RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro.
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.
Takayama
Kazushi
K
Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka, 545-8585, Japan.
Suzuki
Akinobu
A
Manaka
Tomoya
T
Taguchi
Susumu
S
Hashimoto
Yusuke
Y
Imai
Yuuki
Y
Wakitani
Shigeyuki
S
Takaoka
Kunio
K
D020341:Absorbable Implants; D015502:Absorptiometry, Photon; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015519:Bone Density / Q000187:drug effects; D055396:Bone Morphogenetic Protein 2 / Q000008:administration & dosage / Q000494:pharmacology; D019485:Bone Morphogenetic Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D003094:Collagen; D018274:Electroporation; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D014162:Transfection; D000099176:Noggin Protein
D000469:Alkaline Phosphatase; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D003094:Collagen; D015675:Osteocalcin; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000099176:Noggin Protein; C525718:BMP2 protein, human