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: 8168035
• 负责人: Lei Wei
• 金额: $ 19.8万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168035
• 关键词: Adult; Advisory Committees; Arthritis; Attenuated; Binding; Binding Proteins; Birth; Calmodulin; Cartilage; Cartilage Matrix; Caspase; Caspase Inhibitor; Catabolism; Cell Culture Techniques; Cell Nucleus; Centers of Research Excellence; Chondrocytes; Collagen; Comparative Study; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Dental Schools; Development; Dominant-Negative Mutation; Epiphysial cartilage; Erinaceidae; Event; Fluorescence; Funding; Gene Targeting; Genes; Genetic; Goals; Grant; HDAC4 gene; Histologic; Hypertrophy; Imaging Techniques; Immunohistochemistry; In Situ Hybridization; In Vitro; Institution; Interstitial Collagenase; Joints; MAP Kinase Gene; MAP2K6 gene; MAPK11 gene; MAPK14 gene; Matrix Metalloproteinases; Mediating; Medical; Medicine; MicroRNAs; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Monitor; Mus; Nuclear; Operative Surgical Procedures; Osteogenesis; Patients; Pilot Projects; Play; Prevention; Process; Production; Regulation; Relative (related person); Reporting; Research; Research Personnel; Resources; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Source; Staining method; Stains; Stromelysin 1; Tamoxifen; Tissues; United States National Institutes of Health; abstracting; articular cartilage; base; in vivo; mouse model; novel strategies; overexpression; prevent; research study; response; tomography; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-term goal is to understand the molecular mechanisms regulating growth plate development. The last step in maturation of the growth plate is the differentiation of proliferative chondrocytes into hypertrophic chondrocytes, which subsequently undergoes endochondral ossification. Runx 2/Cbfa1 is a transcription factor necessary for chondrocyte differentiation and hypertrophy. HDAC4 functions as a negative regulator of chondrocyte hypertrophy by binding and inhibiting Runx 2/Cbfa1 expression in the nucleus. Our recent findings indicate that HDAC4 nuclear-cytoplasm shuttling and degradation occurs in chondrocytes, allowing chondrocyte differentiation and further hypertrophy. However, the mechanisms underlying HDAC4 shuttling and degradation are unclear. The overall hypothesis includes two parts: Hypothesis 1: HDAC4 nuclear-cytoplasmic shuttling controls chondrocyte differentiation and is dependent on the Ca2+/calmodulin signaling pathway. Specific Aims 1: To determine whether activation of the Ca2+/calmodulin signaling pathway prevents nuclear entry of HDAC4 and enhances the binding of HDAC4 to the cytoplasmic binding protein 14-3-3. This may impair HDAC4-mediated inhibition of chondrocyte differentiation in the nucleus. Hypothesis 2: P38 MAPK activity controls chondrocyte hypertrophy by increasing caspase-regulated degradation of HDAC4, which releases Runx2 from a repressive influence of HDAC4. Specific Aims 2: To determine if caspases induce the degradation of HDAC4 is controlled by p38 MAPK by using constitutively active MKK6 to elevate p38 and dominant negative p38 MAPK to repress p38 in the presence or absence of caspase inhibitors, which in turn increases Runx2 activity. New Experiment " Inducible deletion of Ihh prevents cartilage degeneration in mouse OA model" has been approved by COBRE External Advisory Committee. 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,-3, and 13. Our further pilot study 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们的长期目标是了解调节生长板发育的分子机制。生长板成熟的最后一步是增殖软骨细胞分化为肥大软骨细胞，随后经历软骨内骨化。 Runx 2/Cbfa1 是软骨细胞分化和肥大所必需的转录因子。 HDAC4 通过结合并抑制细胞核中 Runx 2/Cbfa1 的表达，充当软骨细胞肥大的负调节因子。我们最近的研究结果表明，HDAC4 核细胞质穿梭和降解发生在软骨细胞中，从而允许软骨细胞分化和进一步肥大。然而，HDAC4 穿梭和降解的机制尚不清楚。总体假设包括两部分： 假设1：HDAC4核质穿梭控制软骨细胞分化并依赖于Ca2+/钙调蛋白信号通路。 具体目标 1：确定 Ca2+/钙调蛋白信号通路的激活是否会阻止 HDAC4 进入核并增强 HDAC4 与细胞质结合蛋白 14-3-3 的结合。这可能会削弱 HDAC4 介导的细胞核内软骨细胞分化抑制作用。 假设 2：P38 MAPK 活性通过增加 caspase 调节的 HDAC4 降解来控制软骨细胞肥大，从而使 Runx2 免受 HDAC4 的抑制影响。 具体目标 2：确定 caspase 诱导 HDAC4 的降解是否受 p38 MAPK 控制，在存在或不存在 caspase 抑制剂的情况下，使用组成型活性 MKK6 升高 p38，使用显性失活 p38 MAPK 抑制 p38，从而增加 Runx2 活性。 新实验“Ihh的诱导缺失可预防小鼠OA模型中的软骨退化”已获得COBRE外部顾问委员会的批准。 抽象的 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 的过度表达会增加 Indian Hedgehog (Ihh) 和 MMP-1、-3 和 13 的表达。我们进一步的初步研究让我们相信 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。