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  • 學位論文

人類造骨細胞中,CREB與p300對於Cyr61基因的調控

CREB and p300 regulate Cyr61 gene expression in human osteoblastic cells

指導教授 : 林思洸

摘要


Cysteine-rich protein 61﹙Cyr61/CCN1﹚能跟細胞外間質中的一些分子結合,並且具有血管新生;腫瘤生成;促進胚胎發育之功能。最近,已有文獻報導Cyr61 可促進造骨細胞的分化及增生。然而,Cyr61 和骨生成的關係可能還需要更多的研究去釐清。因此,本研究欲評估在人類造骨細胞﹙U2OS﹚中,Oncostatin-M ﹙OSM﹚誘導Cyr61 基因表現上的調節。在細胞遷移實驗中,我們發現了Cyr61 可促進U2OS 的移動;以西方點墨法偵測發現,OSM 同時會誘導Cyr61 的表現以及 cAMPresponsive binding protein﹙CREB﹚的磷酸化;以Luciferase reporter assay觀察啟動子活性發現,CREB 可提高Cyr61 啟動子的活性,且在Site-directed mutagenesis 則透露CREB 能跟Cyr61 啟動子上特定的位置結合;從 Electrophoretic mobility shift assay﹙EMSA﹚顯示在OSM 的刺激下,CREB 會與DNA 產生結合。 另一方面,p300 也可能參與OSM 誘導Cyr61 的過程。Luciferase assay 中,發現了CREB 跟p300 的shRNA 會抑制Cyr61 啟動子的活性。在免疫共沉澱法(Co-immunoprecipitaiton)顯示p300 跟CREB 複合物的形成;此外,Histone acetyltransferase (HAT) assay 也透露在OSM 的刺激下會增加HAT 活性。因此,我們認為OSM 在人類造骨細胞中會刺激CREB 的磷酸化,促進CREB 跟Cyr61 啟動 子結合,同時,召集 p300 一同促進Cyr61 的表現,並造成造骨細胞的移動,所以Cyr61 可能會促進骨生成。

關鍵字

Cyr61 人類造骨細胞

並列摘要


Cysteine-rich protein 61(Cyr61/CCN1) involve in angiogenesis,tumorigenesis, embryogenesis and extracellular matrix production. Cyr61 has been reported to promote osteoblast differentiation recently. However, more information regarding the influences of Cyr61 on osteogenesis is needed. Here, we assessed the transcriptional regulation of oncostatin-M (OSM)-induced Cyr61 gene expression in U2OS, a human osteosarcoma cell line with osteoblastic characters.The effects of Cyr61 on osteoblast migration were also explored. In vitro assay showed stimulation effect of Cyr61 on U2OS migration. Western blot revealed that OSM induced significant Cyr61 synthesis and phsphorylation of cAMP responsive binding protein (CREB). Luciferase assay showed elevated Cyr61 promoter activities following CREB stimulation. Site-directed mutagenesis confirmed that CREB bound directly to specific sites on Cyr61 promoter to trigger gene expression. Electrophoretic mobility shift assay (EMSA) revealed significant CREB/DNA cross-talk after OSM stimulation. Transient transfection analyses demonstrated attenuation of Cyr61 promoter activity by CREB and p300 shRNAs.Co-immunoprecipitaiton confirmed the associations between P300 and CREB.Histone acetyltransferase (HAT) assay also revealed increased HAT activities after OSM treatment. In conclusion, OSM stimulates CREB activation, CREB/DNA cross-talk and recruitment of p300 by CREB in human osteoblasts. OSM also upregulates HAT activity. These effects may induce the transactivation of Cyr61, a novel osteoblast migration factor.

並列關鍵字

Cyr61 human osteoblastic cell

參考文獻


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