RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro

Abstract

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.

Effect of high glucose levels on the calcification of vascular smooth muscle cells by inducing osteoblastic differentiation and intracellular calcium deposition via BMP-2/Cbfα-1 pathway

“…In our experiments, recombinant human Noggin protein was used as the BMP-2 blocking agent, and it was observed not to affect the high glucose-stimulated BMP-2 expression significantly, but to reduce the production of downstream Cbfα-1 and vascular calcification, correspondingly. This is consistent with the mechanisms of Noggin reported by others (Busch et al, 2008;Takayama et al, 2009). Meanwhile, our results also confirmed that high glucose-induced Cbfα-1 expression was regulated by BMP-2, and high glucose levels might induce osteoblastic differentiation and intracellular calcium deposition via the BMP-2/Cbfα-1 pathway.…”

Distraction Osteogenesis and Its Challenges in Bone Regeneration

“…Over-expression of BMP antagonists has been shown to have detrimental effects on various bone parameters (Wu et al, 2003). In contrast, suppression of BMP antagonists using RNA interference caused increased osteogenic differentiation of cultured pre-osteoblastic cells, stromal cells and myoblastic cells (Kwong et al, 2008;Takayama et al, 2009 and may potentiate the effects of endogenous BMPs. Furthermore, as Noggin binds to several BMPs (BMP2, 4, 6 and 7), reducing Noggin expression may lead to increased availability of several, and not one BMP, thus reconstituting the normal biological environment, as compared to the exogenous application of BMPs, where only a single BMP, -either BMP2 or BMP7 can be locally applied.…”

Realizing the potential of gene-based molecular therapies in bone repair

“…After BMP-2 implantation, a small increase in BMD was observed with Noggin siRNA. (90) A subsequent study found similar results when Noggin siRNA was delivered alongside BMP-2 in a synthetic scaffold. (91) For these studies, we estimate that 10 to 16 µg of siRNA was delivered in each implant, which is practical for clinical scale up.…”

“…Abundant in Neuroepithelial Area (ANA), (87) Hoxc8, (88) Protein related to DAN and cerberus (PRDC), (89) Noggin, (90,91) Notch, (92) zinc finger Zfp467, (93) guanine nucleotide-binding protein alpha (GNAS1), (94) and prolyl hydroxylase domain-containing protein 2 (PHD2) (94) have been all shown to enhance osteogenic activity of various cell types in vitro. siRNA has been additionally investigated as a supplement to protein delivery, where siRNA against Noggin have been deployed in support of BMP-induced osteogenesis.…”

“…siRNA has been additionally investigated as a supplement to protein delivery, where siRNA against Noggin have been deployed in support of BMP-induced osteogenesis. (90,91) In one study, the muscle surrounding the implantation site was primed through electroporation of Noggin siRNA. After BMP-2 implantation, a small increase in BMD was observed with Noggin siRNA.…”