The Steroid/Thyroid Hormone Receptor Superfamily

Nurr1是一种转录因子，是核激素受体超家族的成员。为了研究Nurr1的生理作用，我们之前培养了Nurr1基因零突变的小鼠。随后的分析显示，新生幼鼠中央多巴胺能区缺乏神经递质多巴胺、多巴胺生物合成酶、转运体和多巴胺利用受体。我们还表明，神经上皮细胞进行正常的腹化和迁移。这些多巴胺能神经元前体表达一般的神经元标记，如神经元核标记，并投射到纹状体。TUNEL实验显示，Nurr1-null小鼠和野生型小鼠的凋亡细胞数量没有差异。因此，我们的研究结果表明，转录因子Nurr1是中脑多巴胺能神经元终末成熟所必需的，而在缺乏Nurr1基因功能的小鼠出生时，多巴胺能前体的发生、存活和特定靶神经的选择不受影响。为了研究Nurr1的作用，我们建立了一种原代神经元培养方法。最近，我们利用新生儿Nurr1基因敲除的中脑神经元原代培养物，发现在forskolin存在的情况下，神经元群体可以被诱导表达酪氨酸羟化酶（多巴胺生物合成的关键酶），当脑源性神经营养因子和多巴胺联合使用时，酪氨酸羟化酶表达神经元的数量会协同增加。这些数据表明，来自Nurr1基因敲除幼崽的中脑神经元保留了诱导酪氨酸羟化酶表达的能力，即使体内酪氨酸羟化酶表达缺失。因此，福斯克林、脑源性神经营养因子和多巴胺等因子通过独立于Nurr1的途径诱导酪氨酸羟化酶的表达。重组腺病毒为基因表达研究提供了一个通用的系统。利用野生型和突变型Nurr1序列生成重组腺病毒。利用重组腺病毒-Nurr1在PC12细胞中鉴定Nurr1靶基因。这些细胞来源于神经元并合成多巴胺。此外，在这些细胞中，Nurr1的内源性表达可以被多种因素诱导，这表明Nurr1在这些细胞中具有生理作用。用重组病毒感染PC12细胞，然后进行流式激活细胞分选分析，得到同质的感染细胞群。二维凝胶分析显示重组野生Nurr1病毒的存在影响了几个蛋白点。将尝试用质分光光度法鉴定这些斑点的蛋白质组成。结果将提供Nurr1靶基因的信息。为了进一步研究类固醇/甲状腺激素受体超家族的作用机制，我们利用酵母双杂交系统和甲状腺激素受体作为诱饵，从大鼠胚胎脑库中分离出一种新的辅助因子。序列分析显示存在两个富含亮氨酸的基序，这是核受体的辅助因子的特征。该辅助因子在大鼠睾丸精原细胞、间质细胞和海马中高度表达。体外结合实验显示亮氨酸基序的点突变干扰雌激素受体的相互作用。因此，该辅因子抑制了报告基因的雌激素诱导性。这个辅因子与超家族其他成员的相互作用正在调查中。”

Nurr1 is a transcription factor, a member of the superfamily of nuclear hormone receptors. To investigate the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Subsequent analysis revealed the absence of neurotransmitter dopamine, dopamine biosynthesizing enzymes, transporters and receptors for dopamine utilization in the central dopaminergic area of newborn pups. We have also shown that the neuroepithelial cells undergo normal ventralization and migration. These dopaminergic neuron precursors express general neuronal markers such as a neuronal nuclear marker and project to the striatum. The TUNEL assay showed no difference in the number of apoptotic cells between Nurr1-null and wild type mice. Hence, our results show that the transcription factor Nurr1 is required for terminal maturation of mesencephalic dopaminergic neurons, while genesis of dopaminergic precursors, their survival, and selection of specific target innervation are not affected at the time of birth in mice lacking Nurr1 gene function. In order to investigate the role of Nurr1, a procedure for primary neuronal cultures was established. Recently, using primary midbrain neuronal cultures from newborn Nurr1 knockouts, we have shown that a population of neurons could be induced to express tyrosine hydroxylase, a key enzyme in dopamine biosynthesis, in the presence of forskolin with a synergistic increase in the number of tyrosine hydroxylase expression neurons when combined with brain-derived neurotrophic factor and dopamine. These data indicate that midbrain neurons from Nurr1 knockout pups retain the capacity for the induced expression of tyrosine hydroxylase even though in vivo tyrosine hydroylase expression is absent. Thus, the factors, such as forskolin, brain-derived neurotrophic factor, and dopamine induce tyrosine hydroxylase expression via a pathway independent of Nurr1.Recombinant adenoviruses provide a versatile system for gene expression studies. The Nurr1 sequence, wild type and mutated, were used to generate recombinant adenoviruses. Recombinant adenovirus-Nurr1 was employed to identify the Nurr1 target genes in PC12 cells. These cells are of neuronal origin and synthesize dopamine. In addition in these cells endogenous expression of Nurr1 can be induced by several factors suggesting a physiological role of Nurr1 in these cells. The PC12 cells were infected with recombinant virus, followed by flow-activated cell sorting analysis, resulting in a homogenous population of infected cells. Two dimensional gel analyses revealed several protein spots affected by the presence of recombinant wild Nurr1 virus. An attempt will be made to identify the protein composition of these spots using mass spectrophotometry. The results should provide information on Nurr1 target genes. Continuing to investigate the mechanism(s) of action of the steroid/thyroid hormone receptor superfamily, we isolated a novel cofactor from a rat embryonic brain library using a yeast two hybrid system and a thyroid hormone receptor as bait. Sequence analysis revealed the presence of two leucine-rich motifs, a signature of cofactors of the nuclear receptors. This cofactor is highly expressed in rat testis in the spermatogonia and Leydig cells and in the hippocampus. In vitro binding assays revealed that a point mutation in the leucine motif interferes with estrogen receptor interaction. Consequently, this cofactor represses estrogen inducibility of the reporter gene. Interaction of this cofactor with other members of the superfamily is under investigation. "