suppression of macrophage activation. The regulation of cell death and growth is vital for maintaining a balance in the human body. However, during the initiation and/or progression of the autoimmune disease, rheumatoid arthritis (RA), this balance is disrupted. In RA there is an increase in cellular growth and a concomitant decrease in cell death leading to an abnormal increase in the tissue that attaches to the cartilage/bone junction, the synovial lining. During RA, the synovial lining invades and destroys the adjacent cartilage and bone. Analysis of tissue from joints of patients with RA revealed that the number of macrophages, correlated with a worse prognosis. We demonstrated that the death signaling cascade mediated by the pro-death protein Bim is dysfunctional in macrophages from patients with RA. Additionally, we have shown that mice lacking Bim in all cell types develop a worse form of arthritis and that the macrophages from these mice are highly activated, meaning they produce significant amounts of deleterious factors that exacerbate the inflammation. We have developed a potential therapeutic molecule that consists of a portion of Bim, termed BH3 domain. We have now demonstrated that systemic delivery of BH3 peptides to mice prevents the development of inflammatory arthritis with no toxicity to the mice. These studies are the first to show the efficacy of BH3 peptides as a potential molecule for treatment of RA. Our immediate goal is to develop the BH3 peptide therapy as a front line treatment for RA and other rheumatic diseases.