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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的长期目标是了解调控生长板发育的分子机制。生长板成熟的最后一步是增殖的软骨细胞分化成肥大的软骨细胞，其随后经历软骨内骨化。 Runx 2/Cbfa 1是软骨细胞分化和肥大所必需的转录因子。HDAC 4通过结合并抑制细胞核中Runx 2/Cbfa 1的表达，作为软骨细胞肥大的负调节因子。我们最近的研究结果表明，HDAC 4核质穿梭和降解发生在软骨细胞，使软骨细胞分化和进一步肥大。然而，HDAC 4穿梭和降解的机制尚不清楚。总体假设包括两部分： 假设1：HDAC 4核质穿梭控制软骨细胞分化，并依赖于Ca 2 +/钙调蛋白信号通路。 具体目标1：确定Ca 2 +/钙调蛋白信号通路的激活是否阻止HDAC 4进入细胞核并增强HDAC 4与细胞质结合蛋白14-3-3的结合。这可能会损害HDAC 4介导的对细胞核中软骨细胞分化的抑制作用。 假设二：P38 MAPK活性通过增加半胱天冬酶调节的HDAC 4降解来控制软骨细胞肥大，从而从HDAC 4的抑制影响中释放Runx 2。 具体目标2：为了确定半胱天冬酶是否诱导HDAC 4的降解，通过在存在或不存在半胱天冬酶抑制剂的情况下使用组成型活性MKK 6来升高p38和显性负性p38 MAPK来抑制p38，这反过来增加Runx 2活性，从而由p38 MAPK控制。 新的实验“Ihh的诱导性缺失可防止小鼠OA模型中的软骨退化”已获得COBRE外部咨询委员会的批准。 摘要 最近，我们使用微阵列分析OA软骨中的miRNA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、-3和1,-3的表达。我们进一步的试点研究让我们相信，Ihh在OA的发展中起着至关重要的作用。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。 将通过免疫组织化学和原位杂交（ISH）进一步检测II型、IX型和X型胶原以及基质金属蛋白酶（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。

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.