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. ? ?
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