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: 7959903
• 负责人: Lei Wei
• 金额: $ 15.26万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959903
• 关键词: 14-3-3 Proteins; Adult; Affect; Binding; Binding Proteins; Biology; Bone Growth; Calmodulin; Caspase; Caspase Inhibitor; Cell Differentiation process; Cell Nucleus; Centers of Research Excellence; Child; Chondrocytes; Clinical; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Degenerative polyarthritis; Development; Dominant-Negative Mutation; Dwarfism; Epiphysial cartilage; Flowcharts; Funding; Goals; Grant; HDAC4 gene; Health; Hypertrophy; In Situ Hybridization; Institution; Knowledge; Leg; Length; MAP Kinase Gene; MAP2K6 gene; MAPK11 gene; MAPK14 gene; MAPK8 gene; Mediating; Messenger RNA; Molecular; Mus; Nuclear; Organ Culture Techniques; Phosphorylation; Physiological; Prevention; Process; Regulation; Repression; Research; Research Personnel; Resources; Signal Pathway; Signal Transduction; Site; Solutions; Source; Transfection; Transgenic Mice; United States National Institutes of Health; Western Blotting; caspase-2; inhibitor/antagonist; prevent; repaired; research study; skeletal; 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. This aim consists of the four sub-aims. Specific Aim 1-1: We will determine whether HDAC4 shuttling from the nucleus to the cytoplasm is dependent on CaMKIV signaling during chondrocyte differentiation. Specific Aim 1-2: We will determine whether HDAC4 associates with 14-3-3 proteins in a phosphorylation-dependent manner in chondrocytes. Specific Aim 1-3: We will determine whether constitutively active CaMKIV overcomes HDAC4-induced inhibition of Runx2 and allows chondrocyte differentiation to proceed. Specific Aim 1-4: We will determine whether changing CaMKIV levels affects the growth plate proliferation and differentiation in organ culture. Hypothesis 2: P38 MAPK activity controls chondrocyte hypertrophy by increasing caspase-regulated degradation of HDAC4, which releases Runx2 from a repressive influence of HDAC4. This aim consists of the following five sub-aims. The experimental flowchart is described in the figure below. 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. This aim consists of the five sub-aims. Specific Aim 2-1: We will determine whether increase or decrease of chondrocyte p38 activity by transfection of constitutively active MKK6 (CaMKK6) or dominant negative p38 MAPK (DN p38) will affect caspase activity. Specific Aim 2-2: We will determine whether degradation of HDAC4 at Asp-289 site is dependent on caspase 2 and 3 in chondrocytes in the presence and absence of caspase 2 and 3 inhibitors. Specific Aim 2-3: We will determine whether chondrocyte differentiation is regulated by p38 activity. Elevation of chondrocyte p38 activity by constitutively active MKK6 (CaMKK6) and repression of chondrocyte p38 activity by dominant negative p38 MAPK (DN p38) will be employed in the presence or absence of caspase 2 and 3 inhibitors. Specific Aim 2-4: Expression of Runx2 mRNA will be examined by in situ hybridization with p38 MAPK defective growth plates from dominant negative p38 transgenic mice (DN p38). This examination will show whether the expression of Runx2 is dependent on chondrocyte p38 MAPK signaling. Specific Aim 2-5: The activities of MAPK, including p38, ERK, and JNK will be determined by Western blot to detect whether other MAPK are involved in the process. Such experiments will also be performed with p38 MAPK defective chondrocytes from DN p38 mice to determine whether these processes are dependent on chondrocyte p38 MAPK signaling. Significance. Although many factors have been found to impact the function of the growth plate, its biology remains poorly understood. The importance of clearly delineating the physiological controls of the physis cannot be overemphasized. Such knowledge could allow the prevention or correction of a number of bone-growth related problems in adults and children. Leg length discrepancy and dwarfism are just a few clinical dilemmas that may be provided with more elegant solutions if the molecular triggers and stops to the growth plate can be elucidated. The understanding of cell differentiation in growth plate may also be important to the understanding of abnormal chondrocyte differentiation in osteoarthritis.

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。子项目和 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通过结合和抑制细胞核中Runx 2/Cbfa 1的表达，作为软骨细胞肥大的负调节剂发挥作用。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。这可能损害HDAC 4介导的对细胞核中软骨细胞分化的抑制。这一目标由四个次级目标组成。 具体目标1-1：我们将确定HDAC 4从细胞核穿梭到细胞质是否依赖于软骨细胞分化过程中的CaMKIV信号传导。 具体目标1-2：我们将确定HDAC 4是否以磷酸化依赖性方式与软骨细胞中的14-3-3蛋白结合。 具体目标1-3：我们将确定组成型活性CaMKIV是否克服HDAC 4诱导的Runx 2抑制并允许软骨细胞分化进行。 具体目标1-4：我们将确定改变CaMKIV水平是否影响器官培养中生长板的增殖和分化。 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。这一目标包括以下五个次级目标。实验流程图如下图所示。 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。这一目标由五个次级目标组成。 具体目标2-1：我们将确定通过转染组成型活性MKK 6（CaMKK 6）或显性负性p38 MAPK（DN p38）来增加或减少软骨细胞p38活性是否会影响半胱天冬酶活性。 具体目标2-2：我们将确定在存在和不存在胱天蛋白酶2和3抑制剂的情况下，软骨细胞中Asp-289位点的HDAC 4降解是否依赖于胱天蛋白酶2和3。 具体目标2-3：我们将确定软骨细胞分化是否受p38活性调节。在存在或不存在半胱天冬酶2和3抑制剂的情况下，通过组成型活性MKK 6（CaMKK 6）提高软骨细胞p38活性和通过显性负性p38 MAPK（DN p38）抑制软骨细胞p38活性。 具体目标2-4：Runx 2 mRNA的表达将通过与来自显性阴性p38转基因小鼠（DN p38）的p38 MAPK缺陷生长板的原位杂交来检查。该检查将显示Runx 2的表达是否依赖于软骨细胞p38 MAPK信号传导。 具体目标2-5：通过蛋白质印迹法测定MAPK（包括p38、ERK和JNK）的活性，以检测是否有其他MAPK参与该过程。 这些实验也将用来自DN p38小鼠的p38 MAPK缺陷型软骨细胞进行，以确定这些过程是否依赖于软骨细胞p38 MAPK信号传导。 意义尽管已发现许多因素会影响生长板的功能，但对其生物学仍然知之甚少。 清楚地描述生长骨骺板的生理控制的重要性怎么强调也不过分。 这些知识可以预防或纠正成人和儿童的许多骨生长相关问题。腿长不一致和侏儒症只是一些临床难题，如果能够阐明生长板的分子触发和停止，可能会提供更优雅的解决方案。了解生长板中的细胞分化对于了解骨关节炎中软骨细胞的异常分化也很重要。