RI COBRE: REGULATION OF GROWTH PLATE DEVELOPMENT BYNUCLEAR/CYTOPLASMIC FACTORS

RI COBRE：核/细胞质因素对生长板发育的调节

基本信息

批准号：
8360475
负责人：
Lei Wei
金额：
$ 21.6万
依托单位：
RHODE ISLAND HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2012-09-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8360475
关键词：
Adult Arthritis Attenuated Birth Cartilage Cartilage Matrix Catabolism Cell Culture Techniques Centers of Research Excellence Chondrocytes Collagen Comparative Study Data Dental Schools Development Epiphysial cartilage Erinaceidae Event Fluorescence Funding Gene Targeting Genes Genetic Grant Health Histologic Hypertrophy Imaging Techniques Immunohistochemistry In Situ Hybridization In Vitro Interstitial Collagenase Joints Matrix Metalloproteinases Medical Medicine MicroRNAs Microarray Analysis Modeling Molecular Profiling Monitor Mus National Center for Research Resources Operative Surgical Procedures Osteogenesis Patients Play Prevention Principal Investigator Process Production Regulation Relative (related person)Reporting Research Research Infrastructure Resources Roentgen Rays Role Source Staining method Stains Stromelysin 1 Tamoxifen Tissues United States National Institutes of Health abstracting articular cartilage base cost in vivo mouse model novel strategies overexpression prevent repaired response skeletal smoothened signaling pathway tomography

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract Recently, we performed a miRNA expression profile using Microarray analysis in OA cartilage. We found the expression of miRNA-1 is undetectable while miRNA-31 is overexpression in the OA cartilage (154 fold increase) in comparison with the adjacent relative normal cartilage. Overexpression of miRNA-31 increases the expression of Indian Hedgehog (Ihh) and MMP-1 and 13 while knockdown miRNA-31 has opposite effects. Our data let us believe that Ihh plays a critical role in OA development. Our rationale for this focus is based on our and others' findings. Collective evidence include: a) Ihh is a key regulator of chondrocyte hypertrophy and endochondral bone formation [1] [2]; b) Ihh is mainly expressed in the developmental growth plate, and it is almost undetectable in normal adult articular cartilage; c) excessive amounts of Ihh are synthesized by chondrocytes in OA patients; d) Ihh promotes chondrocyte hypertrophy and increases MMP production, which subsequently induces cartilage degeneration; e) Knockdown Ihh in cell culture results in suppression of MMP release. Our comparative study of normal and OA patients indicates that OA cartilage degeneration is accompanied by a chondrocyte response to this damage which involves enhanced Ihh synthesis. The increase of Ihh in OA may involve not only accelerated processes but also the initiation of events that are not ordinarily encountered in healthy cartilage. These findings support the notion that elevated Ihh signaling in the joint may contribute significantly to cartilage matrix degeneration in OA. However, direct genetic evidence for Ihh in OA has not been reported because tissue-specific activation of the Ihh gene (targeted by Col2a1-Cre) died shortly after birth. In this study, we will specifically delete the Ihh gene in chondrocytes in adult mice by generating Ihh conditional activated mice through Col2a1-CreERT2; Ihhfl / Ihhfl (provide by Dr. Beate Lanske, Harvard School of Dental Medicine) to confirm and extend these findings. Hypothesis: Inducible deletion of Ihh prevents cartilage degeneration in OA mouse model Specific Aim: We will determine whether disrupting Ihh signaling pathway in vivo will attenuate OA progression in Col2a1-CreERT2; Ihhfl / Ihhfl mouse OA model induced by surgery. Tamoxifen (TM) will be delivered intraperitoneally for 5 consecutive days to remove Ihh. The resulting changes in OA cartilage will be evaluated by X-ray and histologically by Safranin-O staining, and quantified using the Modified Mankin score. Expression of type II, IX and X collagens and matrix metalloproteinase (MMP), -3, -9, and -13 will be further examined by immunohistochemistry and in situ hybridization (ISH). The change of cartilage degeneration will be monitored using MMPSense probe in vivo by fluorescence-based quantitative tomography, a non-invasive in vivo imaging technique (VisEn Medical). Summary Ihh expression is markedly elevated in OA cartilage. We further demonstrate that Ihh promotes chondrocyte hypertrophy and induces the release of matrix metalloproteinase in vitro. Thus, Ihh may activate cartilage catabolism during arthritis. In this application, we propose to evaluate novel strategies for prevention of Ihh induced cartilage degeneration in arthritis by the direct reduction of Ihh in vivo.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。摘要最近，我们使用微阵列分析对 OA 软骨进行了 miRNA 表达谱分析。我们发现与邻近的相对正常软骨相比，OA 软骨中 miRNA-1 的表达检测不到，而 miRNA-31 过度表达（增加了 154 倍）。 miRNA-31 的过表达会增加 Indian Hedgehog (Ihh) 以及 MMP-1 和 13 的表达，而敲低 miRNA-31 则会产生相反的效果。我们的数据让我们相信 Ihh 在 OA 发展中发挥着关键作用。我们如此关注的理由是基于我们和其他人的发现。集体证据包括： a) Ihh 是软骨细胞肥大和软骨内骨形成的关键调节因子 [1] [2]； b) Ihh主要表达于发育生长板，在正常成人关节软骨中几乎检测不到； c) OA患者的软骨细胞合成过量的Ihh； d) Ihh促进软骨细胞肥大并增加MMP的产生，随后诱导软骨退化； e) 细胞培养物中的敲低 Ihh 导致 MMP 释放的抑制。我们对正常人和 OA 患者的比较研究表明，OA 软骨退化伴随着软骨细胞对这种损伤的反应，其中涉及 Ihh 合成的增强。 OA 中 Ihh 的增加可能不仅涉及加速过程，还涉及健康软骨中通常不会遇到的事件的启动。这些发现支持这样的观点，即关节中 Ihh 信号传导的升高可能会显着导致 OA 中的软骨基质变性。然而，由于 Ihh 基因（Col2a1-Cre 靶向）的组织特异性激活在出生后不久就死亡，因此 Ihh 在 OA 中的直接遗传证据尚未报道。在本研究中，我们将特异性删除成年小鼠软骨细胞中的Ihh基因，通过Col2a1-CreERT2生成Ihh条件激活小鼠； Ihhfl / Ihhfl（由哈佛大学牙科医学院 Beate Lanske 博士提供）证实并扩展了这些发现。假设：Ihh 的诱导缺失可预防 OA 小鼠模型中的软骨退化具体目标：我们将确定破坏体内 Ihh 信号通路是否会减弱 Col2a1-CreERT2 中 OA 的进展；手术诱导的Ihhfl/Ihhfl小鼠OA模型。他莫昔芬 (TM) 将连续 5 天腹腔内给药以消除 Ihh。由此产生的 OA 软骨变化将通过 X 射线和 Safranin-O 染色进行组织学评估，并使用改良 Mankin 评分进行量化。 II型、IX型和X型胶原蛋白以及基质金属蛋白酶(MMP)、-3、-9和-13的表达将通过免疫组织化学和原位杂交(ISH)进一步检查。将使用 MMPSense 探针通过基于荧光的定量断层扫描（一种非侵入性体内成像技术（VisEn Medical））体内监测软骨退变的变化。概括 Ihh 表达在 OA 软骨中显着升高。我们进一步证明 Ihh 促进软骨细胞肥大并诱导体外基质金属蛋白酶的释放。因此，Ihh 可能会在关节炎期间激活软骨分解代谢。在本申请中，我们建议通过直接减少体内 Ihh 来评估预防 Ihh 诱导的关节炎软骨变性的新策略。