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

这个子项目是许多利用 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。我们最近的研究结果表明，HDAC 4的核质穿梭和降解在这一过程中发挥了重要作用。我们特别 推测HDAC 4通过结合和抑制细胞核中Runx 2/Cbfa 1的表达而作为软骨细胞肥大的负调节剂起作用。Runx 2/Cbfa 1是软骨细胞分化和肥大所必需的转录因子。然而，HDAC 4穿梭和降解的机制尚未确定。关于这些机制的总体假设包括两个部分：假设1：HDAC 4核质穿梭控制软骨细胞分化，并依赖于Ca 2 +/钙调素信号通路。具体目的1：确定Ca 2 +/钙调蛋白信号通路的激活是否阻止了 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2：HDAC 4降解由P38 MARK通过增加caspase 2和3的表达来调节。具体目标二：确定是否：2a）半胱天冬酶降解HDAC 4; 2b）p38 MARK调节半胱天冬酶2和3的表达或活性; 2c）通过在p38 MARK缺陷的生长板中的原位杂交，Runx 2表达依赖于p38 MARK; 2d）通过蛋白质印迹，p38、ERK和JNK的活性参与该过程。项目2b将使用活性MKK 6进行测试，以提高p38和显性 p38阴性标记抑制p38。意义生长板或骨骺板决定了人类成熟骨的未来长度和形状。描述生长骨骺的生理控制可以防止儿童致残畸形，而不需要手术干预。一旦更好地理解了影响生长板的分子触发和停止，预防腿长不等、脊柱侧凸、侏儒症、髋关节发育不良和骨关节炎中的异常软骨细胞分化也是可能的。

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 undergo endochondral ossification. Our recent findings indicate that HDAC4 nuclear-cytoplasmic shuttling and degradation plays a major role in this process. Specifically, we suspect that HDAC4 functions as a negative regulator of chondrocyte hypertrophy by binding and inhibiting Runx 2/Cbfa1 expression in the nucleus. Runx 2/Cbfa1 is a transcription factor necessary for chondrocyte differentiation and hypertrophy. However, the mechanisms underlying HDAC4 shuttling and degradation are as yet undetermined. The overall hypothesis about these mechanisms 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: HDAC4 degradation is regulated by P38 MARK by increasing expression of caspases 2 and 3. Specific Aim 2: To determine whether: 2a) caspases degrade HDAC4; 2b) p38 MARK regulates expression or activity of capsases 2 and 3; 2c) Runx2 expression is dependent on p38 MARK by in situ hybridization in p38 MARK defective growth plates; 2d) the activities of p38, ERK, and JNK are involved in the process by western blot. Project 2b will be tested using active MKK6 to elevate p38 and dominant negative p38 MARK to repress p38. Significance. The growth plate, or physis, determines the future length and shape of the human mature bone. Delineating the physiological controls of the physis could prevent disabling deformities in children without the need for surgical interventions. Prevention of leg length discrepancy, scoliosis, dwarfism, hip dysplasia and abnormal chondrocyte differentiation in osteoarthritis might also be possible once molecular triggers and stops affecting the growth plate are better understood.