ATF4 is Downstream Effector of Bone Anabolic Growth Factors

描述（由申请人提供）：成骨细胞的分化和功能由一系列不完全确定的生长因子、细胞因子、激素以及最终按顺序起作用的转录因子控制。我们最近发现，ATF 4及其上游调节因子RSK 2是一种激酶，当突变时，会导致Coffin-Lowry综合征（CLS），这是一种与骨骼表现相关的精神发育迟滞疾病。Atf 4在小鼠中的失活导致严重的骨质疏松症。缺乏Rsk 2的小鼠具有与CLS患者相似的骨骼异常。为了寻找ATF 4和RSK 2的上游调节因子，我们测试了一种重要的骨合成生长因子IGF 1，发现IGF 1诱导RSK 2和ATF 4的磷酸化。我们还观察到，PKA，PTH的下游蛋白激酶，也能够磷酸化ATF 4，并增加其反式激活能力。这些数据表明，IGF 1和PTH通过RSK 2或PKA的作用，以ATF 4为转录因子，对成骨细胞执行合成代谢功能。为了检验这一假设，我们在本申请中提出了以下具体目标： 1.分析IGF 1和PTH是否激活RSK 2或PKA，然后分别在丝氨酸251和254处磷酸化ATF 4，以增强其在成骨细胞中的转录活性。 2.为了测试ATF 4的磷酸化是否通过增加ATF 4在成骨细胞中的稳定性来解释其细胞特异性。 3.建立过表达ATF 4-S251 A和ATF 4-S254 A的转基因小鼠，以研究RSK 2和PKA磷酸化的功能相关性。 从这项研究中获得的知识将提高我们对成骨细胞分化，骨骼发育和骨重塑机制的理解，并有助于未来的治疗药物发现。

DESCRIPTION (provided by applicant): The differentiation and function of osteoblasts are controlled by a series of incompletely defined growth factors, cytokines, hormones, and ultimately transcription factors that act in sequence. We have recently identified that ATF4 and its upstream regulator, RSK2, a kinase that, when mutated, causes Coffin-Lowry syndrome (CLS), a mental retardation condition associated with skeletal manifestations. Inactivation of Atf4 in mice causes severe osteoporosis. Mice lacking Rsk2 have similar skeletal abnormalities to that of CLS patients. In order to search for upstream regulators of ATF4 and RSK2, we tested IGF1, an important bone anabolic growth factor, and found that IGF1 induced the phosphorylation of RSK2 and ATF4. We have also observed that PKA, a downstream protein kinase of PTH, is also able to phosphorylate ATF4 and increase its transactivation ability. These data suggest that IGF1 and PTH use ATF4 as their transcription factor via the action of RSK2 or PKA to execute the anabolic function on osteoblasts. To test this hypothesis, we propose in this application with the following Specific Aims: 1. To analyze whether IGF1 and PTH activate RSK2 or PKA, which then phosphorylate ATF4 at serines 251 and 254, respectively, to enhance its transcription activity in osteoblasts. 2. To test whether phosphorylations of ATF4 accounts for its cell-specificity by increasing ATF4's stability in osteoblasts. 3. To establish transgenic mice overexpressing ATF4-S251A and ATF4-S254A, two mutant forms of ATF4 that are no longer phosphorylated by RSK2 and PKA, to study the functional relevance of RSK2- and PKA-phosphorylation in vivo. The knowledge gained from this study will improve our understanding of the mechanisms governing osteoblast differentiation, skeletal development and bone remodeling throughout life, as well as assist therapeutic drug discoveries in the future.