Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105, USA.
J Biol Chem. 2013 Jan 4;288(1):624-32. doi: 10.1074/jbc.M112.340455. Epub 2012 Nov 19.
Notch receptors regulate osteoblastogenesis, and Notch activation induces cleavage and nuclear translocation of the Notch intracellular domain (NICD), which associates with Epstein-Barr virus latency C-promoter binding factor-1/suppressor of hairless/lag-1 (CSL) and induces transcription of Notch target genes, such as hairy enhancer of split-related with YRPW motif (Hey)1 and Hey2. Nuclear factors of activated T-cells (NFAT) are transcription factors that regulate osteoclastogenesis, but their function in osteoblasts is not clear. Notch inhibits NFATc1 transcription, but interactions between Notch and NFAT are understood poorly. To determine the regulation of NFAT expression by Notch, osteoblasts from Rosa(Notch) mice, where NICD is transcribed following excision of a loxP flanked STOP cassette, were used. Alternatively, wild-type C57BL/6 osteoblasts were exposed to the Notch ligand Delta-like (Dll)1 to induce Notch signaling or to bovine serum albumin as control. In Rosa(Notch) osteoblasts, Notch suppressed NFATc1 expression, increased Nfatc2 mRNA by post-transcriptional mechanisms, and had no effect on NFATc3 and NFATc4 transcripts. Induction of Nfatc2 transcripts by Notch was confirmed in C57BL/6 osteoblasts exposed to Dll1. To investigate NFATc2 function in osteoblasts, constitutively active NFATc2 was overexpressed in Rosa(Notch) osteoblasts. NFATc2 suppressed Notch transactivation and expression of Hey genes. Electrophoretic mobility shift assays revealed that NFATc2 and CSL bind to similar DNA sequences, and chromatin immunoprecipitation indicated that NFATc2 displaced CSL from the Hey2 promoter. The effects of NICD and NFATc2 in Rosa(Notch) osteoblasts were assessed, and both proteins inhibited osteoblast function. In conclusion, Notch stabilizes Nfatc2 transcripts, NFATc2 suppresses Notch signaling, and both proteins inhibit osteoblast function.
Notch 受体调节成骨细胞分化，Notch 的激活诱导 Notch 细胞内结构域（NICD）的切割和核易位，后者与 Epstein-Barr 病毒潜伏期 C-启动子结合因子-1/无毛抑制因子/lag-1（CSL）结合，并诱导 Notch 靶基因的转录，如 hairy enhancer of split-related with YRPW motif（Hey）1 和 Hey2。活化 T 细胞核因子（NFAT）是调节破骨细胞分化的转录因子，但它们在成骨细胞中的功能尚不清楚。Notch 抑制 NFATc1 转录，但 Notch 和 NFAT 之间的相互作用知之甚少。为了确定 Notch 对 NFAT 表达的调节作用，使用了 Rosa（Notch）小鼠的成骨细胞，其中 NICD 在loxP 侧翼的 STOP 盒切除后转录。或者，将野生型 C57BL/6 成骨细胞暴露于 Notch 配体 Delta-like（Dll）1 以诱导 Notch 信号或作为对照的牛血清白蛋白。在 Rosa（Notch）成骨细胞中，Notch 抑制 NFATc1 表达，通过转录后机制增加 Nfatc2 mRNA，并对 NFATc3 和 NFATc4 转录物没有影响。在暴露于 Dll1 的 C57BL/6 成骨细胞中，Notch 诱导 Nfatc2 转录物的产生得到了证实。为了研究 NFATc2 在成骨细胞中的功能，在 Rosa（Notch）成骨细胞中过表达组成型激活的 NFATc2。NFATc2 抑制 Notch 反式激活和 Hey 基因的表达。电泳迁移率变动分析显示，NFATc2 和 CSL 结合到相似的 DNA 序列上，染色质免疫沉淀表明 NFATc2 将 CSL 从 Hey2 启动子上置换下来。评估了 NICD 和 NFATc2 在 Rosa（Notch）成骨细胞中的作用，这两种蛋白都抑制成骨细胞功能。总之，Notch 稳定 Nfatc2 转录物，NFATc2 抑制 Notch 信号，这两种蛋白都抑制成骨细胞功能。
