Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

在过去的几年里，我们的研究重点集中在T细胞发育和激活过程中GC链表达的机制上。除了转录调控机制外，我们还发现了一条新的转录后GC链表达途径，导致可溶GC链的产生。我们在正常人和小鼠血清中都检测到了大量的可溶性GC蛋白，并研究了它在体内平衡和自身免疫条件下的功能。可溶性GC(SGC)是通过外显子跳跃和选择性剪接在开放阅读框中发生移码而产生的。为了进一步评估其在体内的作用，我们培育了在T细胞中过表达可溶性GC的SGC转基因小鼠。这些SGC转基因小鼠在血清中表达高水平的SGC，其T细胞的激活记忆表型百分率增加。重要的是，当SGC TG小鼠在实验性自身免疫性脑脊髓炎(EAE)模型中接受免疫反应性挑战时，SGC TG小鼠的自身免疫反应显著增加，表现为更快、更强和更持久的临床疾病评分。这种增强反应的潜在机制是促炎Th17细胞的生成增加，我们目前正在确定这种增强反应的确切机制。在这项研究中，我们专注于DP胸腺细胞中细胞因子受体的调节，因为这些细胞在所有受试细胞中表达最高水平的SGC。因此，我们认为评估DP细胞特异性核因子的作用，如RORgt，这是理解细胞因子受体表达如何调节的一个合乎逻辑的步骤。因此，我们首先评估了RORgt对DP细胞中IL-7ra和GC链表达的需求。我们发现，在RORgt缺陷的DP胸腺细胞中，IL-7ra的表达仍然受到抑制，但随后发现同一细胞的表面GC链水平显着增加。此外，Western印迹分析表明，随着免疫沉淀GC链分子量的增加，总GC链蛋白水平增加。然而，重要的是，没有RORgt的情况下，GC mRNA的水平没有受到影响，这表明RORgt通过一种新的转录后机制影响GC链的表达。由于GC链是糖基化的，我们考虑了分子量增加是高糖基化的可能性。糖基化有两种主要形式：N-连接和O-连接。利用内切糖苷酶，我们询问了RORgT缺陷的DP细胞中的GC链是否被高糖基化。如果是这样的话，内切糖苷酶治疗应该减少来自RORgt缺陷细胞的GC链的分子量。有趣的是，O-糖苷酶处理对GC相对分子质量没有影响，但内切糖苷酶F(Endo F)处理显著减小了它的大小。这些数据表明，RORgt改变了GC链的糖基化特征，可能是通过影响N-连接的糖基化。差异糖基化GC链的生物学功能是完全未知的。此外，在体内诱导不同GC糖基化的生理信号尚不清楚，我们目前正在解决这些问题。此外，我们已经开始研究其他转录因子的影响，这些转录因子在DP胸腺细胞中高表达，并可能参与细胞因子受体的表达。初步研究结果表明，IL-7ra和GC链的表达在转录和转录后机制中都受到多种因素的调节，这一点的重要性需要进一步的体内模型来评估它们在T细胞发育和激活中的需求和功能。

During the last few of years, we have intensely focused our research on the mechanism of gc-chain expression during T cell development and activation. In addition to transcriptionally controlled mechanisms, we discovered a novel post-transcriptional pathway of gc-chain expression that leads to the generation of soluble gc-chains. We detected soluble gc proteins in significant amounts in both normal human and mouse serum, and we have investigated its function under both homeostatic and autoimmune conditions. Soluble gc (sgc) is generated by exon skipping and a frameshift in the open reading frame by alternative splicing. To further assess its role in vivo, we generated sgc transgenic mice that overexpress soluble gc in T cells. These sgc Tg mice expressed high levels of sgc in serum and their T cells showed an increased percentage of activated memory phenotype. Importantly, when sgc Tg mice were challenged in an experimental autoimmune encephalomyelitis (EAE) model for immune reactivity, sgc Tg mice displayed a significant increase in autoimmune reaction as shown by a faster, stronger and more lasting clinical disease score. The underlying mechanism for such an enhanced reaction turned out to be increased generation of pro-inflammatory Th17 cells, and we are currently in the process of identifying the exact mechanism for this enhanced reactivity. In line of this research, we focused on cytokine receptor regulation in DP thymocytes because these cells expressed the highest level of sgc among all tested cells. Thus, we considered assessing the role of DP cell specific nuclear factors, such as RORgt, a logical step for understanding how cytokine receptor expression is regulated. Consequently, first we assessed the requirement of RORgt for IL-7Ra and gc-chain expression in DP cells. We found that IL-7Ra expression still to be suppressed in RORgt-deficient DP thymocytes but then found that surface gc-chain levels were significantly increased in the same cells. Moreover, Western blot analyses showed an increase in total gc-chain protein levels that was accompanied with increased molecular weight of immunoprecipitated gc-chain. Importantly, however, gc mRNA levels remained unaffected by the absence of RORgt which suggested that RORgt affects gc-chain expression over a novel post-transcriptional mechanism.Since gc-chain is known to be glycosylated, we considered the possibility that the increased molecular weight would be a result of hyper-glycosylations. There are two major forms of glycosylation: N-linked and O-linked. Using endoglycosidases, we asked whether gc-chain in RORgt-deficient DP cells is hyper-glycosylated. If so, endoglycosidase treatment should reduce the molecular weight of gc-chains from RORgt-deficient cells. Interestingly, O-glycosidase treatment did not affect gc molecular weight, but endoglycosidase F (endo F) treatment significantly reduced its size. These data suggest that RORgt alters the glycosylation signature of the gc-chain, potentially by affecting N-linked glycosylation. The biological function of differentially glycosylated gc-chain is completely unknown. Also, the physiological cues that can induce different gc glycosylation in vivo is not known, and we are currently addressing these questions. Furthermore, we have started investigating the effects of other transcription factors that are highly expressed in DP thymocytes and that are potentially involved in cytokine receptor expression. Preliminary findings suggest that both IL-7Ra and gc-chain expression is regulated by multiple factors in both transcriptional and post-transcription mechanism whose importance requires the development of further in vivo models to assess their requirement and function in T cell development and activation.