Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB

Abstract

Disequilibrium between bone-forming osteoblasts and bone-resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancer-binding protein beta (C/EBPbeta) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP(*), LAP) and a short repressive (LIP) isoforms from a single C/EBPbeta transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPbeta null (c/ebpbeta(-/-)) or in LIP knock-in (L/L) osteoclast precursors. C/EBPbeta mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPbeta isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.

Figure 1

Expression of C/EBPβ in bone cells. (A) Immunohistochemistry of the proximal tibia of 4-week-old mice showing expression of C/EBPβ protein (brown precipitate) in the different bone cell types. Lightgreen was used as counterstain. Scale bar, 200 μm. C/EBPβ protein expression in osteoblasts (arrowheads) (B, C), whereas no C/EBPβ protein was detected in differentiated osteoclasts (OC) (arrow), which were identified as large multinucleated cells attached to the bone surface (scale bar, 50 μm) or (C) in osteocytes (arrow, scale bar, 50 μm). Tb, trabeculae; GP, growth plate; BM, bone marrow; Cort, cortical bone (D) Western blot analysis of C/EBPβ isoform expression (LAP^*, LAP and LIP) during osteoblast differentiation (pOB, preosteoblasts; OB, osteoblast; mOB, mature osteoblast) and (E) during osteoclast differentiation of bone marrow derived monocytic precursors on the indicated days (d0, d3, d6) after M-CSF and RANK-L addition. Loading was controlled by analysis of α-tubulin and α-actin expression, respectively. UD, undefined bands, which cross-react with the C/EBPβ antibody.

Figure 2

Generation and characterization of LIP knock-in mice. (A) Schematic representation of the targeting strategy used to generate a knock-in (k.i.) of the LIP isoform of C/EBPβ in the endogenous c/ebpβ locus by homologous recombination in embryonic stem (ES) cells. The structure of the genomic c/ebpβ locus, targeting vector and mutated allele are shown. The c/ebpβ gene is intronless and depicted as filled red box and LIP as dashed red box. The arrow indicates the direction of gene transcription. The DNA fragments and their sizes revealed by Southern blot analysis are indicated by thick coloured lines. B, BamHI; N, NotI; R, R^*, EcoRI; S, SalI; X, XhoI; Xa, XbaI. Neo, neomycin resistance gene; LoxP sites (black triangles); P5′: 5′ probe; P3′: 3′ probe. (B) Southern blot analysis of ES cell DNA. Genomic DNA of targeted ES cells was isolated from two clones and digested with EcoRI and then hybridized with the 5′ probe (in purple). The WT and mutant allele are detected as 4.5 and 3.0 kb fragments, respectively. BamHI digested DNA was hybridized with the 3′ probe (in blue) that detected a 7.0-kb fragment in the WT and a 7.8-kb fragment in the mutant allele. (C) C/EBPβ protein expression in livers isolated from 8-week-old mice. Loading was controlled by analysis of α-tubulin expression. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice; +/L, heterozygous k.i. mice.

Figure 3

Affected bone mass in c/ebpβ mutant mice. (A) Histological analyses (haematoxylin-eosin staining) of tibiae of 8-week-old mice, showing an osteopenic phenotype in c/ebpβ-deficient mice (−/−) and an osteosclerotic phenotype in LIP k.i. mice (L/L), compared with WT (+/+) mice. Scale bar, 100 μm. Bar graph displays the histomorphometric quantification of the bone volume (BV/TV, bone volume/total volume). GP, growth plate; Tb, trabeculae; BM, bone marrow. (B) Images of double calcein labelled bones from WT and c/ebpβ mutant mice showing the mineral apposition rate (MAR) and the bone formation rate/bone surface (BFR/BS). (C) Enhanced osteoclastogenesis in c/ebpβ mutant mice. TRACP staining of osteoclasts (red staining) in tibiae of 8-week-old WT and c/ebpβ mutant mice. Lightgreen was used as counterstain. Scale bar, 20 μm. Bar graph shows the urinary excretion of deoxypyridinoline (DPD) cross-links, reflecting osteoclast activity in vivo. For bone histomorphometric measurements, n=8 per group. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice. Data are presented as mean±s.e.m. ^*P<0.05, ^**P<0.01 versus WT.

Figure 4

Osteoblast differentiation in c/ebpβ mutants. (A) Primary calvarial osteoblast precursor cells were differentiated and stained for alkaline phosphatase (ALP) activity. The bar graph displays the quantification of the percentage ALP positive area per well area. (B) Bone nodule mineralization of primary calvarial osteoblasts determined by alizarin red staining. The bar graph displays the quantification of alizarin red positive mineralized nodules. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice. Data are presented as mean±s.e.m. ^*P<0.05, versus WT.

Figure 5

Osteoblast–osteoclast cross-talk in c/ebpβ mutants. (A) Expression of the osteoclastic regulators RANK-L, OPG and TNFα in primary calvarial osteoblast cultures as determined by real-time RT–PCR. Values represent relative expression levels compared with WT on day 14 (set as 1). Data are presented as mean±s.e.m. of three independent experiments. (B) Number of multinucleated osteoclasts formed from WT bone marrow cells (BM) co-cultured with osteoblasts (OB) from the different genotypes, as indicated. (C) Number of multinucleated osteoclasts formed from bone marrow cells of the C/EBPβ mutant mice (as indicated), co-cultured with WT osteoblasts. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice. Data are presented as mean±s.e.m. ^*P<0.05 versus WT.

Figure 6

c/ebpβ mutations promote osteoclast differentiation. (A) Osteoclast differentiation of bone marrow derived monocytic precursors from WT, c/ebpβ knock-out and LIP k.i. mice and treated with M-CSF and RANK-L. Osteoclasts were stained by TRACP activity (red staining). The bar graph displays the differential quantification of the osteoclasts by number of nuclei per cell (WT cultures set at 100%). (B) Bone resorptive activity of osteoclasts determined by culturing osteoclasts on bovine bone slices and staining resorption pits (arrowheads) with coomassie brilliant blue. The bar graph displays the quantification of the resorption areas expressed as fold of resorbed area in the WT (set at 1). (C) Real-time RT–PCR analysis of expression of the osteoclast markers TRACP, Cathepsin K (CathK), calcitonin receptor (CTR), OSCAR and DC-STAMP in osteoclasts cultured for 6 days in the presence of M-CSF and RANK-L. Values represent relative expression levels compared with WT (set as 1). Data are presented as mean±s.e.m.; n=6 per group. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus WT. (D) Western blot analysis of cleaved-caspase 3 (cl. caspase 3) expression to determine apoptosis in primary osteoclasts cultured for 6 days in the presence of M-CSF and RANK-L. The samples were run on the same gel, but were noncontiguous, as indicated with the black lines. Loading was controlled by α-tubulin expression. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice.

Figure 7

The mammalian target of rapamycin (mTOR) regulates osteoclastogenesis by switching C/EBPβ isoforms. (A) Representative pictures of primary bone marrow derived monocytic precursors from indicated genotypes differentiated into osteoclasts in the absence (solvent) or presence of rapamycin. Osteoclasts were stained for TRACP after 6 days (red staining). Bar graphs show quantification of differentiated osteoclasts (by number of nuclei per cell). The values from WT cultures was set at 100% (indicated as dashed line). Note the difference in scale between the results from WT and mutant cultures. A representative experiment is shown. (B) Western blot analysis of C/EBPβ isoform expression (LAP^*, LAP and LIP) in primary osteoclasts treated with rapamycin (Rap), as indicated. Positive control consisting of mature osteoblasts (mOB) is shown to indicate the different C/EBPβ isoforms. The positive control was run on the same gel, but was noncontiguous, as indicated with the black line. Loading was controlled by α-tubulin expression. (C) Representative pictures of RANK-L-induced osteoclast differentiation in RAW264.7 cells stably expressing the indicated C/EBPβ isoforms or EGFP control. Osteoclasts were stained for TRACP (red staining). Arrowheads indicate small osteoclasts present in the LAP cultures. (D) Western blot analysis of C/EBPβ isoform expression (LAP^*, LAP and LIP) in RAW264.7 cells stably expressing the C/EBPβ isoforms LAP or LIP, or EGFP (as control) and differentiated into osteoclasts. The lanes were run on the same gel, but were noncontiguous as indicated with the black lines. +/+, WT mice; −/−, c/ebpβ^−/− mice; L/L, LIP k.i. mice.

Figure 8

C/EBPβ isoforms regulate osteoclastogenesis through MafB expression (A) Venn diagram and number of differentially regulated genes in RAW264.7 cells induced to differentiate into osteoclasts for 2 days as identified by gene array analysis. Control cells (empty vector) and cells stably expressing LAP were compared with cells treated with rapamycin. (B) Real-time RT–PCR analysis of MafB expression in c/ebpβ^−/− and L/L osteoclasts cultured for 2 days with M-CSF and RANK-L as well as in the absence (grey bars) or presence of rapamycin (green bars), as indicated. Values represent relative expression levels compared with WT (set as 1). (C) Real-time RT–PCR analysis of MafB expression in osteoclasts of WT, c/ebpβ^−/− and L/L mice cultured for 6 days under osteoclastogenic conditions. Values represent relative expression levels compared with WT (set as 1). Data are presented as mean±s.e.m.; n=6 per group. ^*P<0.05, ^**P<0.01 versus WT. (D) Luciferase reporter assay using a mouse MafB promoter reporter. RAW264.7 cells were transfected with empty control (EGFP) or with C/EBPβ isoforms. Values were normalized to CMV-promoter driven Renilla luciferase activity. (E) Luciferase reporter assay using a mouse MafB promoter reporter. RAW264.7 cells were transfected with empty control (EGFP) or LAP expression vector, treated with RANK-L and with rapamycin (green bars), as indicated. (F) C/EBPβ-deficient MEFs were transfected with the mouse MafB promoter reporter and control (empty vector, EGFP) or C/EBPβ (WT) and treated with rapamycin (green bars), as indicated. Data are presented as mean±s.e.m, ^*P<0.05 versus control. (G) Representative pictures of RANK-L-induced osteoclast differentiation of RAW264.7 cells with stable MafB short hairpin interfering RNA (shMafB) or control, in the absence (solvent) or presence of rapamycin, as indicated. Osteoclasts were stained for TRACP (red staining). Bar graphs show quantification of differentiated osteoclasts (number of nuclei per cell), in the absence (solvent) or presence of rapamycin. The values from control cultures in the presence of solvent are set at 100%. A representative experiment is shown. (H) Knock-down of MafB using a shRNA in RAW264.7 cells as determined by western blot analysis. The lanes were run on the same gel, but were noncontiguous. Loading was controlled by analysis of α-tubulin expression.

Figure 9

C/EBPβ as a switch in osteoclastogenesis. Schematic representation of how differences in mTOR activity alters the C/EBPβ isoform ratio to regulate osteoclastogenesis. Rapamycin inhibits mTOR, which causes enhanced expression of LAP. LAP induces expression of MafB. MafB inhibits NFATc1 and other osteoclastic transcriptional regulators (c-Fos and Mitf), which results in the down-regulation of osteoclastic genes including TNFα and the cell fusion genes ATP6v0d2 and DC-STAMP (partially derived from Kim et al, 2007, 2008). Enhanced expression of LAP therefore inhibits osteoclast differentiation, whereas LIP (produced at high mTOR activity) forces osteoclast differentiation.