Interferon-gamma regulation of the human mimecan promoter.
Interferon-gamma regulation of the human mimecan promoter.
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发表时间：
2003-06
期刊：
影响因子：
2.2
通讯作者：
E. Tasheva;G. Conrad
中科院分区：
文献类型：
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作者：
E. Tasheva;G. Conrad
目的：人mimecan/osteoglycin启动子含有多个干扰素刺激反应元件（ISRE）和干扰素γ激活位点（GAS）。ISRE和GAS基序存在于多种干扰素（IFN）诱导基因中。本研究的目的是研究ifn - γ是否影响mimecan基因表达，如果是，确定介导其作用的顺式元件和转录因子。方法采用电泳迁移位移法（EMSA）研究ifn - γ处理细胞的核蛋白是否与含有ISRE位点的人mimecan启动子区域结合。用特异性抗体孵育核提取物来鉴定结合这些位点的转录因子。通过将构建的人类mimecan启动子/荧光素酶报告基因瞬间转染到角膜角质细胞和非角膜细胞中来评估该启动子的转录活性。共转染实验用于研究转录因子在启动子中结合ISRE元件并介导ifn - γ反应的作用。逆转录-聚合酶链反应分析mrna的表达。结果：利用位于人mimecan基因第一个内含子上的两个ISRE位点对应的探针，我们用ifn - γ处理细胞的核提取物检测到特异性的dna -蛋白复合物。通过与未标记探针的竞争，dna -蛋白复合物的形成被取消，其中一个复合物被抗干扰素调节因子-1 （IRF-1）抗体超移。有趣的是，当在EMSA中使用与近端启动子中保守E-box （CACATG）对应的探针和ifn - γ处理细胞的核提取物时，观察到上游刺激因子-1 （USF-1）的结合增加。将含有整个第一个内含子的mimecan启动子结构与IRF-1或IRF-1和USF-1表达质粒共转染，可抑制角膜角质细胞和T-47D细胞中启动子的荧光素酶活性。相比之下，IRF-2或IRF-2和USF-1共转染实验导致相同启动子结构的荧光素酶活性增加。RT-PCR分析表明，ifn - γ能快速、短暂地抑制mimecan的表达，并诱导牛角膜角质细胞中IRF-1和IRF-2 mrna的表达。结论IRF-1结合位于人mimecan基因第一个内含子的ISRE位点，并在转录水平上负调控mimecan的表达。与这些观察结果一致，证明了mimecan和IRF-1在牛角膜角质细胞和非角膜细胞中的表达呈负相关。IRF-2正调控角膜角质细胞中的mimecan转录。由于IRF-1和IRF-2对mimecan转录的影响需要近端启动子中完整的E-box，因此USF-1与IRF-1和IRF-2之间可能存在直接或间接的相互作用。
PURPOSE The human mimecan/osteoglycin promoter contains multiple interferon-stimulated response elements (ISRE) and interferon-gamma-activation sites (GAS). ISRE and GAS motifs are present in a variety of interferon (IFN)-inducible genes. The purpose of this study was to investigate whether IFN-gamma affects mimecan gene expression and, if so, to determine the cis-elements and transcription factors that mediate its action. METHODS Electrophoretic mobility shift assay (EMSA) was used to investigate whether nuclear proteins from IFN-gamma-treated cells bind to regions of the human mimecan promoter containing ISRE sites. Incubation of nuclear extracts with specific antibodies was used to identify transcription factors that bind to these sites. Transcriptional activity of the promoter was evaluated by transient transfections of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Co-transfection experiments were used to study the role of transcription factors that bind ISRE elements in the promoter and mediate the IFN-gamma response. Expression of mRNAs was analyzed by reverse transcription-polymerase chain reaction. RESULTS Using probes that correspond to two ISRE sites located in the first intron of the human mimecan gene, we detected specific DNA-protein complexes with nuclear extracts from IFN-gamma-treated cells. Formation of DNA-protein complexes was abrogated by competition with unlabeled probe and one of the complexes was supershifted by the anti-interferon regulatory factor-1 (IRF-1) antibody. Interestingly, when probe that corresponds to a conserved E-box (CACATG) in the proximal promoter and nuclear extracts from IFN-gamma-treated cells were used in EMSA, increased binding of upstream stimulatory factor-1 (USF-1) was observed. Co-transfection of a mimecan promoter construct that contained the entire first intron with IRF-1, or with both IRF-1 and USF-1 expression plasmids, suppressed luciferase activity of the promoter in corneal keratocytes and T-47D cells. In contrast, co-transfection experiments with IRF-2, or with both IRF-2 and USF-1, led to increased luciferase activity of the same promoter construct. RT-PCR analyses demonstrate that IFN-gamma rapidly and transiently suppresses mimecan expression and induces IRF-1 and IRF-2 mRNAs in bovine corneal keratocytes. CONCLUSIONS IRF-1 binds to ISRE sites, located in the first intron of the human mimecan gene, and negatively regulates mimecan expression at the level of transcription. Consistent with these observations, an inverse correlation between the expression of mimecan and IRF-1 in bovine corneal keratocytes and non-corneal cells was demonstrated. IRF-2 positively regulates mimecan transcription in corneal keratocytes. Because the intact E-box in the proximal promoter was required for IRF-1 and IRF-2 effects on mimecan transcription, potential direct or indirect interactions between USF-1 and IRF-1 and IRF-2 are likely.