Nuclear receptors: action, functions, and roles in disea

视黄醇相关孤儿受体g(RORg)是核受体超家族的一员。为了确定RORg的生理功能，通过靶向干扰产生了RORg功能缺陷的小鼠。RORg-/-小鼠缺乏外周和肠系膜淋巴结以及Peyer？S斑块，提示RORg的表达是淋巴器官发生所必需的。虽然脾增大了，但它的结构正常。外周血中CD3+和CD4+淋巴细胞的数量分别减少6倍和10倍，而循环中的B细胞数量正常。RORg-/-小鼠胸腺中的胸腺细胞比wt小鼠少74.4±8.9%。流式细胞仪分析显示CD4+CD8+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四倍。观察到膜联蛋白V阳性细胞的增加支持了这一点。培养中的RORg-/-胸腺细胞表现出“自发”凋亡率的显著增加。这种增加在很大程度上与CD4+CD8+胸腺细胞有关，至少部分可能与抗凋亡基因Bcl-xl的表达水平大幅下降有关。流式细胞仪分析显示，RORg-/-小鼠胸腺细胞中处于细胞周期S期的细胞百分比增加了6倍。我们的观察表明，RORg对淋巴器官的发生是必不可少的，并在胸腺生成中起着重要的调节作用。最近，我们发现RORg-/-小鼠对T细胞淋巴瘤的发生高度易感。ROR的表达改变导致胸腺细胞分化的失控，增加双阴性胸腺细胞的增殖，进而增加肿瘤形成的频率。最近的研究表明，ROR受体在多种炎症反应中起着调节作用。RORA小鼠对内毒素诱导的炎症更敏感。微阵列分析证明了Rors在类固醇和药物新陈代谢中的作用。 2.TAK1：核孤儿受体TAK1既是转录的正性调节因子，也是负性转录调节因子，但对影响其活性的因素知之甚少。酵母双杂交分析以TAK1的配体结合域为诱饵，鉴定出一种新的TAK1相互作用蛋白，称为TIP27。TIP27是一个27kD的核蛋白，含有两个锌指基序。用TIP27和几个缺失突变体进行的共聚焦显微镜观察表明，TIP27定位于细胞核，并且含有可能的核定位信号的羧基末端对其核定位很重要。TIP27mRNA在几个成体组织中都有表达，但在精子发生的特定阶段被诱导的睾丸中表达最高。哺乳动物双杂交分析表明，TIP27与TAK1特异地相互作用，而不与其他几种核受体相互作用。缺失突变分析表明，TIP27的Asp39和Lys79之间的区域，称为TAK1-相互作用域(TID)，是其与TAK1相互作用的关键。此外，研究还表明，与TIP27的最佳相互作用需要从H3到羧基末端的TAK1-LBD。下拉分析表明TIP27与TAK1在物理上相互作用，并支持TID的重要性。TIP27是TAK1依赖于DR1的转录激活的强抑制因子。这种抑制不涉及对TAK1同源二聚或DR1结合的抑制，但似乎是由于抑制了共激活子的招募。我们的研究表明，TIP27是TAK1转录激活的有效抑制因子，因此可能在TAK1对多种生理功能的调节中发挥关键作用。一代TAK1基因敲除小鼠揭示了目前正在研究的几种表型。核蛋白RAP80与雌激素受体α以激动剂依赖的方式相互作用。此外，RAP80还与DNA修复有关。RAP80在肿瘤发生中的作用正在研究中。

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. The nuclear protein RAP80 was shown to interact with the estrogen receptor alpha in an agonist dependent manner. In addition, RAP80 was implicated in DNA repair. The role of RAP80 in tumorigenesis is being investigated.