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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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将增殖的软骨细胞分化为肥大的软骨细胞，随后经历软骨内成骨。RUNX 2/Cbfa1是软骨细胞分化和肥大所必需的转录因子。HDAC4通过结合和抑制核内RUNX-2/Cbfa1的表达，发挥软骨细胞肥大的负性调节作用。我们最近的发现表明，HDAC4在软骨细胞中发生核浆穿梭和降解，使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。总体假设包括两个部分： 假设1：HDAC4核质穿梭控制软骨细胞分化，并依赖于钙/钙调蛋白信号通路。 具体目标1：确定钙/钙调蛋白信号通路的激活是否阻止HDAC4的核进入，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介导的对细胞核内软骨细胞分化的抑制。 假设2：p38MAPK活性通过增加caspase调节的HDAC4的降解来控制软骨细胞肥大，从而从HDAC4的抑制影响中释放Runx2。 特异性目的2：确定caspase是否诱导HDAC4的降解是由p38MAPK控制的，在caspase抑制剂存在或不存在的情况下，利用结构性活性的MKK6上调p38，显性负的p38MAPK抑制p38，进而增加Runx2的活性。 Cobre外部咨询委员会批准了一项新的实验--“诱导IHH缺失预防小鼠骨关节炎模型软骨退变”。 摘要 最近，我们利用微阵列分析技术在骨性关节炎软骨中进行了miRNA表达谱分析。我们发现在OA软骨中没有检测到miRNA-1的表达，而miRNA-31在OA软骨中过表达(与邻近的相对正常软骨相比增加了154倍)。MiRNA-31的过表达增加了印度Hedgehog(IHH)和MMP-1、-3和13的表达。我们进一步的初步研究使我们相信IHH在OA的发生发展中起着关键作用。我们关注这一点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主要在发育中的生长板表达，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多的IHH；d)IHH促进软骨细胞肥大，增加MMP的产生，从而导致软骨退化；e)在细胞培养中抑制IHH的释放。 我们对正常和骨性关节炎患者的对比研究表明，骨性关节炎软骨退化伴随着软骨细胞对这种损伤的反应，这涉及到IHH合成的增强。骨性关节炎中IHH的增加可能不仅涉及加速的过程，而且还涉及正常软骨中不常见的事件的启动。这些发现支持这样一种观点，即关节中IHH信号的升高可能是骨关节炎软骨基质退变的重要原因。然而，由于IHH基因的组织特异性激活(以Col2a1-Cre为靶点)在出生后不久就死亡，因此还没有直接的遗传学证据在OA中被报道。在这项研究中，我们将通过Col2a1-CreERT2；Ihhfl/Ihhfl(由哈佛大学牙科医学院Beate Lanske博士提供)产生IHH条件激活小鼠，从而特异性地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。 假说：诱导性IHH缺失可防止骨性关节炎小鼠模型软骨退变 具体目的：我们将确定体内阻断IHH信号通路是否会延缓手术诱导的Col2a1-CreERT2；Ihhf1/Ihhf1小鼠OA模型的进展。他莫昔芬(TM)将连续5天通过腹膜腔给药，以消除IHH。由此产生的骨性关节炎软骨的变化将通过X射线和组织学通过藏红花-O染色进行评估，并使用改良的Mankin评分进行量化。免疫组织化学和原位杂交将进一步检测II型、IX型和X型胶原以及基质金属蛋白酶(MMPs)、-3、-9和-13的表达。软骨退变的变化将使用MMPSense探针通过基于荧光的定量断层扫描进行体内监测，这是一种非侵入性体内成像技术(Visen Medical)。 摘要 IHH在骨性关节炎软骨中的表达明显升高。我们进一步证明，IHH在体外促进软骨细胞肥大，并诱导基质金属蛋白酶的释放。因此，IHH可能在关节炎过程中激活软骨分解代谢。在这项应用中，我们建议评估通过体内直接减少IHH来预防IHH诱导的关节炎软骨退化的新策略。