Sambandam Yuvaraj, Sundaram Kumaran, Saigusa Takamitsu, Balasubramanian Sundaravadivel, Reddy Sakamuri V
Department of Pediatrics/Endocrinology, Darby Children's Research Institute, USA.
Division of Nephrology, University of Alabama at Birmingham, AL, USA.
Bone. 2017 Aug;101:236-244. doi: 10.1016/j.bone.2017.05.013. Epub 2017 May 12.
Paget's disease of bone (PDB) is marked by the focal activity of abnormal osteoclasts (OCLs) with excess bone resorption. We previously detected measles virus nucleocapsid protein (MVNP) transcripts in OCLs from patients with PDB. Also, MVNP stimulates pagetic OCL formation in vitro and in vivo. However, the mechanism by which MVNP induces excess OCLs/bone resorption activity in PDB is unclear. Microarray analysis identified MVNP induction of NFAM1 (NFAT activating protein with ITAM motif 1) expression. Therefore, we hypothesize that MVNP induction of NFAM1 enhances OCL differentiation and bone resorption in PDB. MVNP transduced normal human PBMC showed an increased NFAM1 mRNA expression without RANKL treatment. Further, bone marrow cells from patients with PDB demonstrated elevated levels of NFAM1 mRNA expression. Interestingly, shRNA suppression of NFAM1 inhibits MVNP induced OCL differentiation and bone resorption activity in mouse bone marrow cultures. Live cell widefield fluorescence microscopy analysis revealed that MVNP induced intracellular Ca oscillations and levels were significantly reduced in NFAM1 suppressed preosteoclasts. Further, western blot analysis demonstrates that shRNA against NFAM1 inhibits MVNP stimulated PLCγ, calcineurin, and Syk activation in preosteoclast cells. Furthermore, NFAM1 expression controls NFATc1, a critical transcription factor expression and nuclear translocation in MVNP transuded preosteoclast cells. Thus, our results suggest that MVNP modulation of the NFAM1 signaling axis plays an essential role in pagetic OCL formation and bone resorption activity.
骨佩吉特病（PDB）的特征是异常破骨细胞（OCLs）的局灶性活动以及过度的骨吸收。我们之前在PDB患者的OCLs中检测到麻疹病毒核衣壳蛋白（MVNP）转录本。此外，MVNP在体外和体内均可刺激佩吉特OCL的形成。然而，MVNP在PDB中诱导过量OCLs/骨吸收活性的机制尚不清楚。微阵列分析确定了MVNP对NFAM1（具有ITAM基序的NFAT激活蛋白1）表达的诱导作用。因此，我们推测MVNP对NFAM1的诱导增强了PDB中OCL的分化和骨吸收。转导了MVNP的正常人外周血单核细胞（PBMC）在未进行RANKL处理的情况下显示NFAM1 mRNA表达增加。此外，PDB患者的骨髓细胞显示NFAM1 mRNA表达水平升高。有趣的是，shRNA抑制NFAM1可抑制小鼠骨髓培养物中MVNP诱导的OCL分化和骨吸收活性。活细胞宽场荧光显微镜分析显示，MVNP诱导的细胞内钙振荡在NFAM1抑制的前破骨细胞中显著降低。此外，蛋白质印迹分析表明，针对NFAM1的shRNA可抑制前破骨细胞中MVNP刺激的PLCγ、钙调神经磷酸酶和Syk激活。此外，NFAM1的表达控制NFATc1，这是MVNP转导的前破骨细胞中一种关键转录因子的表达和核转位。因此，我们的结果表明，MVNP对NFAM1信号轴的调节在佩吉特OCL形成和骨吸收活性中起重要作用。
