AGGRECAN-hG1 AND LP TRANSGENIC MICE AS MODELS OF OSTEOARTHRITIS (OA)

AGGRECAN-hG1 和 LP 转基因小鼠作为骨关节炎 (OA) 模型

• 批准号: 7436245
• 负责人: TIBOR T. GLANT
• 金额: $ 18.71万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-10 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7436245
• 关键词: Age; Age-Months; Aging; Aging-Related Process; Arthritis; Backcrossings; Binding; Binding Sites; Biochemical; Biomechanics; CD44 Antigens; Cartilage; Chondrocytes; Collagen Type II; Complex; Core Protein; Degenerative polyarthritis; Embryo; Event; Exhibits; Figs - dietary; Genetic; Glycosaminoglycans; Goals; Hand; Hip Joint; Homeostasis; Human; Hyaluronan; Immune Tolerance; Inbred BALB C Mice; Individual; Joints; Knee; Knock-out; Lead; Life; Measures; Modeling; Mus; Numbers; Osteoarthrosis Deformans; Perinatal; Physiological; Process; Protein Deficiency; Protein Overexpression; Proteoglycan; Purpose; Recombinants; Resistance; Rheumatoid Arthritis; Role; Side; Simulate; Solid; Structure; Surface; Testing; Time; Tissues; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Model; Transgenic Organisms; Vertebral column; Water; Weight-Bearing state; Work; age related; aggrecan; articular cartilage; base; biglycan; dalton; desire; in vivo; juvenile animal; link protein; loss of function; normal aging; repaired; resilience; size; transgene expression

DESCRIPTION (provided by applicant): The G1 domain of cartilage proteoglycan (PG) aggrecan is responsible for the binding to hyaluronan (HA), and this macromolecular interaction is stabilized with cartilage link protein (LP). This interaction (in normal cartilage) forms multimillion-Dalton-size aggregates entrapped within the type II collagen meshwork, and the glycosaminoglycan (GAG)-substituted regions of the aggrecan core protein retain water responsible for the compression-resisting resilience of cartilage. Therefore, our working hypothesis is that the in vivo constitutive (over)expression of recombinant HA-binding molecules (such as cartilage LP and G1 domain of aggrecan) should disrupt the natural in vivo homeostasis of cartilage, as the recombinant molecules compete with the endogenous (wild-type) molecules for HA-binding sites. We have generated transgenic mice overexpressing either LP or G1 domain (hG1) of human aggrecan in cartilage. We selected high and low transgene expression lines of each, and backcrossed them into BALB/c background. Here, we propose to test our hypothesis that joint/cartilage abnormalities might be due to the consequence of the slow, but continuous accumulation of the G1 domain and LP in aging mice, and these HA-binding structures may competitively inhibit desired repair processes in cartilage by occupying the binding sites on the HA backbone from newly synthesized aggrecan and LP. A similar process may occur in humans, which is slow in normal aging tissue, but it is "accelerated" in osteoarthritic cartilage. The two transgenic models (hG1-Tg and LP-Tg), although generated for different purposes, may be useful models to test this hypothesis. We will simultaneously perform morphological, biochemical (transcriptional and translational) and biomechanical studies of knee and hip joints of these transgenic animals. We will identify the consequence of the constitutive (over)expression of individual (hG1 or LP) transgene, and the combination (co-expression) of both HA-binding proteins during the aging processes of transgenic animals. The ultimate goal of this proposal is to gather sufficient information on the in vivo role of HA-binding proteins and create a solid basis for subsequent transgenic and knockout approaches for studying osteoarthritic events in cartilage. Cartilage proteoglycan (PG) aggrecan binds to hyaluronan (HA), and this aggrecan-HA interaction is stabilized by a third component called "link protein" (LP). The major physiological function of aggrecan is to immobilize water, via its glycosaminoglycan (GAG) side chains, providing resilience for the weight-bearing articular cartilage. Although the differences between normal aging and osteoarthritic tissues are significant, there are also similarities in (patho)physiological mechanisms. For example, degradation products are either lost (GAG-binding domains) or accumulate (HA-binding domain) in both aging and osteoarthritic cartilages, which results in cartilage degradation and loss of function. Although resident chondrocytes attempt to repair cartilage damage, the newly synthesized aggrecan is also lost as virtually no binding site is available on the G1/LP-saturated HA backbone. To date, no systemic study has been performed for measuring simultaneously the expression and synthesis of HA, aggrecan and LP. We propose to investigate the in vivo consequence of manipulating aggrecan, LP and HA-binding interactions through transgenic approaches. We have generated two different transgenic colonies constitutively (over)expressing either the G1 domain of human aggrecan, and LP. We propose to study "accelerated aging" in cartilage, which may lead to early OA-like abnormalities. We propose to achieve this goal by in vivo overexpression of the aggrecan G1 domain (the HA-binding region) and LP in transgenic mice. These transgene products should compete for the binding sites on HA with naturally synthesized aggrecan and LP. Transgenic animals overexpressing G1 domain and LP will be intercrossed to create double transgenic mice, possibly simulating various degrees of aging and/or osteoarthritic processes.

描述（由申请人提供）：软骨蛋白聚糖（PG）聚集蛋白聚糖的G1结构域负责与透明质酸（HA）结合，并且这种大分子相互作用与软骨连接蛋白（LP）稳定。这种相互作用（在正常软骨中）形成了截留在II型胶原网络内的数百万道尔顿大小的聚集体，并且聚集蛋白聚糖核心蛋白的糖胺聚糖（GAG）取代区域保留了负责软骨抗压弹性的水。因此，我们的工作假设是，重组HA结合分子（如软骨LP和聚集蛋白聚糖的G1结构域）的体内组成型（过）表达应破坏软骨的天然体内稳态，因为重组分子与内源性（野生型）分子竞争HA结合位点。我们已经产生了转基因小鼠过度表达LP或G1结构域（hG 1）的人聚集蛋白聚糖在软骨。我们选择了每个基因的高和低转基因表达系，并将它们回交到BALB/c背景中。在这里，我们建议测试我们的假设，关节/软骨异常可能是由于缓慢的后果，但在老龄小鼠中的G1域和LP的连续积累，这些HA结合结构可能会竞争性地抑制所需的修复过程中软骨通过占领结合位点的HA骨干从新合成的聚集蛋白聚糖和LP。类似的过程也可能发生在人类身上，在正常的老化组织中是缓慢的，但在骨关节炎软骨中是“加速的”。两种转基因模型（hG 1-Tg和LP-Tg），虽然产生的目的不同，可能是有用的模型来检验这一假设。我们将同时对这些转基因动物的膝关节和髋关节进行形态学、生物化学（转录和翻译）和生物力学研究。我们将鉴定单个（hG 1或LP）转基因的组成性（过）表达的结果，以及转基因动物的衰老过程中两种HA结合蛋白的组合（共表达）。该建议的最终目标是收集足够的信息，在体内的HA结合蛋白的作用，并创建一个坚实的基础，为随后的转基因和敲除方法研究软骨中的骨关节炎事件。胶原蛋白聚糖（PG）聚集蛋白聚糖与透明质酸（HA）结合，并且这种聚集蛋白聚糖-HA相互作用由称为“连接蛋白”（LP）的第三组分稳定。聚集蛋白聚糖的主要生理功能是通过其糖胺聚糖（GAG）侧链脱水，为承重关节软骨提供弹性。虽然正常老化和骨关节炎组织之间的差异是显着的，也有相似之处（病理）生理机制。例如，降解产物在老化和骨关节炎软骨中丢失（GAG结合结构域）或积累（HA结合结构域），这导致软骨降解和功能丧失。虽然常驻软骨细胞试图修复软骨损伤，但新合成的聚集蛋白聚糖也会丢失，因为在G1/LP饱和的HA骨架上几乎没有结合位点。迄今为止，还没有进行系统的研究来同时测量HA、聚集蛋白聚糖和LP的表达和合成。我们建议通过转基因方法研究操纵聚集蛋白聚糖，LP和HA结合相互作用的体内后果。我们已经产生了两个不同的转基因菌落组成型（过）表达的G1域的人聚集蛋白聚糖，和LP。我们建议研究软骨的“加速老化”，这可能导致早期OA样异常。我们建议通过在转基因小鼠中过表达聚集蛋白聚糖G1结构域（HA结合区）和LP来实现这一目标。这些转基因产物应与天然合成的聚集蛋白聚糖和LP竞争HA上的结合位点。将过表达G1结构域和LP的转基因动物杂交以产生双转基因小鼠，可能模拟不同程度的衰老和/或骨关节炎过程。