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

• 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
• 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

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 摘要最近，我们利用微阵列技术分析了OA软骨中miRNA的表达谱。我们发现，与邻近的相对正常软骨相比，在OA软骨中miRNA-1的表达是不可检测的，而miRNA-31是过表达的（154倍增加）。miRNA-31的过表达增加了Indian Hedgehog（Ihh）和MMP-1和13的表达，而敲低miRNA-31具有相反的作用。我们的数据让我们相信Ihh在OA发展中起着关键作用。我们之所以关注这一点，是基于我们和其他人的调查结果。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主要在发育生长板中表达，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，从而诱导软骨退变; e）在细胞培养物中敲低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软骨退变伴随着软骨细胞对这种损伤的反应，这涉及增强Ihh的合成。OA中Ihh的增加可能不仅涉及加速的过程，而且还涉及健康软骨中通常不会遇到的事件的启动。这些发现支持了关节中升高的Ihh信号可能显著促进OA中软骨基质变性的观点。然而，OA中Ihh的直接遗传证据尚未报道，因为Ihh基因的组织特异性激活（由Col 2a 1-Cre靶向）在出生后不久死亡。在这项研究中，我们将通过Col 2a 1-CreERT 2; Ihhfl / Ihhfl（由哈佛牙科医学院的Beate Lanske博士提供）产生Ihh条件激活小鼠来特异性删除成年小鼠软骨细胞中的Ihh基因，以证实和扩展这些发现。 假设：诱导性Ihh缺失可防止OA小鼠模型中的软骨退变 具体目标：我们将确定在体内破坏Ihh信号通路是否会减弱手术诱导的Col 2a 1-CreERT 2; Ihhfl / Ihhfl小鼠OA模型中的OA进展。他莫昔芬（TM）将连续5天腹膜内递送以去除Ihh。 将通过X射线和番红-O染色进行组织学评价，并使用改良Mankin评分进行定量。 通过免疫组织化学和原位杂交（ISH）进一步检测II、IX和X型胶原和基质金属蛋白酶（MMP）、-3、-9和-13的表达。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））在体内监测软骨退化的变化。 总结 Ihh表达在OA软骨中显著升高。我们进一步证明，IHH促进软骨细胞肥大，并诱导释放基质金属蛋白酶在体外。因此，Ihh可能会激活关节炎期间的软骨catastrophic。在本申请中，我们建议评估新的策略，用于预防IHH诱导的关节炎软骨退化的直接减少IHH在体内。