Epigenetic regulation of Nedd9 gene during osteoclastogenesis.

<p>(A) Smad2/3 binding and histone modification changes of <i>Nedd9</i> gene during osteoclastogenesis. Histone modification patterns in BMMs changed from K4(+)K27(+) to K4(+)K27(-) patterns by TGF-ß and returned to K4(+)K27(+) patterns after RANKL treatment. (B) <i>Nedd9</i> mRNA expression after TGF-ß or RANKL stimulation. The expression increased by TGF-ß stimulation and was reduced after RANKL treatment. The expression remained at low levels in the presence of SB431542.</p

Identification of Smad2/3 binding sites.

<p>(A) BMMs were treated with 2 ng/ml TGF-ß for 1.5 h and cells were subjected to ChIP-seq analysis using anti-Smad2/3 antibody. Three known TGF-ß target genes (<i>Cdkn1a</i>, <i>Serpine1</i>, <i>and Smad7</i>) and a negative control gene (<i>Hprt1</i>) were analyzed as representative examples. Smad2/3-binding regions (SBRs; peak signal ratio ≥8) and the peak position of each SBR are shown by black bars. (B) Eight positive regions and two negative regions for Smad2/3 binding were selected from ChIP-seq data and validated by realtime PCR. ChIP using mouse IgG was used as control. Values are presented as n-fold enrichment over Hprt1. (C) Average Smad2/3 signal profile around transcriptional start site (TSS) in ChIP-seq analysis. Smad2/3 binding was enriched around TSS.</p

Impaired osteoclastogenesis in Nedd9-/- BMMs.

<p>(A) BMMs were isolated from <i>Nedd9</i> knockout (KO) and wild-type (WT) mice and cultured in the presence of RANKL with or without TGF-ß. Osteoclastogenesis was evaluated by TRAP staining (A), the number of multi-nuclear osteoclasts (B) and the expression of Cathepsin K protein (C).</p

Nedd9 is critical for osteoclast differentiation.

<p>(A) Realtime PCR analysis of effects of retroviral overexpression of <i>Nedd9</i> gene on RANKL-induced osteoclastogenesis, as indicated by expression of <i>Nedd9</i> and <i>Cathepsin K</i> mRNAs. (B) Effects of SB431542 (SB) treatment on osteoclastogenesis, as evaluated by TRAP staining, in <i>Nedd9</i>-overexpressing cells treated with RANKL. Overexpression of <i>Nedd9</i> increased RANKL-induced osteoclastogenesis. SB431542 suppressed osteoclastogenesis, which was partly recovered by <i>Nedd9</i> overexpression. Cultures were stained by TRAP. Bars = 100 μm. (C) The expression of <i>Nedd9</i> gene as determined by realtime PCR. (D) The number of TRAP positive osteoclasts was significantly increased by <i>Nedd9</i> overexpression, and the suppression of osteoclastogenesis by SB431542 was partly suppressed by <i>Nedd9</i> overexpression. *<i>P</i> < 0.05.</p