Essential role of RSK2 in c-Fos–dependent osteosarcoma development
Jean-Pierre David, … , Michael Amling, Erwin F. Wagner
Jean-Pierre David, … , Michael Amling, Erwin F. Wagner
Published March 1, 2005
Citation Information: J Clin Invest. 2005;115(3):664-672. https://doi.org/10.1172/JCI22877.
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Categories: Article Bone biology

Essential role of RSK2 in c-Fos–dependent osteosarcoma development

Abstract

Inactivation of the growth factor–regulated S6 kinase RSK2 causes Coffin-Lowry syndrome in humans, an X-linked mental retardation condition associated with progressive skeletal abnormalities. Here we show that mice lacking RSK2 develop a progressive skeletal disease, osteopenia due to impaired osteoblast function and normal osteoclast differentiation. The phenotype is associated with decreased expression of Phex, an endopeptidase regulating bone mineralization. This defect is probably not mediated by RSK2-dependent phosphorylation of c-Fos on serine 362 in the C-terminus. However, in the absence of RSK2, c-Fos–dependent osteosarcoma formation is impaired. The lack of c-Fos phosphorylation leads to reduced c-Fos protein levels, which are thought to be responsible for decreased proliferation and increased apoptosis of transformed osteoblasts. Therefore, RSK2-dependent stabilization of c-Fos is essential for osteosarcoma formation in mice and may also be important for human osteosarcomas.

Authors

Jean-Pierre David, Denis Mehic, Latifa Bakiri, Arndt F. Schilling, Vice Mandic, Matthias Priemel, Maria Helena Idarraga, Markus O. Reschke, Oskar Hoffmann, Michael Amling, Erwin F. Wagner

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Figure 1

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c-Fos phosphorylation by RSK2 is not required for osteoclast differentia...
c-Fos phosphorylation by RSK2 is not required for osteoclast differentiation. (A) Characterization of the specific antibody directed against c-Fos phospho–serine 362. The position of the putative RSK2 phosphorylation site in the C-terminal domain of c-Fos is indicated in red. Other proposed phosphorylation sites, kinases that phosphorylate them, and the relative positions of the DNA binding and dimerization domain (bZIP) are also indicated. The following were analyzed by Western blot: c-Fos expression and phosphorylation on serine 362 in c-fos–deficient 3T3 fibroblasts re-expressing wild-type c-Fos (WT) or mutated forms in which serine 362 has been replaced by alanine (362A) and/or serine 374 replaced by alanine (AA and 374A). A c-Fos osteosarcoma cell line (C3) and the parental 3T3 (KO) were used as controls. *Nonspecific reacting bands. (B) c-Fos phosphorylation in M-CSF–stimulated M-BMMs. Immunostaining using the phospho–serine 362 c-Fos antibody. Arrowheads indicate immuno-positive nuclei, visualized using DAPI. Magnification, ×20. (C) RSK2 activation in M-CSF–stimulated M-BMMs. Immunoprecipitation kinase assay using an antibody directed against RSK2 and histone H3 as a substrate. (D) c-Fos expression and phosphorylation on serine 362 in Rsk2–/y M-BMMs compared with wild-type. RSK2 activation was analyzed using anti–phospho-RSK2; β-actin was used as loading control. (E) Expression of c-fos mRNA in M-BMMs lacking RSK2, analyzed by RNase protection assay; gapdh was used as loading control. (F) Left: Differentiation, induced by M-CSF and RANKL, of Rsk2–/y or wild-type monocytes into multinucleated osteoclasts (TRAP-positive cells). Right: Osteoclastogenic properties of calvarial osteoblasts isolated from Rsk2–/y or wild-type newborn littermates were analyzed by coculture with wild-type bone marrow cells stimulated by 1,25-dihydroxyvitamin D3 (VitD3) and dexamethasone (Dex).