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Cited by 501

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Nuclear Factor of Activated T-cells (NFAT) Rescues Osteoclastogenesis in Precursors Lacking c-Fos

Matsuo, Koichi, Galson, Deborah L., Zhao, Chen, Peng, Lan, Laplace, Catherine, Wang, Kent Z.Q., Bachler, Marcus A., Amano, Hitoshi, Aburatani, Hiroyuki, Ishikawa, Hiromichi, Wagner, Erwin F.·2004·Journal of Biological Chemistry
DOI 10.1074/jbc.m313973200
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CC-BY · From the PDF
FIG. 2 c-Fos binds to the Nfatc1 promoter. A , schematic presentation of putative Fos ( open ovals ) and NFAT ( closed rectangles ) binding sites in the mouse Nfatc1 promoter (27, 28). The numbering is relative to the proximal transcriptional start site directed by promoter P1. Distal ( dis. ) and proximal ( prox. ) blocks of homology are shown in thick lines . Arrows , PCR primers. B , chromatin immunoprecipitation assay. Non-adherent Fos -/ -splenocytes and wild-type bone marrow cells were treated with or without RANKL for 20 h before cross-linking. Precipitation was performed with IgG or anti-c-Fos antibody ( 𝛂 Fos ), and the amounts of precipitated DNA relative to total input DNA were quantified by a real-time PCR for Nfatc1 ( top ) and Acp5 ( bottom ; see Fig. 3 A for PCR primers).
FIG. 2 c-Fos binds to the Nfatc1 promoter. A , schematic presentation of putative Fos ( open ovals ) and NFAT ( closed rectangles ) binding sites in the mouse Nfatc1 promoter (27, 28). The numbering is relative to the proximal transcriptional start site directed by promoter P1. Distal ( dis. ) and proximal ( prox. ) blocks of homology are shown in thick lines . Arrows , PCR primers. B , chromatin immunoprecipitation assay. Non-adherent Fos -/ -splenocytes and wild-type bone marrow cells were treated with or without RANKL for 20 h before cross-linking. Precipitation was performed with IgG or anti-c-Fos antibody ( 𝛂 Fos ), and the amounts of precipitated DNA relative to total input DNA were quantified by a real-time PCR for Nfatc1 ( top ) and Acp5 ( bottom ; see Fig. 3 A for PCR primers).CC-BY · From the PDF
FIG. 3 Identification of NFAT/AP-1 site in the Acp5 promoter. A , conserved Acp5 promoter sequences in mouse ( M ), human ( H ), and pig ( P ). Asterisks indicate nucleotides identical to the 15-bp mouse IL-2 distal NFAT/AP-1 site. Arrows indicate PCR primers for the chromatin immunoprecipitation assay in Fig. 2 B . B , EMSA using Acp5 -120 ( 5 ), IL-2 distal NFAT/AP-1 site ( I ) and AP-1 consensus ( A ) or mutant ( m ) sites for probes and competitors ( Comp. ). Nuclear extracts containing NFAT binding activity were prepared from osteoclastogenic co-culture. C , transient transfection assay in RAW264.7 cells using wild-type ( WT ) and mutant ( MUT ) mouse Acp5 promoter-luciferase constructs. The activator plasmid expressing 𝚫 NFAT ( 𝚫 ) was co-transfected. RLU , relative light units (normalized to co-transfected Renilla luciferase activity and relative to the unstimulated wild-type promoter). D , the Acp5 promoter-luciferase construct was activated by c-Fos expression vector ( Fos ) and suppressed by Fos small interfering RNA vectors ( RNAi1 , RNAi2 ) in transient co-transfection in RAW264.7 cells.
FIG. 3 Identification of NFAT/AP-1 site in the Acp5 promoter. A , conserved Acp5 promoter sequences in mouse ( M ), human ( H ), and pig ( P ). Asterisks indicate nucleotides identical to the 15-bp mouse IL-2 distal NFAT/AP-1 site. Arrows indicate PCR primers for the chromatin immunoprecipitation assay in Fig. 2 B . B , EMSA using Acp5 -120 ( 5 ), IL-2 distal NFAT/AP-1 site ( I ) and AP-1 consensus ( A ) or mutant ( m ) sites for probes and competitors ( Comp. ). Nuclear extracts containing NFAT binding activity were prepared from osteoclastogenic co-culture. C , transient transfection assay in RAW264.7 cells using wild-type ( WT ) and mutant ( MUT ) mouse Acp5 promoter-luciferase constructs. The activator plasmid expressing 𝚫 NFAT ( 𝚫 ) was co-transfected. RLU , relative light units (normalized to co-transfected Renilla luciferase activity and relative to the unstimulated wild-type promoter). D , the Acp5 promoter-luciferase construct was activated by c-Fos expression vector ( Fos ) and suppressed by Fos small interfering RNA vectors ( RNAi1 , RNAi2 ) in transient co-transfection in RAW264.7 cells.CC-BY · From the PDF
FIG. 5 Rescue of osteoclast formation with 𝚫 NFAT. A , TRAP staining of osteoclastogenic co-cultures derived from wild-type ( + / + ) splenocytes and GFP and 𝚫 NFAT virus-infected Fos -/ -splenocytes ( -/ -). Below , GFP fluorescence and TRAP stain are shown in higher magnification. The bars represent 100 𝛍 m. B , bone resorption assay. Infected cells were co-cultured with ST2-T cells on bovine bone slices for 10 days, and pit formation was examined in a double-blind study with backscattered electron imaging. The bars represent 0.75 mm. Arrows indicate bone resorption pits. C , rescue activity of 𝚫 NFAT virus relative to that of c-Fos virus. st , osteoclastogenic co-cultures using ST2-T cells. M + R , osteoblast-free cultures using soluble M-CSF and RANKL. Fos -/ -splenocytes were infected with GFP, c-Fos, or 𝚫 NFAT viruses. Calcr transcripts were quantitated by real-time PCR; TRAP-positive multinucleated cells ( MNC ) were counted, and the resorbed area (%) was measured on bone surfaces.
FIG. 5 Rescue of osteoclast formation with 𝚫 NFAT. A , TRAP staining of osteoclastogenic co-cultures derived from wild-type ( + / + ) splenocytes and GFP and 𝚫 NFAT virus-infected Fos -/ -splenocytes ( -/ -). Below , GFP fluorescence and TRAP stain are shown in higher magnification. The bars represent 100 𝛍 m. B , bone resorption assay. Infected cells were co-cultured with ST2-T cells on bovine bone slices for 10 days, and pit formation was examined in a double-blind study with backscattered electron imaging. The bars represent 0.75 mm. Arrows indicate bone resorption pits. C , rescue activity of 𝚫 NFAT virus relative to that of c-Fos virus. st , osteoclastogenic co-cultures using ST2-T cells. M + R , osteoblast-free cultures using soluble M-CSF and RANKL. Fos -/ -splenocytes were infected with GFP, c-Fos, or 𝚫 NFAT viruses. Calcr transcripts were quantitated by real-time PCR; TRAP-positive multinucleated cells ( MNC ) were counted, and the resorbed area (%) was measured on bone surfaces.CC-BY · From the PDF
FIG. 6 Rescue of osteoclast formation with 𝚫 NFAT in the absence of RANKL. A , transient transfection of 𝚫 NFAT-expression vector induces endogenous Acp5 and Calcr expression in RAW264.7 cells untreated or treated with 30 ng/ml RANKL as indicated and harvested at the days depicted. Osteoclasts generated in co-culture served as positive control ( lane 8 ) and no RNA as negative control ( lane 1 ). Transcripts were analyzed by reverse transcriptase-PCR with primers for Acp5 , Calcr (all isoforms), and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ). The reverse transcripts-PCR products for Calcr were also detected by hybridization with an internal probe ( hyb ). B , rescue of TRAP-positive Fos -/ -cells by 𝚫 NFAT requires RANKL. M-CSF-dependent macrophages derived from wild-type and Fos -/ -splenocytes ( Sp + / + and Sp -/ -) and wild-type bone marrow cells ( Bm + / + ) were infected with the GFP 𝚫 NFAT virus and then were cultured for 3 more days in the presence or absence of RANKL. The bottom row shows expression of GFP 𝚫 NFAT fusion protein.
FIG. 6 Rescue of osteoclast formation with 𝚫 NFAT in the absence of RANKL. A , transient transfection of 𝚫 NFAT-expression vector induces endogenous Acp5 and Calcr expression in RAW264.7 cells untreated or treated with 30 ng/ml RANKL as indicated and harvested at the days depicted. Osteoclasts generated in co-culture served as positive control ( lane 8 ) and no RNA as negative control ( lane 1 ). Transcripts were analyzed by reverse transcriptase-PCR with primers for Acp5 , Calcr (all isoforms), and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ). The reverse transcripts-PCR products for Calcr were also detected by hybridization with an internal probe ( hyb ). B , rescue of TRAP-positive Fos -/ -cells by 𝚫 NFAT requires RANKL. M-CSF-dependent macrophages derived from wild-type and Fos -/ -splenocytes ( Sp + / + and Sp -/ -) and wild-type bone marrow cells ( Bm + / + ) were infected with the GFP 𝚫 NFAT virus and then were cultured for 3 more days in the presence or absence of RANKL. The bottom row shows expression of GFP 𝚫 NFAT fusion protein.CC-BY · From the PDF

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Metadata

Authors
Matsuo, K., Galson, D. L., Zhao, C., Peng, L., Laplace, C., Wang, K. Z. Q., Bachler, M. A., Amano, H., Aburatani, H., Ishikawa, H., & Wagner, E. F.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2004-06
Volume
279
Issue
25
Pages
26475-26480
ISSN
0021-9258
DOI
10.1074/jbc.m313973200
Cited by
501
References
5
Language
en
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