【作者】 唐伟；

【导师】 于修平； 刘传聚；

【作者基本信息】 山东大学 ， 病原生物学， 2011， 博士

【摘要】 类风湿性关节炎是一种慢性系统性炎性失调,自身免疫在其慢性和进行性方面发挥着关键作用,因此类风湿性关节炎被认为是一种全身性自身免疫性疾病。细胞因子作为炎性介质对自身免疫性疾病的发生和发展具有决定性作用,自身免疫性疾病的所有阶段均由细胞因子控制。肿瘤坏死因子(Tumour necrosis factor, TNF)作为一种重要的免疫调控细胞因子在免疫细胞或非免疫细胞的生长、分化以及死亡等病理生理过程的重要功能已被广泛接受。TNF分泌失调可导致多种人类疾病包括重性抑郁、阿尔茨海默病、肿瘤和自身免疫性疾病(如：类风湿性关节炎、克罗恩病和溃疡性大肠炎等)。TNFα自身免疫性疾病和炎症性疾病中为上游的关键调控因子,在炎症的发生中位于核心地位,以TNFα为靶位的TNF拮抗剂类蛋白药物已经应用于临床治疗类风湿性关节炎等自身免疫性疾病。美国食品药品管理局批准上市的五种中和TNFα的拮抗剂的治疗原理均为选择性结合TNF,用于治疗包括类风湿性关节炎、银屑病关节炎、斑块型银屑病、强直性脊柱炎和克罗恩病等自身免疫性疾病,抑制TNF信号途径的药物是世界上最畅销的生物制剂。TNFα单克隆抗体Remicade和Humira及’TNFR2-Fc融合蛋白Enbrel均位列2009年重组蛋白药物世界销售额前十位。然而,TNF拮抗剂治疗在免疫病理方面的弊端,以及近年来对TNF功能复杂性和自身免疫性疾病模型中两类TNF受体独立功能的深入了解,提示TNF受体活性和信号途径在确认新药作用靶位和研发安全、高效药物中更为重要,促使自身免疫性疾病治疗转向针对TNF受体的新策略。针对受体的拮抗剂已经在临床应用,FDA批准的蛋白质药物Actemra是抑制IL-6受体的人源化单克隆抗体,目前主要应用于轻度至重度类风湿性关节炎的治疗,证明选择性针刘细胞因子受体的治疗可以获得高效的临床效果。生长因子Progranulin(PGRN)能够抑制[NFα诱导的中性粒细胞活化,抑制氧化物与蛋白酶的释放,因此PGRN是炎性细胞因子TNFα的潜在抑制剂。PGRN基因敲除小鼠清除胞内细菌感染能力下降、炎症反应增强、PGRN敲除的巨噬细胞经诱导,抗炎细胞因子释放减少、炎性细胞因子释放增多；均提示PGRN的抗炎能力。PGRN在多种生理和病理条件下具有重要的功能,但是其结合受体仍未被鉴定,导致对PGRN的活性及机制不能被深入阐明。本研究利用酵母双杂交系统筛选生长因子PGRN相互作用蛋白时发现PGRN能够结合TNFR2.通过一系列蛋白质相互作用分析证实PGRN与TNFRl和TNFR2的直接结合,并抑制TNFα与TNFRs的结合及TNFα的生物功能;利用Ⅱ型胶原蛋白诱导的关节炎和TNF转基因小鼠炎症性关节炎模型发现PGRN缺失造成小鼠对关节炎诱导因素极度敏感,并促进TNF转基因小鼠炎症性关节炎的自动发生,说明内源性PGRN对小鼠类风湿性关节炎的保护性,PGRN的抗炎效果通过其重组蛋白处理补偿并逆转PGRN基因敲除造成炎症性关节炎敏感性,以及抑制TNF转基因小鼠的关节炎恶化得到印证。PGRN/TNFR2结合区段分析基础上,构建的重组蛋白Atsttrin(Antagonist of TNF/TNFR Signaling via Targeting to TNF Receptors),可选择性结合TNFR2并抑制TNF.TNFRs的结合及TNFα诱导的炎症反应；PGRN和Atsttrin有效抑制胶原蛋白抗体诱导的关节炎、Ⅱ型胶原蛋白诱导的关节炎和TNF转基因小鼠模型的炎症性关节炎的发生与进展。本研究首次鉴定了TNFR2为生长因子PGRN的结合受体,回答了PGRN研究领域的一个重要科学问题,证实了PGRN通过结合TNFRl和TNFR2抑制TNF的活性,解释了PGRN抗TNFα诱导的炎症反应的具体机制,并为PGRN多种生物学功能及其相关分子机制研究奠定了基础;证实了PGRN为一种新型的TNFα-TNFR信号途径拮抗剂,并揭示了抑制这一炎症主要信号途径的新策略。更重要的是,本研究构建了来源于PGRN的具有抗炎功能的重组蛋白Atsttrin,证明了以PGRN为模板设计针对TNFRs的炎症与自身免疫性疾病治疗药物的可行性。由于TNFα-TNFRs信号途径涉及多种疾病进程,对这一新型TNFα-TNFRs信号途径拮抗剂的鉴定和研究,将推动各种TNF介导的类风湿性关节炎等炎症性自身免疫疾病的药物革新。本研究将PGRN与TNF-TNFRs两个重要的热门研究领域真正地联系起来；由于PGRN涉及炎症反应、神经系统疾病、胚胎发育、肿瘤、宿主防御、损伤修复等生理功能与重要疾病,而TNF-TNFRs在多种生命活动中的核心地位和长期以来科学家对]NF-TNFRs功能与信号途径研究的关注,因此PGRN结合TNFRs的观点将对上述领域产生重大影响,并为多种生理、病理进程的机制提供新的解释。骨质疏松症、类风湿性关节炎、牙周炎、多发性骨髓瘤和转移瘤等成年性骨骼疾病都是由于过度的破骨细胞活性引起的,导致倾向于骨吸收的骨再造失调。目前世界上有超过7千万骨质疏松患者,面临着骨折等风险,因而对破骨细胞活性的调控机制研究具有重要的临床意义。破骨细胞功能主要通过骨保护素(osteoprotegerin, OPG)、RANKL (receptor activator of nuclear factorκB ligand)和RANK (receptor activator of nuclear factorκB)共同调节。RANKL激活RANK促进破骨细胞的形成及其骨吸收功能的活化；活化的破骨细胞在骨表面进行不断侵蚀引起骨丢失,导致骨质疏松症。绝经后女性中骨质疏松症发病率较高,主要是机体雌激素水平迅速下降及长期雌激素缺乏所致,破骨细胞分化对雌激素缺乏的应答是细胞因子驱动的;除RANKL外,TNF可在雌激素缺乏条件下促进破骨细胞分化。类风湿性关节炎作为一种慢性炎症性关节疾病,具有持续性炎症诱导的骨吸收。在类风湿性关节炎发病过程中,TNFα、IL-1和IL-6等炎性细胞因子维持炎症环境的持续。RANK/RANKL/OPG信号途径也有效地参与了类风湿性关节的病理过程。因此,骨质疏松症中破骨细胞分化与激活需要RANKL-RANK和INF-TNFRs的协同刺激。绝经后骨质疏松症是由雌激素缺乏引起的,具有器官局限性自身免疫失调特征的炎症性疾病。骨质疏松症的发病机制中免疫系统,尤其是T细胞在其中具有重要功能,雌激素缺乏或炎症可诱导产TNF的T细胞增殖及其产TNF能力的增强；类风湿性关节炎、自身免疫性甲状腺疾病等自身免疫性疾病中常伴有骨质疏松,促使越来越多人认为骨质疏松症属于自身免疫性疾病。在PGRN结合TNFRs基础上,本研究调查了PGRN与]INFRSF若干成员的结合及结合能力,发现PGRN能够微弱的结合RANK,在蛋白水平证实PGRN直接结合RANK并抑制RANKL诱导破骨细胞分化。在对PGRN基因敲除小鼠骨密度调查中,发现老年PGRN基因敲除雌性小鼠可自发产生较同龄野生型雌性小鼠更加严重的骨丢失,更早产生了骨质疏松；而卵巢切除诱导的小鼠雌激素缺乏模型中,内源性PGRN表达缺失可提高OVX小鼠的骨质疏松的严重程度。在PGRN/RANK结合区段分析基础上,构建重组蛋白Arstrin (Antagonist of RANKL/RANK Signaling via Targeting to RANK),可结合RANK并有效地抑制RANKL-RANK的结合及RANKL诱导的破骨细胞分化；动物水平调查显示,PGRN、Arstrin和Atsttrin有效抑制雌激素缺乏诱导的骨质疏松和TNF转基因诱导炎症性骨丢失小鼠模型中骨质疏松。本研究首次报道了老年PGRN基因敲除雌性小鼠易患骨质疏松症的现象,提示了PGRN与破骨细胞分化和骨质疏松症的直接关系,以及内源性PGRN对骨骼完整性的保护作用；证实PGRN与RANK结合是PGRN抑制破骨细胞分化的主要机制,并在动物水平证实PGRN、PGRN来源的工程蛋白Arstrin和Atsttrin对雌激素缺失诱导的骨质疏松和TNF转基因小鼠炎症性骨丢失的有效抑制功能,为骨质疏松症药物开发提供新的候选蛋白。TNF-TNFRs与RANKL-RANK同属于TNF/TNFR超家族。该家族成员为多种结构、功能相关的配基和TNFR样受体。TNF/TNFR超家族的重要成员在免疫细胞中表达,TNFRSF成员经其配基活化,导致细胞增殖、存活、分化及应答细胞的凋亡。随着关于TNF/TNFR家族成员的知识及其在宿主防御、炎症、凋亡、自身免疫性疾病和器官发生等生命进程中的核心生物功能的不断丰富,一些TNF/TNFR家族成员现已成为人类疾病如自身免疫失调、发炎性肠炎、骨质疏松和肿瘤等的重要药物治疗靶位。PGRN对TNF-TNFRs与RANKL-RANK的配基-受体结合及信号途径的影响,以及类风湿性关节炎、骨质疏松症等自身免疫性疾病的防治能力,从一个侧面提示PGRN与TNF/TNFR超家族的密切相关性。基于PGRN和][NF/TNFRSF涉及的诸多生理、病理性进程与疾病,本研究将很大程度上推动PGRN(?)TNF/TNFRSF在自身免疫性疾病、炎症与免疫应答、免疫耐受与逃逸、细胞增殖与凋亡、肿瘤及神经退行性疾病等多种重大生物医学领域的理论研究和临床应用。更多还原

【Abstract】 Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder, in which autoimmunity plays a critical role in the progress of the disease. RA is thus recognized as a systemic autoimmune disease. As the mediator of imflammation, cytokines control all stages of progress of autoimmune diseases, including the onset and development of imflammtion.Tumour necrosis factor (TNF) is a key cytokine involved in the proliferation, differenciation and death of immune- and nonimmune-cells. Disorder of TNF production results in many kinds of human diseases, such as major depression, Alzheimer’s disease, cancer, and autoimmune diseases including rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. TNFa is an upstream master regulator in autoimmune and inflammatory diseases, and a central mediator in inflammation. Anti-TNF therapy has been applied in the clinical treatment of autoimmune diseases, such as rheumatoid arthritis.Five TNF antagonists, specificly binding with TNFa, have been approved by US Food and Drug Administration (FDA) to treat selected autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, ankylosing spondylitis, and Crohn’s disease. Anti-TNF therapeutics are the best-selling biological agents in the world. Remicade and Humira (monoclonal antibodies of TNFa), and Enbrel (TNFR2-Fc fusion protein) are all the top 10 biological agents in 2009. Recently, anti-TNFs carry black-box warning labels regarding the increased risk of serious infections associated with the immunosuppressive activity of these drugs. The new strategy of autoimmune diseases treatment is now focusing to TNFRs blockage due to the recent understanding of the complex function of TNF and the independent singnaling of TNFR1 and TNFR2 in autoimmune disease models, which reveal that activation and signaling of TNFRs are more important in the recognization of target site and development of the safer and more effective new drugs. Drugs such as anakinra (Kineret) and tocilizumab (Actemra), which target IL-1 and IL-6 receptors, respectively, have demonstrated that the selective targeting on cytokine receptors can deliver a highly effective clinical outcome.Progranulin (PGRN), a growth factor, can inhibit the activation of neutrophil, and release of oxide and protease induced by TNFa, therefore, PGRN is considered as a potential TNF inhibitor. The recent study of PGRN-deficient mice showed reduced bacteria infection elimination and enhanced inflammatory reaction. Pro-inflammatory cytokine production increased, whereas anti-inflammatory cytokines production decreased, in PGRN-deficient macrophages after stimulation. All these data suggest the anti-inflammation activity of PGRN in vivo. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the activity of PGRN and understand the mechanisms involved have been significantly hampered by the unidentification of its binding receptor(s). To address this issue, we performed a global genetic screen that led to the identification of TNFR2 as the first known PGRN-associated receptor.In this study, the direct binding of PGRN with TNFR1 or TNFR2 are proved via a series of protein interaction assays and the interaction of PGRN and TNFRs block the function of TNFa. The function evaluation of PGRN in vivo by using the models of collagen-induced arthritis and inflammatory arthritis of TNF transgenic mice revealed that PGRN-deficient mice were susceptible to collagen-induced arthritis, and enhanced the spontaneous occurrence of inflammatory arthritis in collagen-challenged PGRN-deficient mice. This indicated the protection of endogenous PGRN against rheumatoid arthritis of mice. Deletion of the PGRN gene exacerbates, whereas recombinant PGRN prevent, the spontaneous development of inflammatory arthritis in TNF-Tg mice. Base on the binding segments analysis of PGRN and TNFR2, Atsttrin (Antagonist of TNF/TNFR Signaling via Targeting to TNF Receptors), a bioengineered PGRN-derived protein, is constructed. Atsttrin selectively binds to TNFR2 and exhibits effective blockade of the TNF/TNFR interaction, which in turn inhibits the inflammatory reaction induced by TNFa. PGRN and Atsttrin both effectively prevent the onset and progression of arthritis in TNF-Tg transgenic mice, collagen antibody- and collagen-induced arthritis models.In summary, this study identifies the first PGRN-binding receptor, TNFR2, also a wellknown receptor of TNFa, which will provide a foundation for future discoveries relating to this growth factor. In addition, PGRN was also identified as a novel antagonist of the TNFa-TNFR pathway, and uncovers a new strategy for inhibiting this cardinal pathway of inflammation. More importantly, we have discovered and produced an engineered PGRN-derived protein, Atsttrin, which effectively prevents the onset and progression of inflammatory arthritis in several preclinical animal models. These data suggest the feasibility for the anti-inflammatory therapeutics design depending on PGRN targetting TNFRs. With the consideration that TNFa-TNFR signaling is involved in a plethora of disease processes, the identification and manipulation of this new antagonist of the TNF/TNFR pathway may lead to innovative therapeutics for various TNFa-mediated pathologies and conditions, such as rheumatoid arthritis. This study linked the study of PGRN and TNF-TNFRs, two hot research areas, and will provide new explaination to many kinds of physio- and pathological process.Most adult skeletal diseases are due to excess osteoclastic activity, leading to an imbalance in bone remodelling which favours resorption. Such diseases would include osteoporosis, periodontal disease, rheumatoid arthritis, multiple myeloma and metastatic cancers. For individuals with osteoporosis, bone fractures represent life-threatening events, and today there are excess of 70 million people worldwide at risk. Recent breakthrough in our understanding of osteoclast differentiation and activation have come from the analysis of a family of biologically related tumour necrosis factor (TNF) receptor (TNFR)/TNF-like proteins:osteoprotegerin (OPG), receptor activator of nuclear factor (NF)-kB (RANK) and RANK ligand (RANKL), which together regulate osteoclast function.RANKL binds to and activates RANK, and in turn promotes osteoclast differentiation and activation. Continue erosion in the bone surface of activated osteoclasts result in bone loss, lead to osteoporosis. Increasing risk of osteoporosis appears in postmenopausal women, which results from the estrogen deficiency. Cytokines, such as RANKL and TNFa costimulate osteoclastogenesis to respond to estrogen deficiency. Other than estrogen deficiency, rheumatoid arthritis with persistent inflammation can also result in bone resorption. Cytokines including TNFa, IL-1, and IL-6 are involved in the maintenance of inflammatory environment. RANK/RANKL/OPG signaling pathway also actively involved in the pathological process of rheumatoid arthritis. RANKL-RANK and TNF-TNFRs signaling pathway both play critical roles in osteoclastogenesis and activation.Postmenopausal osteoporosis is regarded as the product of an inflammatory disease bearing many characteristics of an organlimited autoimmune disorder, triggered by estrogen deficiency. Immune system, especially T cells are involved in the pathogenesis of osteoporosis. Estrogen deficiency and inflammation both can stimulate the proliferation and TNF production of TNF-producing T cell. Rheumatoid arthritis and autoimmune thyroid diseases usually accompany with osteoporosis. All these evidences classify osteoporosis as an autoimmune disease.In the study of PGRN binding TNFR superfamily members, we indentified RANK, TNFRSF 11 A, is also associated with PGRN in a lower affinity compared with the binding of PGRN to TNFRs. The direct binding of PGRN to RANK can effectively block the interaction of RANKL and RANK, by which inhibits RANKL induced osteoclastogenesis. An exciting phenomenon is that aged PGRN-deficient female mice developed severer bone loss compared with the even-aged wild type female mice. Deletion of PGRN expression exacerbates the severity of osteoporosis in ovariectomized mice model. A bioengineered PGRN-derived protein named Arstrin (Antagonist of RANKL/RANK Signaling via Targeting to RANK) is constructed depending on a PGRN-RANK binding domain assay. Arstrin strongly blocks the interaction of RANKL and RANK, and in turn inhibits RANKL induced osteocalstogenesis. PGRN, Arstrin, and Atsttrin all demonstrate effective inhibition in estrogen deficiency induced osteoporosis and TNF transgene induced bone loss mice models.In summary, this study firstly described the severer osteoporosis in aged PGRN-deficient female mice, and pointed out the relationship between PGRN and osteoclastogenesis and osteoporosis. This study also proves the protective effect of endogenous PGRN in maintenance of skeletal integrality. Binding of PGRN to RANK is one of the major inhibitory mechanisms of PGRN in osteoclastogenesis. Finally, The high inhibition of PGRN and PGRN derived Arstrin and Atsttrin are confirmed in animal models, which suggest that these biological agents can be candidates of anti-osteoporosis therapeutics target RANKL-RANK pathway.TNF-TNFRs and RANKL-RANK belong to TNF/TNFR superfamily that contains more than 40 types of ligands and TNFR-like receptors with relative sturcture and function. Almost all important members are expressed in immune cells. Activated TNFRSF members by their ligands lead to proliferation, survival, differentiation, and apoptosis of responding cell. Following the understanding of TNF/TNFRSF members in host defense, inflammation, apoptosis, autoimmune diseases, and organogenesis, some of them have become important therapeutic targets of human diseases, such as autoimmune disorder, inflammatory bowel disease, osteoporosis, and cancer.The ability of PGRN in blocking interaction and signaling transduction of TNF-TNFRs and RANKL-RANK, as well as the distinguished therapeutic ability of PGRN and bioengineered PGRN derived protein in autoimmune diseases, such as rheumatoid arthritis and osteoporosis, are implying a close relationship between PGRN and TNF/TNFR superfamily. Based on the various physio-, pathological processes and diseases in which PGRN and TNF/TNFRSF are involved, this study will contribute to the theoretical research and clinical application of PGRN and TNF/TNFRSF in biomedicine projects, including autoimmune diseases, inflammation and immune response, immune tolerance and escape, cell proliferation and apoptosis, cancer, and neural degenerative diseases.更多还原