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doi: 10.1242/10.1242/jcs.00082

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JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms

Jean-Pierre David¹, Kanaga Sabapathy^1,*, Oskar Hoffmann², Maria H. Idarraga¹ and Erwin F. Wagner^1,

¹ Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria
² Institute fur Pharmakologie und Toxikologie, Pharmazie-Zentrum, Althanstrasse 14, A-1090 Vienna, Austria
^* Present address: National Cancer Center, Division of Cellular and Molecular Research, 11 Hospital Drive, 169610 Singapore, Singapore

View larger version (54K): [in a new window]   Fig. 1. Osteoclastogenesis is impaired in Jnk1-/- but not Jnk2-/- mice. Bone marrow hematopoietic precursors were isolated from Jnk1-/- (A,C,E), Jnk2-/- (B,D,F) and wild-type littermates and co-cultured with wild-type primary osteoblasts in osteoclastogenic conditions. Osteoclastogenesis was monitored by TRAP staining (red color). (A,B) TRAP staining after 6 days of culture; the arrows indicate multinucleated TRAP-positive cells. (C,D) Time course of osteoclast differentiation. (E,F) Osteoclast activity measured by pit assay on bovine cortical bone slices. The method for statistical analysis is described in the materials and methods, the stars indicate the statistical significance: (***) P<0.0001, (**) P<0.001, (*) P<0.005, (n) number of slices analyzed.

View larger version (32K): [in a new window]   Fig. 2. JNK1 but not JNK2 is activated by RANKL in osteoclast progenitors. (A) The expression of JNK isoforms was analyzed in bone marrow monocytes (BMMs) isolated from wild-type, Jnk1-/- and Jnk2-/- mice and cultured for 3 days in the presence of M-CSF alone (20 ng/ml) (-) or M-CSF and RANKL (50 ng/ml) (+). The expression of JNK isoforms was determined by western blotting using an antibody that recognizes both JNK1 and JNK2. (B,C) Time-dependent activation of JNK by RANKL. M-CSF-dependent monocytes were isolated from wild-type, Jnk1-/- and Jnk2-/- mice and treated with RANKL (50 ng/ml) for the indicated times. The kinase activity was assayed following JNK immunoprecipitation using GST-N-terminal-c-Jun fusion protein as a substrate. The phosphorylation of GST-c-Jun was quantified after SDS PAGE separation and phosphoimager analysis. (D) JNK1 immunodepletion. Wild-type M-CSF-dependent monocytes were treated with RANKL (50 ng/ml) for 15 minutes. JNK1 and JNK2 were sequentially immunoprecipitated, and JNK activity was assayed in both fractions. (E) Activation of JNK2 in Jnk1-/- BMMs. Wild-type and Jnk1-/- M-CSF-dependent monocytes were exposed to UV irradiation (40 J/m2). Kinase activity was measured following JNK immunoprecipitation.

View larger version (23K): [in a new window]   Fig. 3. TRAF2 upregulation by RANKL is abolished in Jnk1-/- osteoclast progenitors. Purified BMMs were isolated and treated for 3 days in the absence (-) or presence (+) of RANKL (50 ng/ml). The expression of TRAF2 (A) or TRAF6 (B) protein was analyzed by western blotting. TRAF2 expression was also analyzed 1 hour following UV irradiation of JNK1-deficient BMMs (UV +). Loading was controlled by reblotting with an anti- actin antibody. (C) The expression of TRAF2 mRNA was analyzed by northern blotting. RNA loading was controlled by probing with GAPDH.

View larger version (30K): [in a new window]   Fig. 4. c-Jun phosphorylation by osteoclastogenic signals is JNK1 dependent. (A) The phosphorylation of c-Jun in BMMs was monitored by western blotting using a specific antibody directed against the phosphorylated form of c-Jun (phospho-Ser 63), 1 hour after the addition of M-CSF (20 ng/ml), RANKL (50 ng/ml), TNF- (50ng/ml) or UV irradiation (40 J/m2). (B) The expression of c-Jun was analyzed in M-CSF-(20ng/ml) and RANKL- (50 ng/ml) stimulated BMMs isolated from wild-type (wt) or Jnk1-/- mice. (C) The activation of p38 and ERK in response to M-CSF and RANKL was analyzed by western blotting using specific anti-phosphoantibodies. (D) The activation of NF-B was analyzed by EMSA using nuclear extracts isolated from BMMs treated for 30 minutes with RANKL (+) compared with untreated cells (-).

View larger version (24K): [in a new window]   Fig. 5. Osteoclastogenesis is affected in cells from JunAA/JunAA mice and in cells lacking c-Jun but not lacking JunD. (A) Co-culture of JunAA/JunAA or wild-type hematopoietic precursors with wild-type osteoblasts. (B) Osteoclast differentiation of bone marrow precursors treated with M-CSF (20 ng/ml) and RANKL (50 ng/ml), or M-CSF (20 ng/ml) and TNF- (50 ng/ml) (C). The data represent results obtained from two experiments (B) or with pairs of littermate mice (C). (D) The function of JunD in osteoclastogenesis was addressed by direct stimulation of bone-marrow precursors isolated from wild-type, JunD+/- or JunD-/- mice by M-CSF and RANKL. (E) RANKL-mediated differentiation of double mutated cells JunD-/-; JunAA/JunAA compared with JunD+/-; JunAA/JunAA cells. (F) RANKL-mediated differentiation of BMMs lacking c-Jun (/) compared with wild-type (wt) or heterozygous controls (/wt). Osteoclast differentiation was monitored by TRAP staining from day 3 to 6 (A) or at day 4 (C,D,E,F) of culture. The deletion of c-Jun was measured by PCR analysis of DNA isolated from the cultured cells (data not shown). The stars indicate the statistical significance: (***) P<0.0001, (**) P<0.001; (ns) non-significant.

View larger version (21K): [in a new window]   Fig. 6. Increased apoptosis in RANKL-treated Jnk1-/- BMMs. (A) TUNEL staining of BMMs isolated from Jnk1-/- and wild-type mice cultured in the presence of M-CSF or M-CSF and RANKL for 2 days and restimulated for 12 hours by the indicated cytokines. (B,C) Quantification of the apoptotic index in BMMs isolated from Jnk1-/- or Jnk2-/- male (B) or Jnk1-/- female (C) mice compared with wild-type (wt) mice. (D) Quantification of the apoptotic index in BMMs isolated from JunAA/JunAA compared with JunAA/wt male mice. The stars indicate the statistical significance: (***) P<0.0001, (**) P<0.001; (ns) non-significant.