ATF4 is Downstream Effector of Bone Anabolic Growth Factors

描述(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、激素和最终的转录因子顺序作用控制。我们最近发现了ATF4及其上游调节因子RSK2，一种当突变时会导致Coffin-Lowry综合征(CLS)的激酶，这是一种与骨骼症状相关的智力低下状况。ATF4在小鼠体内的失活会导致严重的骨质疏松。缺乏RSK2的小鼠的骨骼异常与CLS患者相似。为了寻找ATF4和RSK2的上游调控因子，我们检测了重要的骨合成生长因子IGF1，发现IGF1诱导了RSK2和ATF4的磷酸化。我们还观察到，PTH下游的蛋白激酶PKA也能够磷酸化ATF4，增加其反式激活能力。这些结果表明，IGF1和PTH通过RSK2或PKA的作用，以ATF4为转录因子，对成骨细胞执行合成代谢功能。为了验证这一假设，我们在本申请中提出了以下具体目标： 1.分析IGF1和PTH是否激活RSK2或PKA，进而分别在251和254位磷酸化ATF4，以增强其在成骨细胞中的转录活性。 2.检测ATF4的磷酸化是否通过增加成骨细胞中ATF4‘S的稳定性来解释其细胞特异性。 3.建立过表达RSK2和PKA不再磷酸化的ATF4突变形式ATF4-S251A和ATF4-S254A的转基因小鼠，研究RSK2-和PKA-磷酸化在体内的功能相关性。 通过本研究获得的知识将加深我们对成骨细胞分化、骨骼发育和终生骨重塑机制的理解，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.