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.

Figure 1

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362 in c-fos–deficient 3T3 fibroblasts re-expressing wild-type c-Fos (WT) or mutated forms in which serine 362 has been replaced by alanine (362A) and/or serine 374 replaced by alanine (AA and 374A).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).

Figure 2

Rsk2-deficient mice are osteopenic. (A) Histological analysis of vertebral bodies of 2-, 4-, and 28-week-old male wild-type and Rsk2–/y littermates and quantification of the bone volume (BV) relative to total volume (TV). (B) In vivo quantification of osteoclast, osteoblast, and osteocyte numbers in 28-week-old mice. (C) Decreased femoral thickness (left panel) and decreased biomechanical competence (right panel) of bones lacking Rsk2. Bone diameters in millimeters are indicated in the lower left corners. (D) Determination of the bone-formation rate. Rsk2-deficient and wild-type littermates were injected twice with calcein at 1-week intervals, and the distance between the 2 lines of fluorochrome (left panel) was measured for assessment of bone-formation rate (BFR) (right panel). (E) Toluidine blue staining of growth plates from 2-week-old wild-type and Rsk2–/y littermates (left panel), and quantitation of growth plate width in 2-, 4-, and 28-week-old mice. *P < 0.05. Magnification, ×25.

Figure 3

Molecular and cellular analysis of the bone defects in Rsk2-deficient mice. (A) Analysis of type I collagen (col I), osteocalcin (oc), osteopontin (op), and Phex expression in the cortical bone of 8-week-old wild-type and Rsk2–/y littermates. (B) Analysis of osteoid volume in 28-week-old wild-type and Rsk2–/y littermates compared with Hyp–/y mice (24 weeks old). *P < 0.05. (C) Analysis of RSK2 and ERK activation in primary osteoblast cultures stimulated by IGF-1. RSK2 and ERK activation was analyzed by blotting with phospho-specific antibodies, and the loading was controlled by reblotting with total ERK antibody. (D) Proliferation curve of primary Rsk2-deficient osteoblasts compared with wild-type. (E) Determination of proliferation and apoptotic indices in Rsk2-deficient and wild-type osteoblasts in vitro. Arrowheads indicate TUNEL-positive cells. Quantifications are indicated in the lower left corners. Magnification, ×10. (F) In vitro differentiation of Rsk2-deficient osteoblasts compared with wild-type. Alkaline phosphatase activity (ALP) and bone nodule mineralization by alizarin red staining (ALZ) are shown. Arrowheads indicate alizarin red–positive bone nodules. Magnification, ×5. (G) Effect of IGF-1 on bone nodule mineralization by Rsk2-deficient osteoblasts compared with wild-type.

Figure 4

RSK2 is essential for c-Fos–induced tumor progression. (A) X-ray analysis of 7-month-old H2-c-fosLTR and H2-c-fosLTR/Rsk2–/y littermates. Histological analyses of bone sections from vertebral bodies and tibiae. White arrowheads indicate the osteosarcomas. Magnification, ×25. (B) Kinetic analysis of the development of osteosarcomas in H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y littermates; n indicates the number of tumors measured in tibiae at the indicated age. (C) Top: Microcomputed tomography analysis of vertebrae of 7-month-old H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y littermates. Magnification, ×5. Bottom: Quantification of tumor incidence (percentage of affected vertebrae) and tumor burden (tumor volume relative to tissue volume) in vertebral bodies; n indicates the number of tumor-affected vertebrae measured for tumor burden. (D) Top: Analysis of osteoid volume in tumors of wild-type and Rsk2–/y littermates. Magnification, ×200. Bottom: Quantification of the osteoid volume in the tumors and in the cortex. *P < 0.05.

Figure 5

Molecular and cellular analysis of RSK2 function in c-Fos–induced osteosarcomas. (A) In vivo analysis of RSK2 expression and c-Fos expression and phosphorylation on serine 362 in sections of osteosarcomas isolated from H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y mice. Top row: toluidine blue staining; second row, RSK2; third row, c-Fos; bottom row, P-Ser362. b, bone; arrows, osteoblasts. (B) Determination of proliferation and apoptotic indices in tumor section of H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y littermates. Proliferation was determined by counting of the proportion of Ki67-positive cells within the tumor, and the apoptotic indices were determined by counting of the proportion of TUNEL-positive cells (arrowheads) within the tumor. *P < 0.05. (C) Left: Western blot analysis of c-Fos expression and phosphorylation on serine 362 in primary osteoblasts (OBs) and in cell lines (P1.15, P1.4, P1, K11, R3, and C3) established from c-Fos–induced osteosarcomas of various origins (P, pelvis; K, long bone; R, ribs; C, calvaria). Right: Western blot analysis of c-Fos expression and phosphorylation in primary cells isolated from tumors of H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y littermates and Northern blot analysis of c-Fos expression in long bones of H2-c-fosLTR/Rsk2+/y and H2-c-fosLTR/Rsk2–/y littermates. Loading was controlled by analysis of β-actin and fox expression, respectively. Double asterisks indicate nonspecific bands. (D) Effect of inhibition of proteasome activity by MG132 on c-Fos expression in primary cells isolated from H2-c-fosLTR/Rsk2–/y tumors (left) and in a cell line established from H2-c-fosLTR/Rsk2–/y tumors (right). (E) Effect of RSK2 inhibitor (RO 31-8220) on c-Fos expression in primary cells isolated from tumors of H2-c-fosLTR/Rsk2+/y mice. (F) Effect of RSK2 inhibitor on apoptosis and proliferation of primary cells isolated from tumors of H2-c-fosLTR/Rsk2+/y mice.