QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online July 23, 2008
doi: 10.1242/10.1242/jcs.028217

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chang, E.-J. Articles by Kim, H.-H. Search for Related Content PubMed PubMed Citation Articles by Chang, E.-J. Articles by Kim, H.-H. Social Bookmarking                         What's this?

The JNK-dependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation

¹ Department of Cell and Developmental Biology, BK21 Program, and DRI, Seoul National University, Seoul 110-749, Korea
² Seoul National University Biomedical Informatics, Seoul National University College of Medicine, Seoul 110-799, Korea

View larger version (35K): [in this window] [in a new window]   Fig. 1. Reversion of committed pOCs by blocking JNK activity. (A) Experimental scheme of osteoclastogenesis from mouse BMMs. Non-adherent bone marrow cells (BMCs) were cultured with M-CSF (30 ng/ml) for 3 days to generate BMMs. BMMs were cultured with M-CSF (30 ng/ml) plus RANKL (100 ng/ml) for 2 days to generate pOCs. (A-C) pOCs were incubated for 48 hours with the NF-B inhibitor SN50 (SN, 20 µM), JNK inhibitor SP600125 (SP, 10 µM), MEK inhibitor PD98059 (PD, 10 µM), p38 inhibitor SB203580 (SB, 10 µM) or the vehicle (Vh, 0.1% DMSO) in the presence of M-CSF (30 ng/ml) and the indicated combination of RANKL (R, 100 ng/ml), TNF (T, 20 ng/ml) and IL1 (I, 10 ng/ml). Cells were stained and the number of TRAP+ MNCs was counted. (A,D-F) pOCs were incubated for 24 hours with the inhibitor as above in the presence of M-CSF and RANKL (+MR). Cells were either stained for TRAP (D,E) or incubated with CCK-8 (F) to assess the percentage of TRAP+ cells and the viability of cells, respectively. (A,G) pOCs were treated for 24 hours with SP600125 at the indicated concentration (G) together with M-CSF and RANKL. (A,H) pOCs were incubated for 24 hours with or without SP600125 (10 µM) in the presence of M-CSF and RANKL, and mRNA levels of TRAP, calcitonin receptor (CTR) and MMP9 were analyzed by real-time PCR. (C,E-H) Error bars represent s.d. from triplicate samples. Similar results were obtained in two other experiments. txt, treatment.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Stage-dependent induction of phenotypic reversion by JNK inhibition. (A) BMMs were cultured with M-CSF (30 ng/ml) plus RANKL (100 ng/ml). After 2 or 3 days, SP600125 (+SP) or vehicle (–) was added and cells were further incubated for 24 hours before TRAP staining. pOCs were also stained for comparison. (B) RAW264.7 cells were cultured with RANKL (100 ng/ml). Cells were treated with SP600215 at the indicated day or vehicle at day 2 for 24 hours and were then stained for TRAP. (C) RAW264.7 cells cultured with RANKL (100 ng/ml) for 2 (left) or 3 (right) days were transfected with wild type (WT) or dominant negative (DN) JNK. Cells were further incubated with RANKL for 24 hours and the percentage of TRAP+ cells was determined. Data are the mean ± s.d. and are representative of three independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Expression changes of macrophage surface markers by JNK-inhibitor treatment. BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 2 days to generate pOCs. pOCs were further incubated with M-CSF and RANKL for 24 hours in the absence (–SP) or presence (+SP) of SP600125. (A-D) Cells were stained with fluorescein-conjugated anti-CD11b (A), -CD68 (B), -F4/80 (C) or CD14 (D) as surface markers of macrophages and/or monocytes, and were then subjected to flow-cytometry analyses. The x-axes represent fluorescence intensity for FITC-zymosan-positive cells. Bar graphs show the percentage of cells positive for each surface marker. (E) Histograms represent the MFI of the stained cells. Similar results were observed in another experiment.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Determination of phagocytic activity and dendritic differentiation potential. (A-C) BMMs, pOCs and pOCs treated with (+SP) or without (–SP) SP for 24 hours were incubated with FITC-labeled zymosan for 45 minutes as described in the Materials and Methods. After washing, cells were permeabilized and incubated with propidium iodide. (A) Cells that have taken up zymosan appear yellow because of overlap of green (FITC-zymosan) and red (propidium iodide) fluorescences in confocal microscopy when images were merged. (B) The cells were also analyzed for FITC fluorescence by flow cytometry. n.c., negative control cells incubated without FITC-zymosan and stained with FITC-Ig. The x-axes represent fluorescence intensity for FITC-zymosan-positive cells. (C) Histograms show the percentage of phagocytic cells determined by flow cytometry. Error bars represent s.d. from triplicate samples. Results are representative of three independent experiments. (D,E) BMMs, and –SP and +SP cells were stained for CD11c and F4/80 before and after culture in dendritic-differentiation medium as described in the Materials and Methods. The percentage of dendritic cells (DCs) (F4/80–CD11c+) and macrophage (M) population (F4/80+CD11c–) was determined by flow cytometry.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Reversion of human PBMC-derived pOCs by JNK blockade. (A,B) PBMCs were cultured with M-CSF (100 ng/ml) and RANKL (100 ng/ml) for 3 days to the pOC stage. pOCs were treated with (+SP) or without (–SP) SP600125 (10 µM) for 24 hours and TRAP+ cells were counted. (C,D) PBMCs, PBMC-derived pOCs and pOCs treated with (+SP) or without (–SP) SP for 24 hours were incubated with FITC-zymosan and subjected to flow cytometry as described in the Materials and Methods. (C) n.c., negative control cells incubated without FITC-zymosan and stained with FITC-Ig. The x-axes represent fluorescence intensity for FITC-zymosan-positive cells. (B,D) Error bars represent s.d. from triplicate samples. Similar results were obtained in three independent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Involvement of NFATc1 in the reversion of differentiation by JNK inhibition. (A) RAW264.7 cells were transfected with NFAT-reporter luciferase plasmid and stimulated with RANKL for 24 hours in the presence or absence of SP600125 (SP, 10 µM). Luciferase activity was assessed with cell lysates. (B,C) BMM-derived pOCs were treated with or without SP600125 (10 µM) for 24 hours. The mRNA (B) and protein (C) levels of NFATc1 were determined by reverse transcriptase (RT)-PCR and western blotting analyses, respectively. (D-F) pOCs were infected with NFATc1 siRNA or control siRNA (Luc) retroviruses. (D) The reduced level of NFATc1 was determined by western blotting. (E) Infected cells were cultured with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for 24 hours before TRAP-staining. (F) The percentage of TRAP+ cells was assessed. (G,H) pOCs were infected with active NFATc1 or control retroviruses and incubated with or without SP600125 (10 µM) in the presence of RANKL and M-CSF for 24 hours. The percentage of TRAP+ cells was determined. (A,F,H) Data are mean ± s.d. of triplicate samples. All results are representative of three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Role of CaMK in the JNK-dependent maintenance of the committed state. (A) Relative signal intensity of CaMKIV from the DNA microarray analysis of three different human samples (1-3) described in the Materials and Methods is presented. (B) Mouse BMM-derived pOCs were treated with (+SP) or without (–SP) SP600125 (10 µM) for 24 hours in the presence of RANKL (100 ng/ml) and M-CSF (30 ng/ml). Expression of CaMKII, CaMKII and CaMKIV was examined by real-time PCR. (C-E) pOCs were transfected with oligonucleotides for CamKII and/or CamKIV siRNA. (C) Knockdown of CaMK expression was confirmed by reverse transcriptase (RT)-PCR. (D) Cells were TRAP-stained and the percentage of TRAP+ cells was determined. (E) Cells were also evaluated for phagocytic activity with FITC-labeled zymosan. (B,D,E) Error bars represent s.d. from triplicate samples. Results are representative of three independent experiments.

View larger version (31K): [in this window] [in a new window]   Fig. 8. Modulation of NFATc1 level by CaMK in pOCs. (A) Mouse BMM-derived pOCs were transfected with oligonucleotides for CamKII and/or CamKIV siRNA, and the protein level of NFATc1 was analyzed by western blotting. (B-D) pOCs were treated for 24 hours with or without KN93 at the indicated concentrations in the presence of RANKL and M-CSF. NFATc1 protein level (B), the percentage of TRAP+ cells (C) and phagocytic activity (D) were determined. (C,D) Error bars represent s.d. from triplicate samples. Results are representative of three independent experiments. txt, treatment.

View larger version (62K): [in this window] [in a new window]   Fig. 9. A diagram illustrating the role of JNK, CaMK and NFATc1 in maintaining the committed state during osteoclastogenesis by RANKL. JNK stimulates AP-1 transcription-factor activity and upregulates CaMKII and CaMKIV in committed pOCs. The elevated CaMK level in pOCs leads, through a mechanism yet to be elucidated, to a sustained increase in NFATc1 level, which is required to keep TRAP gene expression `on' in committed pOCs.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter