From the Department of Pathology, University of Alabama, Birmingham, Alabama 35294.
From the Department of Pathology, University of Alabama, Birmingham, Alabama 35294
J Biol Chem. 2018 Jan 26;293(4):1480-1492. doi: 10.1074/jbc.M116.736009. Epub 2017 Nov 9.
Binding of receptor activator of NF-κB ligand (RANKL) to its receptor RANK on osteoclast (OC) precursors up-regulates c-Fos and CCAAT/enhancer-binding protein-α (C/EBPα), two critical OC transcription factors. However, the effects of c-Fos and C/EBPα on osteoclastogenesis have not been compared. Herein, we demonstrate that overexpression of c-Fos or C/EBPα in OC precursors up-regulates OC genes and initiates osteoclastogenesis independently of RANKL. However, although C/EBPα up-regulated c-Fos, c-Fos failed to up-regulate C/EBPα in OC precursors. Consistently, C/EBPα overexpression more strongly promoted OC differentiation than did c-Fos overexpression. RANK has a cytoplasmic IVVY (IVVY) motif that is essential for osteoclastogenesis, and we found that mutation of the IVVY motif blocked OC differentiation by partly inhibiting expression of C/EBPα but not expression of c-Fos. We therefore hypothesized that C/EBPα overexpression might rescue osteoclastogenesis in cells expressing the mutated IVVY motif. However, overexpression of C/EBPα or c-Fos failed to stimulate osteoclastogenesis in the mutant cells. Notably, the IVVY motif mutation abrogated OC gene expression compared with a vector control, suggesting that the IVVY motif might counteract OC inhibitors during osteoclastogenesis. Consistently, the IVVY motif mutant triggered up-regulation of recombinant recognition sequence-binding protein at the Jκ site (RBP-J) protein, a potent OC inhibitor. Mechanistically, C/EBPα or c-Fos overexpression in the mutant cells failed to control the up-regulated RBP-J expression, leading to suppression of OC genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or c-Fos overexpression with C/EBPα exhibiting a stronger osteoclastogenic effect. Collectively, our findings indicate that C/EBPα is a stronger inducer of OC differentiation than c-Fos, partly via C/EBPα regulation by the RANK IVVY motif.
受体激活核因子-κB 配体（RANKL）与破骨细胞（OC）前体上的受体 RANK 的结合上调了两个关键的 OC 转录因子 c-Fos 和 CCAAT/增强子结合蛋白-α（C/EBPα）。然而，c-Fos 和 C/EBPα 对破骨细胞生成的影响尚未进行比较。在此，我们证明 OC 前体中 c-Fos 或 C/EBPα 的过表达可上调 OC 基因并独立于 RANKL 启动破骨细胞生成。然而，尽管 C/EBPα 上调了 c-Fos，但 c-Fos 未能在上调 OC 前体中的 C/EBPα。一致地，C/EBPα 的过表达比 c-Fos 的过表达更强烈地促进 OC 分化。RANK 具有细胞质 IVVY（IVVY）基序，该基序对破骨细胞生成至关重要，我们发现该 IVVY 基序的突变通过部分抑制 C/EBPα的表达而不是 c-Fos 的表达来阻断 OC 分化。因此，我们假设 C/EBPα 的过表达可能挽救表达突变的 IVVY 基序的细胞中的破骨细胞生成。然而，C/EBPα 或 c-Fos 的过表达未能刺激突变细胞中的破骨细胞生成。值得注意的是，与载体对照相比，IVVY 基序突变消除了 OC 基因的表达，表明在破骨细胞生成过程中，IVVY 基序可能与 OC 抑制剂拮抗。一致地，IVVY 基序突变体触发了 Jκ 位点（RBP-J）重组识别序列结合蛋白的上调，这是一种有效的 OC 抑制剂。从机制上讲，在突变细胞中过表达 C/EBPα 或 c-Fos 未能控制上调的 RBP-J 表达，导致 OC 基因的抑制。相应地，在突变细胞中沉默 RBP-J 可挽救 C/EBPα 或 c-Fos 过表达的破骨细胞生成，其中 C/EBPα 表现出更强的破骨细胞生成作用。总之，我们的研究结果表明，C/EBPα 是比 c-Fos 更强的 OC 分化诱导剂，部分通过 RANK IVVY 基序对 C/EBPα 的调节。
