| 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. |
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