A Study Of The Mechanism Of ROG On Down-regulate The Expression Of T Lymphocyte Cytokines

As the core mechanism of asthma is the down-regulation of the ratio of Th1/Th2 cytokines and as there exists a reciprocal suppression role between Th1 and Th2 cytokines, suppression of the transcription factor GATA-3, which can enhance the expression of Th2 cytokines, or stimulation of the transcription factor T-bet, which can enhance the expression of Th1 cytokines, are effective ways to reverse the descended ratio of Th1/Th2 cytokines in asthma. Previous studies founded that the newly discovered transcription factor, ROG, a suppressor of GATA-3, could simultaneously suppress the expression of Th1 and Th2 cytokines, but the mechanism was unknown. We believe that, elucidation the mechanism of ROG on down-regulate the expression of T lymphocyte cytokines could help to comprehend the mechanism of disequilibrium of T lymphocyte cytokines in asthma.ROG is a new member of zinic finger transcription factor family. In early studies, it was found that the expression of ROG was markly upregulated in CD4~+T cells closely after being stimulated by anti-CD3. Upregution of ROG could markly suppress the transactivation role of GATA-3 in activating the promoters of IL-4 and IL-5, which makes the denomination of ROG In subquent studies, it was founded that expression of Th1 and Th2 cytokines could both be suppressed by transfection of ROG gene. While ROG could suppress the expression of Th2 cytokines completely or partly by suppression of GATA-3, an unknown molecule might serve as the other target of ROG, which could suppresss the expression of T cell cytokines, at least the Th1 cytokines.Based on the current understanding of the expression mechanism of T lymphocyte cytokines, possible target of ROG in regulating the expression of T cell cytokines might include CD3 (The first signal on T cell membrane), CD28, ICOS, CTLA-4 (The second signal on T cell membrane), and CD45 (A molecule of signal transduction pathway in T cell cytoplasm). After an allround analysis of the features of these molecules, we hypothesized that, ROG might suppress the expression of T cell cytokines directly or indirectly by suppress CD3, CD28, or ICOS, which could upregulate the expression of T lymphocyte cytokines, or by indirectly suppress CTLA-4 or CD45, which can downregulate the expression of T lymphocyte cytokines.In order to confirm the above-proposed hypothesis and reveal the relative mechanism, we carried out the following experiments: Firstly, mRNA and protein levels of ROG, CD3, CD28, ICOS, CTLA-4 and CD45 in Th1 and Th2 cells of initial status were studied by sybergreen real-time quantitive RT-PCR and Western Blot. Secondly, mRNA and protein levels of CD3, CD28, ICOS, CTLA-4 and CD45 in Th1 and Th2 cells with upregulated expression of ROG, achieved by ROG-pcDNA3.1 nucleofection, were studied by sybergreen real-time quantitive RT-PCR and Western Blot. Finally, mRNA and protein levels of CD3, CD28, ICOS, CTLA-4 and CD45 in Th1 and Th2 cells with downregulated expression of ROG, achieved by ROG-siRNA nucleofection, were studied by sybergreen real-time quantitive RT-PCR and Western Blot.Results were as following: the mRNA and protein levels of ROG are relatively low in Th1 and Th2 cells of initial status (P<0.01). R0G-pcDNA3.1 nucleofection could lead to a upregulation of ROG mRNA (P<0.01) and protein (P<0.01) and a downregulation of ICOS mRNA (P<0.01) and protein (P<0.01), followed by a downregulation of T cell cytokines both in Th1 and Th2 cells (P<0.01), and ROG-siRNA nucleofection could lead to a downregulation of ROG mRNA (P<0.01) and protein (P<0.01) and a upregulation of ICOS mRNA (P<0.01) and protein (P<0.01), followed by a upregulation of T lymphocyte cytokines both in Th1 and Th2 cells (P<0.01). While during the changing of the ROG protein level, the mRNA and protein levels of CD3, CD28, CTLA-4, and CD45 did not change both in Th1 and Th2 cells (P>0.05).From these results, we conclude that, ROG could down-regulate the expression of Th1 and Th2 cytokines by down-regulative the expression of ICOS. By bioinformatics analysis, we found that there exists a GATA-1 area, which share a 76% homology with GATA-3 in the promoter area of ICOS, which confirm the reliability of the results of our experiments.Our study could help to more exactly understand the role of ROG on negatively regulating the expression of T cell cytokines and help to reveal the mechanism of disequilibrium of T cell cytokines, which may set an experimental and theoretical foundation in finding a target for gene treatment of asthma.