Nuclear receptors--Action, functions, & roles in disease

I. ROR γ：类维生素A相关孤儿受体g（RORg）是核受体超家族的成员。为了鉴定RORg的生理功能，通过靶向破坏产生RORg功能缺陷的小鼠。RORg -/-小鼠缺乏外周和肠系膜淋巴结，Peyer？表明RORg的表达是淋巴结器官形成所必需的。虽然脾脏增大，但结构正常。外周血CD 3+和CD 4+淋巴细胞的数量分别减少6倍和10倍，而循环B细胞的数量是正常的。RORg-/-小鼠的胸腺含有比wt小鼠少74.4 ± 8.9%的胸腺细胞。流式细胞术分析显示CD 4 + CD 8+亚群减少。TUNEL染色显示RORg-/-小鼠胸腺皮质中的凋亡细胞增加了4倍。这得到了所观察到的膜联蛋白V阳性细胞增加的支持。置于培养物中的RORg-/-胸腺细胞表现出“自发”细胞凋亡率的急剧增加。这种增加在很大程度上与CD 4 + CD 8+胸腺细胞有关，并且可能至少部分与抗凋亡基因Bcl-XL的表达水平大大降低有关。流式细胞术分析表明，在RORg-/-小鼠胸腺细胞中，细胞周期S期细胞的百分比增加了6倍。我们的观察表明，RORg是必不可少的淋巴器官的发生，并发挥重要的调节作用，在胸腺生成。最近，我们发现RORg-/-小鼠对T细胞淋巴瘤的发展高度敏感。ROR的表达改变导致胸腺细胞分化失调，双阴性胸腺细胞增殖增加，这反过来又增加了肿瘤形成的频率。近年来研究表明ROR受体在多种炎症反应中起调节作用。RORa小鼠对内毒素诱导的炎症更敏感。微阵列分析显示ROR在类固醇和药物代谢中的作用。 二.任务1：核孤儿受体TAK 1作为转录的正调控因子和负调控因子发挥作用;然而对介导其活性的因子知之甚少。利用TAK 1的配体结合结构域作为诱饵的酵母双杂交分析鉴定了一种新的TAK 1相互作用蛋白，称为TIP 27。TIP 27是一个27 kD的核蛋白，含有两个锌指基序。使用TIP 27和几个缺失突变体的共聚焦显微镜显示，TIP 27定位于细胞核和羧基末端含有一个假定的核定位信号是重要的核定位。TIP 27 mRNA在几种成体组织中表达，但在睾丸中表达最高，在睾丸中它在精子发生的特定阶段被诱导。哺乳动物双杂交分析表明，TIP 27与TAK 1特异性相互作用，而不与其他几种核受体测试。缺失突变分析确定TIP 27的Asp 39和Lys 79之间的区域，称为TAK 1相互作用结构域（TID），是其与TAK 1相互作用的关键。此外，它证明了从H3到羧基末端的TAK 1-LBD是与TIP 27最佳相互作用所需的。下拉分析表明，TIP 27与TAK 1物理相互作用，并支持TID的重要性。TIP 27是TAK 1对DR 1依赖性转录激活的强阻遏物。这种抑制不涉及TAK 1同源二聚化或DR 1结合的抑制，但似乎是由于共激活剂募集的抑制。我们的研究表明，TIP 27是一个有效的抑制转录激活的TAK 1，因此，可能发挥了重要作用，在几个生理功能的调节TAK 1。TAK 1基因敲除小鼠的产生揭示了目前正在研究的几种表型。

I. RORgamma: The retinoid-related orphan receptor g (RORg) is a member of the nuclear receptor superfamily. To identify the physiological functions of RORg, mice deficient in RORg function were generated by targeted disruption. RORg -/- mice lack peripheral and mesenteric lymph nodes and Peyer?s patches indicating that RORg expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, while the number of circulating B cells is normal. The thymus of RORg-/- mice contains 74.4+ 8.9% fewer thymocytes than that of wt mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. TUNEL staining demonstrated a four-fold increase in apoptotic cells in the cortex of the thymus of RORg-/- mice. This was supported by the observed increase in annexin V-positive cells. RORg-/- thymocytes placed in culture exhibit a dramatic increase in the rate of "spontaneous" apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may at least in part be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a six-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORg-/- mice. Our observations indicate that RORg is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Recently, we found that RORg-/- mice are highly susceptible to the development of T-cells lymphomas. Altered expression of ROR leads to deregulation of the differentiation of thymocytes, increased proliferation of double negative thymocytes, which in turn increases the frequency of tumor formation. Recent studies have demonstrated that ROR receptors play a regulatory role in various inflammatory respeonses. RORa mice are more susceptible to endotoxin-induced inflammation. Microarray analysis demonstarted a role for RORs in steroid and drug metabolism. II. TAK1: The nuclear orphan receptor TAK1 functions as a positive as well as a negative regulator of transcription; however little is know about factors mediating its activity. Yeast two-hybrid analysis using the ligand binding domain of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27. TIP27 is a 27 kD nuclear protein that contains two zinc finger motifs. Confocal microscopy using TIP27 and several deletion mutants showed that TIP27 localized to the nucleus and that the carboxyl terminus containing a putative nuclear localization signal is important for its nuclear localization. TIP27 mRNA is expressed in several adult tissues but is most highly expressed in testis where it is induced at a specific stage of spermatogenesis. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. Deletion mutation analysis determined that the region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1. Moreover, it demonstrated that the TAK1-LBD from H3 till the carboxyl terminus is required for optimal interaction with TIP27. Pull-down assays demonstrated that TIP27 physically interacts with TAK1 and supported the importance of the TID. TIP27 is a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve inhibition of TAK1 homodimerization or DR1 binding but appears to due to an inhibition of the recruitment of co-activators. Our studies indicate that TIP27 is an effective repressor of transcriptional activation by TAK1 and, therefore, may play a critical role in the regulation of several physiological functions by TAK1. Generation of TAK1 knockout mice revealed several phenotypes that are currently being investigated.