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

描述（由申请人提供）：软骨蛋白聚糖（PG）聚集蛋白的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）形成了数百万dalton大小的聚集体，聚集体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G1结构域）的体内组成性（过）表达应该会破坏软骨的体内自然稳态，因为重组分子与内源性（野生型）分子竞争ha结合位点。我们在软骨中培养了过表达人聚集蛋白LP或G1结构域（hG1）的转基因小鼠。分别选择高、低转基因表达系，回交BALB/c背景。在这里，我们提出验证我们的假设，即关节/软骨异常可能是由于衰老小鼠中G1结构域和LP缓慢但持续积累的结果，这些HA结合结构可能通过占领新合成的聚集蛋白和LP在HA主链上的结合位点来竞争性地抑制软骨中所需的修复过程。类似的过程可能发生在人类身上，在正常的衰老组织中是缓慢的，但在骨关节炎软骨中是“加速”的。两种转基因模型（hG1-Tg和LP-Tg）虽然产生的目的不同，但可能是检验这一假设的有用模型。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、生化（转录和翻译）和生物力学研究。我们将确定个体（hG1或LP）转基因的组成（过）表达的结果，以及在转基因动物衰老过程中两种ha结合蛋白的组合（共表达）。本提案的最终目标是收集关于ha结合蛋白在体内作用的足够信息，并为后续的转基因和基因敲除方法研究软骨骨关节炎事件奠定坚实的基础。软骨蛋白聚糖（PG）聚集蛋白与透明质酸（HA）结合，这种聚集蛋白与透明质酸的相互作用被称为“连接蛋白”（LP）的第三种成分所稳定。聚集蛋白的主要生理功能是通过其糖胺聚糖（GAG）侧链固定水，为负重关节软骨提供弹性。尽管正常衰老和骨关节炎组织之间存在显著差异，但在（病理）生理机制上也存在相似之处。例如，老化和骨关节炎软骨中的降解产物要么丢失（gag结合结构域），要么积累（ha结合结构域），从而导致软骨降解和功能丧失。尽管存在的软骨细胞试图修复软骨损伤，但由于G1/ lp饱和的HA主干上几乎没有可用的结合位点，新合成的聚集蛋白也会丢失。迄今为止，还没有系统的研究同时测量HA， aggrecan和LP的表达和合成。我们建议通过转基因方法研究操纵聚合蛋白、LP和ha结合相互作用的体内结果。我们已经产生了两个不同的转基因菌落组成（过）表达人类聚集蛋白G1结构域和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体内过表达聚合蛋白G1结构域（ha结合区）和LP来实现这一目标。这些转基因产物应该与天然合成的聚集蛋白和LP争夺HA上的结合位点。过表达G1结构域和LP的转基因动物将交叉产生双转基因小鼠，可能模拟不同程度的衰老和/或骨关节炎过程。

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.