National Institute of Radiological Sciences, Chiba, Japan.
Exp Cell Res. 2010 Apr 15;316(7):1127-36. doi: 10.1016/j.yexcr.2010.01.023. Epub 2010 Jan 29.
Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.
丝裂原活化蛋白激酶（MAPKs）调节成骨细胞的增殖和分化。Nemo 的脊椎同源物，Nemo 样激酶（NLK），是一种非典型的 MAPK，它靶向几种信号转导成分，包括 T 细胞因子/淋巴增强因子（TCF/Lef1）转录因子。最近的研究表明，NLK 与组蛋白 H3-K9 甲基转移酶 SETDB1 形成复合物，并抑制过氧化物酶体增殖物激活受体（PPAR）-γ：在间充质细胞系 ST2 中的作用。在这里，我们研究了 NLK 是否调节成骨细胞分化。我们表明，NLK mRNA 在体内表达于胚胎第 18.5 天（E18.5）的小鼠颅骨成骨细胞中。通过使用逆转录病毒载体，我们在原代颅骨成骨细胞（pOB 细胞）和间充质细胞系 ST2 中进行了 NLK 的强制表达。野生型 NLK（NLK-WT）抑制碱性磷酸酶活性和骨标记基因如碱性磷酸酶、I 型原胶原、runx2、osterix、骨桥蛋白和骨钙素的表达。NLK-WT 还降低了 pOB 和 ST2 细胞中 I 型胶原蛋白蛋白的表达。此外，在过表达 NLK-WT 的 pOB 细胞中，矿化结节形成减少。相比之下，NLK 的激酶缺失形式（NLK-KN）在 pOB 和 ST2 细胞中未抑制或部分抑制碱性磷酸酶活性和骨标记基因表达。NLK-KN 并未抑制 pOB 细胞中的结节形成。除了强制表达外，siRNA 抑制内源性 NLK 表达增加了 pOB 和 ST2 细胞中的骨标记基因表达。最后，基因启动子的转录活性分析表明，NLK-WT 抑制了 Wnt1 对 TOP flash 启动子的激活和 Runx2 对骨钙素启动子的激活。总之，这些结果表明 NLK 负调节成骨细胞分化。
