Response of Major Histocompatibility Complex (MHC) to DEX & PCN in Non-APC(pilot)

合成类固醇，孕烯醇酮-16-α-碳腈(PCN)，几十年来一直用于探索一系列假定的肝脏防御系统在环境“应激”的情况下被激活。以往的研究是在PCN的控制下探索基因的全域结构域，采用差异显示技术检测PCN或地塞米松(DEX)选择性诱导的大鼠肝脏mRNA的种类。这些研究揭示了RT1.B(I)β的诱导，它是主要组织相容II类(MHC)基因家族的成员，通常只在抗原提呈细胞(APC)中发现。从大鼠肝脏或培养的肝细胞中获得的初步数据证实，在DEX或PCN处理后的3-6小时内，RT1.B(I)βmRNA及其伴随基因RT1.B(I)α的数量很容易被诱导，而在脾(APC部位)则没有被诱导。使用糖皮质激素的经典剂量反应实验一直以来 在免疫组织中发现MHCII基因被抑制。然而，干扰素干扰素在许多组织和细胞中是最有效的MHCU基因诱导剂之一，但在肝细胞中不是。糖皮质激素对大鼠肝脏中MHCII基因的意外诱导，就其可能的作用机制提出了一系列问题。 这样的效果。在毒性应激期间，一组基因受到协调控制，以维持实质组织的动态平衡，这一概念现在必须扩展到包括免疫系统。由于大鼠和小鼠MHC-II组分之间有很高的相似性(在cDNA序列中同源性为91%)，我们认为这种大鼠肝脏诱导现象也会发生在小鼠身上。由于MHCII小鼠启动子是众所周知的，特征也很清楚，而且非常相似 对于人类来说，这将是研究类固醇治疗对MHCII调节的一个很好的和首选的模型。第二类反式激活因子(CIITA)已被认为是MHCII基因转录的关键调控因子。已经描述了三种不同类型的启动子(I、III和IV)来调节小鼠的CIITA转录。这项研究旨在确定是否可以通过类固醇治疗在小鼠身上实现MHC-II基因的诱导，就像在大鼠中一样。如果 这样我们就有了一个很好的模型来研究非APC中MHCII诱导的机制。我们将立即确定MHC II诱导是依赖还是独立于CIITA，或者是否通过新的机制。我们还将说明哪个CIITA启动子参与了肝和肺特定组织的这种诱导。如果MHC II基因对类固醇诱导小鼠无效，我们计划继续我们的 并对该物种中的启动子进行充分的表征，以阐明与这一现象相关的机制。这些实验的结果对于理解类固醇对MHC调节的影响是至关重要的，因为许多类固醇如地塞米松被用于治疗某些自身免疫性疾病(特征是 MHCII基因的过度表达)相当高的剂量，高达1000毫克/天。这项建议旨在阐明DEX和PCN是否通过其在小鼠和大鼠中的已知反式激活途径或通过独立于CIITA的不同途径在非APC中诱导MHC II类基因。

The synthetic steroid, pregnenolone-16-alpha carbonitrile (PCN), has served for decades to probe for a postulated series of hepatic defenses activated under situations of environmental "stress". Previous research conducted to explore the full domain of genes under the control of PCN employed differential display to detect rat liver mRNA species selectively induced by PCN or by Dexamethasone(DEX). These studies revealed the induction of RT1.B (I)beta a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells (APC). Preliminary data obtained from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B (I)beta mRNA and also of its companion gene, RT1.B (I)alpha, became readily induced within 3-6 h following DEX or PCN treatment, whereas no induction was observed in spleen (site of APC). Classical dose response experiments using glucocorticoids have always revealed inhibition of MHCII genes in immune tissues. However, gamma interferon IFNgamma is one of the most potent inducers of MHCU genes in many tissues and cells but not in hepatocytes. This unexpected induction of MHCII genes in the rat liver by glucocorticoids has opened a series of questions in terms of the possible mechanisms responsible for such effect. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system. Due to the high similarities between rat and mice MHC-II components (91% homology in cDNA sequence) we believe that this rat liver induction phenomenon would also take place in mice. Since the MHCII mouse promoter is well known, fairly well characterized, and is very similar to humans, it would be an excellent and preferred model for studying MHCII regulation due to steroid treatment. The class II transactivator (CIITA) has been accepted as the key regulator for the transcription of MHCII genes. Three different types of promoters (I, III, and IV) have been described to regulate CIITA transcription in mice. This research is designed to determine if induction of MHC-II genes can be achieved by steroid treatment in mice as it is in rats. If this is achieved then we have an excellent model to study the mechanisms involved in MHCII induction in non-APC. We will immediately establish whether MHC II induction is dependent or independent on CIITA or whether it is through a new mechanism. We will also address which CIITA promoter is involved in this induction in the specific tissues of liver and lung. In the event that MHC II genes are refractory to steroid induction in mice we plan to continue with our rat model and fully characterize the promoter in this species in order to elucidate the mechanism associated with this phenomenon. The results of these experimentsare of utmost importance for understanding the effect of steroids on MHC regulation since many steroids such as dexamethasone are used to treat certain autoimmune diseases (characterized by over-expression of MHCII genes) at considerably high doses of up to 1000mg/day. This proposal is designed to elucidate if DEX and PCN are inducing the MHC class II genes in non-APC through its known transactivation pathway in mice and rat or through a different route independent of CIITA.