Cartilage Molecular Genetics

软骨分子遗传学

• 批准号: 7592469
• 负责人: David J Hall
• 金额: $ 130.67万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592469
• 关键词: Affect; Age; American; Apoptotic; Area; Arthritis; Attention; BIRC4 gene; Biology; Caloric Restriction; Cartilage; Cartilage Diseases; Caspase; Cell Death; Cell Proliferation; Cell Survival; Cells; Cessation of life; Chondrocytes; Chromatin; Chronic; Class; Data; Degenerative polyarthritis; Disease; Down-Regulation; Enhancers; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Event; Extracellular Matrix; Factor Analysis; Family member; Future; Gene Expression; Gene Expression Regulation; Genes; Goals; HDAC1 gene; HDAC2 gene; Hela Cells; Histone Deacetylase; Histones; Human; Integrins; Knowledge; Lead; Ligaments; Light; Longevity; Mediating; Messenger RNA; Molecular; Molecular Genetics; Mutation; Nicotinamide adenine dinucleotide; Osteoarthrosis Deformans; Other Finding; Patients; Plant Roots; Play; Population; Protein Family; Proteins; RGD (sequence); Regulation; Research; Role; Side; Signal Transduction; Skeletal system; Structure; Tendon structure; Treatment Protocols; Tumor Necrosis Factor-alpha; Work; X-linked IAP; articular cartilage; caspase-3; early onset; enzyme pathway; extracellular; human TNF protein; inhibitor-of-apoptosis protein; inhibitor/antagonist; malformation; mammalian COMP; survivin

a) Background The work in my lab has revolved around the study of chondrocyte gene expression, cell proliferation and viability with attention focused on the changes that take place in human cartilage that lead to the arthritic diseases. We have sought to identify and analyze the factors that affect these aspects of cartilage biology. We have explored the roles of cartilage oligomeric matrix protein (COMP) and histone deacetylases in regulating chondrocyte viability and gene expression. We have shown that these proteins affect chondrocytes in distinct ways. For example COMP enhances survival and proliferation by regulating expression of a unique set of antiapoptotic proteins. We have also found that distinct histone deacetylase are potent regulators of cartilage specific gene expression. These results provide us with the background to our present studies, enabling us to fill the gaps in our knowledge about the role of critical extracellular and intracellular proteins in cartilage biology. b) Objectives of Present Studies Currently our objectives are two-fold. The first objective is to define the role of histone deacetylateses in chondrocyte gene epxression. The second objective is to identify the mechanism by which specific histone deacetylase enzymes regulate chondrocyte survival. c) Results During the Past Year The results of the past year are grouped according to the Objectives outlined above. (1) Analysis of the structure and function of the COMP Gene. Cartilage oligomeric matrix protein (COMP) is a specific component of cartilage, tendon and ligament. Mutations within the COMP gene lead to skeletal malformations and early onset arthritis. While COMP appears to play a crucial role in cartilage biology its exact role is unknown. We have analyzed the role of COMP in regulating cell survival and gene expression. We have utilized human chondrocytes in these studies. We find that COMP protects these cells against death, either in the presence or absence of TNF alpha (Tumor Necrosis Factor alpha) and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (Inhibitor of Apoptosis Protein) family of anti-apoptotic proteins since the levels of XIAP, Survivin, cIAP1 and cIAP2 are significantly elevated in the COMP expressing cells and downregulation of Survivin and XIAP protein levels by siRNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However in both HeLa cells and chondrocytes, COMP induced the Survivin mRNA level by 5-fold. Thus Survivin is the first gene identified to be upregulated transcriptionally by COMP. The carboxy terminal half of the protein comprising the Type 3 repeats and the RDG sequence (CaCTD domain) was sufficient to promote survival and to elevate the levels of the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and Survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death and should help to shed light on the contribution of this important ECM molecule to cartilage biology and osteoarthritis. (2) We have been able to identify the prominent histone deacetylase enzymes (HDAC) in primary human chondrocytes. We find that these enzymes are potent regulators of chondrocyte specific gene expression such that when select HDACs are potent enhancers and inhibitors of cartilage gene expression and chondrocyte survival. For example, SirT1 is a class III histone deacetylase that plays a key role in extension of life span during caloric restriction. However, little is know of the role it plays in human chondrocyte biology. We find that the HDAC SirT1, and NAD dependent enzyme can play a very positive role in the regulation of cartilage expression and cell survival by modifying chromatin and that the NAD salvage pathway enzyme NAMPT, is significantly decreased in OA cartilage. Thus NAMPT may be an important new marker for OA. In addition, we find that other HDACs, namely HDAC1 and HDAC2, are potent repressors of cartilage gene expression. Further we find that HDAC1 and HDAC2 are elevated in OA cartilage. Thus a misregulation of HDAC enzymes that may lead to OA d) Conclusions and Significance With Future Directions In the last two to three decades, much progress has been made in the discovery of the causes and treatments of the various forms of arthritis. However, the initiating events for these diseases are not well understood and many of the treatment regimens are suboptimal at best. Since arthritis is a very debilitating disease that affects a large proportion of the population, much effort should be made at uncovering the root cause of the disease and identifying new treatments. I believe we have made excellent progress in defining new areas of research in cartilage biology and potential treatments of arthritic diseases. We have explored the role of COMP, a prominent cartilage protein, in the role of cell death and survival and are attempting to determine its role in the long-term progression of arthritis. Further, we have explored the role of HDACs and their corresponding inhibitors in chondrocyte gene expression and as a potential treatment of OA. The role of HDACs in cartilage biology is a completely untouched area that holds much promise for a better understanding of the regulatory mechanisms underlying chondrocyte gene expression and in the treatment of cartilage diseases through the use of specific HDAC inhibitors.

一）背景 我实验室的工作围绕软骨细胞基因表达、细胞增殖和活力的研究，重点关注人类软骨中发生的导致关节炎疾病的变化。 我们试图识别和分析影响软骨生物学这些方面的因素。 我们探索了软骨寡聚基质蛋白（COMP）和组蛋白脱乙酰酶在调节软骨细胞活力和基因表达中的作用。 我们已经证明这些蛋白质以不同的方式影响软骨细胞。 例如，COMP 通过调节一组独特的抗凋亡蛋白的表达来增强存活和增殖。 我们还发现独特的组蛋白脱乙酰酶是软骨特异性基因表达的有效调节剂。 这些结果为我们当前的研究提供了背景，使我们能够填补关于关键细胞外和细胞内蛋白质在软骨生物学中的作用的知识空白。 b) 当前研究的目的 目前我们的目标有两个。 第一个目标是确定组蛋白脱乙酰化在软骨细胞基因表达中的作用。 第二个目标是确定特定组蛋白脱乙酰酶调节软骨细胞存活的机制。 c) 过去一年的业绩 去年的结果根据上述目标进行分组。 (1)COMP基因的结构和功能分析。软骨寡聚基质蛋白（COMP）是软骨、肌腱和韧带的特定成分。 COMP 基因内的突变会导致骨骼畸形和早发性关节炎。 虽然 COMP 似乎在软骨生物学中发挥着至关重要的作用，但其确切作用尚不清楚。 我们分析了 COMP 在调节细胞存活和基因表达中的作用。 我们在这些研究中使用了人类软骨细胞。 我们发现，无论存在或不存在 TNF α（肿瘤坏死因子 α），COMP 都能保护这些细胞免于死亡，并且能够阻断 caspase 3（一种关键效应 caspase）的激活。这种效应似乎是由抗凋亡蛋白 IAP（凋亡蛋白抑制剂）家族介导的，因为 COMP 表达细胞中 XIAP、Survivin、cIAP1 和 cIAP2 的水平显着升高，并且 siRNA 下调了 Survivin 和 XIAP 蛋白水平阻断 COMP 增强生存的能力。 大多数 IAP 家族成员的 mRNA 并未因 COMP 而增加，表明翻译/翻译后机制参与了它们的诱导。然而，在 HeLa 细胞和软骨细胞中，COMP 均将 Survivin mRNA 水平诱导了 5 倍。 因此，Survivin 是第一个被鉴定为可被 COMP 上调转录的基因。 包含 3 型重复序列和 RDG 序列（CaCTD 结构域）的蛋白质羧基末端一半足以促进存活并提高 IAP 水平。 此外，RGD 肽能够阻断 COMP 的促生存作用以及 XIAP 和生存素的诱导，表明生存可能是通过整合素信号传导介导的。这些数据指出了 COMP 在保护细胞免于死亡方面的新作用，并应有助于阐明这种重要的 ECM 分子对软骨生物学和骨关节炎的贡献。 (2) 我们已经能够在原代人软骨细胞中鉴定出重要的组蛋白脱乙酰酶 (HDAC)。 我们发现这些酶是软骨细胞特异性基因表达的有效调节剂，因此当选择 HDAC 时，它们是软骨基因表达和软骨细胞存活的有效增强剂和抑制剂。 例如，SirT1 是一种 III 类组蛋白脱乙酰酶，在热量限制期间在延长寿命方面发挥着关键作用。 然而，人们对其在人类软骨细胞生物学中所起的作用知之甚少。我们发现 HDAC SirT1 和 NAD 依赖性酶可以通过修饰染色质在软骨表达和细胞存活的调节中发挥非常积极的作用，并且 NAD 挽救途径酶 NAMPT 在 OA 软骨中显着减少。 因此，NAMPT 可能是 OA 的一个重要的新标志物。 此外，我们发现其他 HDAC，即 HDAC1 和 HDAC2，是软骨基因表达的有效抑制因子。 此外，我们发现 OA 软骨中的 HDAC1 和 HDAC2 升高。 因此，HDAC 酶的失调可能导致 OA d) 结论和意义及未来方向 在过去的两到三十年中，在发现各种形式的关节炎的病因和治疗方法方面取得了很大进展。 然而，这些疾病的起始事件尚不清楚，许多治疗方案充其量也不是最理想的。 由于关节炎是一种使人非常衰弱的疾病，影响很大一部分人口，因此应该付出很大努力来揭示该疾病的根本原因并确定新的治疗方法。 我相信我们在定义软骨生物学的新研究领域和关节炎疾病的潜在治疗方面已经取得了巨大进展。 我们探索了 COMP（一种重要的软骨蛋白）在细胞死亡和存活中的作用，并试图确定其在关节炎长期进展中的作用。 此外，我们还探索了 HDAC 及其相应抑制剂在软骨细胞基因表达中的作用以及作为 OA 潜在治疗方法的作用。 HDAC 在软骨生物学中的作用是一个完全未被触及的领域，它为更好地了解软骨细胞基因表达的调控机制以及通过使用特定 HDAC 抑制剂治疗软骨疾病带来了很大希望。