Class II transactivator抑制人類造骨細胞Cyr61基因表現的機制

Abstract

類風濕性關節炎是一種自體免疫細胞攻擊軟骨組織造成關節部分的慢性發炎反應，病人常因骨骼及軟組織受破壞影響關節的正常功能。此病的盛行率約為1％。2006年，Christian S. Haas 利用cDNA microarray分析病人檢體，發現一個血管新生因子cysteine-rich， angiogenic inducer 61，Cyr61 （a known angiogenic factor） 的表現有顯著差異，然而其在疾病的過程扮演著正面或負面的角色仍然不清楚。我們實驗室透過在成骨細胞（U2OS， an osteoblast-like osteosarcoma cell line） 加入有活性的Cyr61合成蛋白以及轉染表現質體Cyr61/pCDNA3.1均發現，這個蛋白質可以促進細胞爬行（migration）的能力。此外，一個能夠促進成骨細胞分化及成熟的蛋白質，抑瘤素 Oncostatin M，OSM （IL-6家族成員之一），我們實驗室也發現它能夠透過一連串細胞內訊號導致磷酸化CREB （cAMP response element-binding）蛋白質促進Cyr61的表現。確認了成骨細胞Cyr61基因的表現模式後，本文旨在研究是否有其他扮演調控的因子參與其中。CIITA （MHC Class II transactivatoir），是表現MHC class II重要的調控因子，但是，在表現其他基因，例如collagen type I，則被認為是一個抑制者。透過西方墨點轉漬法以及promoter activity的分析，我們確認了CIITA在Cyr61表現上扮演著抑制的角色。CIITA沒有DNA binding domain而需要透過其他轉錄因子，例如CREB，NF-Y及RFX等，結合在特定的位置。於是，我們推論透過CREB的幫助，CIITA整併到Cyr61 promoter處。接著，我們把Cyr61 promoter region的CRE site （CREB binding site）進行突變，也的確發現了CIITA抑制mutant Cyr61 promoter activity的能力明顯變弱。對於CIITA弱化Cyr61基因表現可能的機轉，目前我們發現CIITA的存在會抑制CREB的磷酸化，不僅影響CREB與Cyr61 promoter的結合也阻礙了CREB 與p300之間直接的互動。研究成果展現了CIITA在Cyr61基因表現過程當中扮演抑制者可能的機制與角色。也更加清楚OSM所誘導的Cyr61轉錄的作用機制。

AIMS: To evaluate the possible role of human Cyr61 in rheumatoid arthritis and the regulation mechanisms of Cyr61 gene expression by class II transactivator in human osteoblasts. METHODS AND RESULTS: 1.） We assessed the transcriptional regulation of oncostatin-M （OSM）-induced Cyr61 gene expression in U2OS, a human osteosarcoma cell line with osteoblastic characters. Western blot revealed that OSM induced obvious Cyr61 synthesis and phsphorylation of cAMP responsive binding protein （CREB）. Luciferase assay showed elevated Cyr61 promoter activities following CREB over-expression. Electrophoretic mobility shift assay （EMSA） revealed significant CREB/DNA cross-talk after OSM stimulation. But, Dominant negative p300 showed no significant difference in inducing Cyr61 expression versus vector only in luciferase assay. 2.） We studied the regulation of class transcactivator （CIITA）. Western blot revealed that CIITA expression reduced Cyr61 expression and attenuated CREB activity. Luciferase assay showed CIITA regulates Cyr61 expressionin a dose-dependent manner CIITA and suppression on Cyr61 gene expression is CRE-dependent. CO-IP data indicated that CIITA reduced the binding between CREB / p300. 3.） In RA animal model we saw clearly ankylosis, bone resorption and granulation tissue infiltration. IHC data showed us Cyr61 expression in tissue-specific manner. CONCLUSION: CREB phosphorylation and HAT activity of p300 was critical for Cyr61 gene expression. Class II transactivator suppressed Cyr61 transcription by sequester CREB and result in attenuating CREB / p300 binding. In vivo examination showed that Cyr61 expression in rheumatoid arthritis rat is cell and tissue specific. INPACT OF THIS STUDY: This study provided the regulation mechanisms of Cyr61 gene expression in human osteoblasts and indicated a close relationship between Cyr61 expression and rheumatoid arthritis pathology.