Takayama Kazushi, Suzuki Akinobu, Manaka Tomoya, Taguchi Susumu, Hashimoto Yusuke, Imai Yuuki, Wakitani Shigeyuki, Takaoka Kunio
Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka, 545-8585, Japan.
J Bone Miner Metab. 2009;27(4):402-11. doi: 10.1007/s00774-009-0054-x. Epub 2009 Feb 28.
Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.
头蛋白是骨形态发生蛋白（BMPs）的主要细胞外拮抗剂，它与BMPs结合，阻断其与BMP特异性受体的结合，并负向调节BMP诱导的成骨细胞分化。在本研究中，我们通过转染小干扰RNA（siRNA）沉默头蛋白，研究其对体外BMP诱导的成骨细胞分化以及重组人BMP-2（rhBMP-2）诱导的体内异位骨形成的影响。通过实时RT-PCR分析确定，在成肌细胞系C2C12细胞中，rhBMP-2可剂量和时间依赖性地上调头蛋白mRNA表达。转染头蛋白siRNA沉默头蛋白表达可抑制BMP刺激的头蛋白表达，从而加速BMP诱导的成骨细胞分化。对于体内头蛋白沉默，将siRNA局部注射到背部肌肉中，并通过电穿孔转染到局部细胞中，在该处已手术植入保留rhBMP-2（5微克）的胶原盘。术后2周从实验组和对照组小鼠中取出植入物，通过放射学和组织学方法进行分析。结果，沉默头蛋白可使rhBMP-2异位诱导的小骨的骨矿物质含量显著增加。我们的研究结果表明，沉默头蛋白可通过消除BMP作用的负向调节，增强体外BMP反应性细胞的成骨细胞分化以及体内rhBMP-2诱导的新骨形成。RNA干扰可能有助于增强BMP在促进受损骨修复中形成新骨（骨痂）的作用。
