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

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。这一目标由四个子目标组成。 具体目标1-1：我们将确定在软骨细胞分化过程中，HDAC4是否依赖于CaMKIV信号从细胞核到细胞质。 具体目标1-2：我们将确定HDAC4是否以磷酸化依赖的方式与软骨细胞中的14-3-3蛋白结合。 具体目标1-3：我们将确定结构性活性CaMKIV是否能克服HDAC4对Runx2的抑制并允许软骨细胞分化。 具体目标1-4：我们将确定改变CaMKIV水平是否会影响器官培养中生长板的增殖和分化。 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。这一目标包括以下五个分目标。实验流程图如下图所示。 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。这一目标由五个子目标组成。 具体目标2-1：我们将确定通过转染结构性活性的MKK6(CaMKK6)或显性阴性的p38MAPK(DN P38)来增加或降低软骨细胞p38活性是否会影响caspase活性。 具体目标2-2：我们将确定在存在和不存在caspase 2和3抑制剂的情况下，软骨细胞中hdac4在Asp-289位点的降解是否依赖于caspase 2和3。 具体目标2-3：我们将确定软骨细胞分化是否受p38活性的调节。在caspase 2和3抑制剂存在或不存在的情况下，通过结构性激活的MKK6(CaMKK6)提高软骨细胞p38的活性，通过显性阴性的p38MAPK(DN P38)抑制软骨细胞p38的活性。 特定目的2-4：用p38 MAPK缺陷生长板与显性阴性p38转基因小鼠(DN P38)的生长板进行原位杂交，检测Runx2基因的表达。这项检查将显示Runx2的表达是否依赖于软骨细胞p38MAPK信号。 具体目的2-5：用Western印迹法检测MAPK的活性，包括p38、ERK和JNK，以检测是否有其他MAPK参与这一过程。这些实验也将对来自dnp38小鼠的p38MAPK缺陷软骨细胞进行，以确定这些过程是否依赖于软骨细胞p38MAPK信号。 意义重大。虽然已经发现许多因素影响生长板的功能，但对其生物学仍知之甚少。清楚地描述骨赘的生理控制的重要性怎么强调都不为过。这样的知识可以预防或纠正成人和儿童的一些与骨骼生长相关的问题。如果生长板的分子触发和停止被阐明，腿长不一致和侏儒症只是几个临床难题，可能会提供更好的解决方案。了解生长板中的细胞分化对于理解骨关节炎中软骨细胞的异常分化也很重要。

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.