CIS REGULATORY MOTIFS IN ADULT ARTICULAR CHONDROYTES

描述（由申请人提供）：本补充是对2009年美国再投资和复苏法案的回应，特别是对竞争性修订申请的复苏法案基金的回应（NOT-OD-09-058）。这是我们资助的父母资助R01 AR 050847 （NIH/NIAMS）题为“成人关节软骨细胞中的Cis调控基序”的补充。亲本资助的具体目的是：(1)研究Site-1蛋白酶（S1P）在体内软骨形成和软骨细胞表型维持中的作用；(2)探讨S1P对体外增生性软骨细胞分化和基质合成的影响。S1P在内质网（ER）膜结合的潜在转录因子转化为自由和活性核形式的过程中起着重要作用。它激活在维持脂质稳态中起作用的转录因子SREBP；它还能激活在内质网应激反应中起作用的转录因子ATF6。一项对S1Pcko小鼠（具有软骨特异性缺失S1P的小鼠）表型的研究表明，由于软骨缺乏主要的软骨蛋白，胶原型IIB (Col IIB)，小鼠表现出软骨发育不良。软骨细胞也表现出内质网应激，并伴随着重要的脂质生成途径的下调和内质网中Col IIB的包裹。本增刊旨在研究软骨细胞内质网应激反应调控途径，提供新的就业机会，并将涉及华盛顿大学、范德比尔特大学和哈佛医学院的合作努力。在SPECIFIC AIM 1中，我们将研究软骨细胞中脂质生成途径的下调是否会导致内质网应激和Col IIB在内质网中的包裹。我们将通过气相色谱/质谱法测量软骨细胞内质膜的胆固醇/脂肪酸组成，并使用电子顺磁共振波谱法分析内质膜的流动性。内质网将从S1Pcko小鼠的软骨细胞中获得，从他莫昔芬注射的S1Pf/f；在体外用表达Cre重组酶的腺病毒处理S1Pf/f小鼠的软骨细胞来删除S1P。在SPECIFIC AIM 2中，我们将分析功能性内质网应激反应途径对软骨和骨骼发育的重要性。对于这个分析，我们将构造Xbp1f/f；Col2-Cre小鼠特异性地在软骨细胞中删除Xbp1 （x- box结合蛋白1）功能（参与内质网应激反应）。Xbp1f / f;我们将通过形态学、组织学和免疫荧光研究系统地研究Col2-Cre小鼠，以研究Xbp1缺失对软骨和骨骼发育的影响。在SPECIFIC AIM 3中，我们将使用我们实验室开发的胫骨骨折愈合模型，研究S1P和Xbp1对出生后小鼠软骨和骨骼发育的需求。制定了在两年内完成所有三个具体目标的时间表。

DESCRIPTION (provided by applicant): This Supplement is in response to the American Reinvestment and Recovery Act of 2009, specifically in response to Recovery Act Funds for Competitive Revision Applications (NOT-OD-09-058). It is a Supplement to our funded parent grant R01 AR 050847 (NIH/NIAMS) titled "Cis Regulatory Motifs in Adult Articular Chondrocytes". The specific aims of the parent grant are (1) Investigate the role of Site-1 protease (S1P) in chondrogenesis and maintenance of the chondrocyte phenotype in vivo; and (2) Investigate the role of S1P on hypertrophic chondrocyte differentiation and matrix synthesis in vitro. S1P plays a fundamental role in the processing of endoplasmic reticulum (ER) membrane-bound latent transcription factors to their free and active nuclear form. It activates the transcription factor SREBP that plays a role in maintaining lipid homeostasis; it also activates ATF6, a transcription factor with a role in ER stress response. A study of the S1Pcko mice (mice with cartilage-specific deletion of S1P) phenotype show that the mice exhibit chondrodysplasia as the cartilage suffers from lack of the major cartilage protein, collagen type IIB (Col IIB). The chondrocytes also exhibit ER stress coupled with a down-regulation of important lipogenic pathways and entrapment of Col IIB in the ER. This Supplement is designed to investigate ER stress response regulatory pathways in chondrocytes, provide newer employment opportunities, and will involve the collaborative efforts of Washington University, Vanderbilt University, and Harvard Medical School. In SPECIFIC AIM 1, we will investigate whether down-regulation of lipogenic pathways in chondrocytes causes ER stress and the entrapment of Col IIB in the ER. We will measure the cholesterol/fatty acid composition of the chondrocytic ER membrane by gas chromatography/mass spectrometry and use electron paramagnetic resonance spectroscopy to analyze ER membrane fluidity. The ER will be obtained from chondrocytes from S1Pcko mice, from tamoxifen-injected S1Pf/f;CreERT mice, and from chondrocytes from S1Pf/f mice treated with Cre recombinase-expressing adenoviruses to delete S1P in vitro. In SPECIFIC AIM 2, we will analyze the importance of a functional ER stress response pathway to cartilage and skeletal development. For this analysis we will construct Xbp1f/f;Col2-Cre mice to delete Xbp1 (X-box-binding protein 1) function (which is involved in ER stress response) specifically in chondrocytes. The Xbp1f/f;Col2-Cre mice will be systematically studied through morphological, histological, and immunofluorescence studies to study the impact of Xbp1 deletion in cartilage and skeletal development. In SPECIFIC AIM 3, we will use a tibial fracture-healing model developed in our laboratory, to study the requirement of S1P and Xbp1 for cartilage and skeletal development in postnatal mice. A time-line has been developed to complete all three specific aims within two years. PUBLIC HEALTH RELEVANCE: An inability to respond to endoplasmic reticulum stress has been shown in recent years to be linked to debilitating human diseases such as diabetes, the neurodegenerative diseases retinitis pigmentosa and alzheimer's, atherosclerosis, and cancer. This proposal will focus on understanding the causes of endoplasmic reticulum stress in chondrocytes and analyze the importance of an endoplasmic reticulum stress response to cartilage and skeletal development. The knowledge gained from this study may allow for better strategies in designing tissue-engineered cartilage that would allow for the replacement of degenerate joint cartilage as seen during osteoarthritis.