Study on regulation of osteogenesis through BMP antagonists

在这项研究中，我们旨在了解BMP拮抗剂如何调节成骨。为此，我们首先研究了多少BMP拮抗剂在成骨细胞中表达以响应BMP，然后研究了每种拮抗剂在成骨中的功能。1)寻找BMP配体诱导的BMP拮抗剂BMP2.5.7和5 + 7分别处理成骨细胞系MC3TE1细胞。然后，我们使用每种特定引物检测BMP拮抗剂的mRNA表达。Noggin, Sost （Sclerostin）和PRDC在BMP反应中特异性表达，而DAN， Tsg, USAG1， cereberus和Gremlin的表达水平没有改变。2) SostIn的表达和功能原位杂交研究表明，sostmrna在体内定位于颅骨成骨前端，接近成骨表达区。在成骨细胞分化过程中，Sost mRNA表达增加，这种增加被Noggin感染成骨细胞的腺病毒所阻断。抑制成骨导致Sost mRNA在成骨细胞中的表达降低。3)利用siRNA抑制Noggin和PRDC的表达我们设计了几种siRNA抑制Noggin和PRDC的表达。其中，Noggin2 siRNA比Noggin4更有效，PRDC 880 siRNA比PRDC 373和367更有效。siRNA抑制Noggin未改变成骨细胞ALP活性和表达。4) PRDC的表达和功能。通过原位杂交，我们发现PRDC mRNA在骨形成过程中在椎骨和颅骨原基中表达。利用腺病毒在成骨细胞中过表达PRDC，可抑制成骨细胞ALP和骨钙素mRNA的表达。表达腺病毒PRDC也能抑制ALP活性和矿化结节数量。5)sirdc抑制PRDC PRDC抑制内源性表达PRDC导致成骨细胞ALP活性和表达升高。sirna也增加了矿化结核的数量。

In this study, we aimed at understanding how BMP antagonists regulate osteogenesis. For this purpose, we first examined how many BMP antagonists are expressed in response to BMP in osteoblasts and then investigated function of each antagonist in osteogenesis.1)Search for BMP antagonists induced by BMP ligandsOsteoblastic cell line MC3TE1 cells were treated with BMP2.5.7, and 5 plus 7. We then examined mRNA expression of BMP antagonists using each specific primers. Noggin, Sost (Sclerostin) and PRDC were specifically expressed in response to BMP whereas expression levels of DAN, Tsg, USAG1, cereberus and Gremlin were not altered.2)Expression and function of SostIn situ hybridization study revealed that Sost mRNA is localized to osteogenic front of calvaria in close to the area of osterix expression in vivo. During osteoblastic differentiation, Sost mRNA was increased and this increase was blocked by adenoviral infection of Noggin into osteoblasts. Suppression of osterix resulted in decrease in Sost mRNA expression in osteoblasts.3)Suppression of Noggin and PRDC expression using siRNAWe designed several siRNA to suppress expression of Noggin and PRDC. Of the siRNAs, Noggin2 siRNA was more effective than Noggin4 and PRDC 880 siRNA is more effective than PRDC 373 and 367.Suppression of Noggin by siRNA did not change ALP activity and expression in osteoblast.4)Expression and function of PRDC.By in situ hybridization, we found that PRDC mRNA was expressed in primordia of vertebrae and skull during skeletogenesis. Over expression of PRDC using adenovirus in osteoblasts resulted in suppression of mRNA expression of ALP and osteocalcin in osteoblasts. ALP activity and numbers of mineralized nodules were also suppressed by PRDC expressing adenovirus5)Suppression of PRDC by siRNASuppression of endogenous expression PRDC by PRDC 880 siRNA resulted in elevation of ALP activity and expression in osteoblast. Numbers of mineralized nodules were also increased by si RNA.