Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c-Fos in RANKL-Induced Osteoclastogenesis

Abstract

We have previously reported that transforming growth factor b (TGF-b) plays an essential role in receptor activator of nuclear factorkB ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-b, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF-b signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF-b regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos.

Acute transcriptional up-regulation specific to osteoblasts/osteoclasts in medaka fish immediately after exposure to microgravity

“…Omata et al . reported that c-fos and Smads can activate osteoclasts, in which Smads are involved in the TGF-β signaling pathway39, a pathway potentially activated by mechanical stress.…”

Smad-dependent mechanisms of inflammatory bone destruction

“…Activated p-R-Smad2/3 generates an active Smad complex by binding to Co-Smad4, which translocates into the nucleus and induces the transcription of several target genes. In this context, the cooperation of the activated Smad complex with the co-transcription factor c-Fos is necessary for nuclear translocation and subsequent DNA binding [39]. In detail, TGF-β enhances the RANKL-mediated translocation of the activated Smad complex (p-R-Smad2/3 and p-c-Fos) into the nucleus, followed by c-Fos-mediated binding of the activated complex to the Nfatc1 gene.…”

“…In detail, TGF-β enhances the RANKL-mediated translocation of the activated Smad complex (p-R-Smad2/3 and p-c-Fos) into the nucleus, followed by c-Fos-mediated binding of the activated complex to the Nfatc1 gene. This then drives the expression of NFATc1, which is an essential factor in the regulation of osteoclast differentiation [39]. Furthermore, TRAF6 binds to Smad3 via its MH2 domain, which is important for the RANKL/RANK signal transduction [20].…”

Osteoclasts—Key Players in Skeletal Health and Disease

“…It also promotes signaling downstream of RANK via interactions between TRAF6, an adaptor for RANK, and Smad3, a transcription factor downstream of TGF-β receptor (228). A more recent study by the Tanaka group using genome-wide analysis of chromatin highlighted cooperativity of Smad2/3 with c-Fos in the promotion of NFATc1 expression (229). MiTF, another transcription factor important for osteoclastogenesis, is also enhanced by TGF-β treatment (230) likely due to activation of p38 (231), providing further mechanisms for promotion of differentiation.…”