Mechanisms and biological consequences of the nuclear receptor CAR activation

CAR激活机制：这个系统的独特之处在于，外源性物质不直接与CAR结合以激活它。我们先前确定内源性CAR的苏氨酸48在小鼠原代肝细胞中被磷酸化，苯巴比妥处理使该苏氨酸去磷酸化，激活CAR并将其易位到细胞核中。我们现在已经鉴定了蛋白磷酸酶2A作为使CAR的苏氨酸48去磷酸化的酶。此外，活化的C-激酶1受体（receptor for activated C-kinase 1，RACK 1）被表征为激活PP 2A核心酶以使CAR的苏氨酸48去磷酸化的必需调节亚基。我们还将生长因子-MEK 1/2-ERK 1/2途径定义为抑制苏氨酸48去磷酸化的细胞信号传导机制，从而抑制CAR活化。活化的P-ERK 1/2直接与磷酸化的CAR相互作用，并阻止PP 2A与CAR相互作用并使苏氨酸48去磷酸化。苯巴比妥处理导致P-ERK 1/2失活，并使PP 2A能够使小鼠原代肝细胞中的苏氨酸48去磷酸化。我们的研究已经将这种去磷酸化定义为CAR激活和功能的主要调节剂。 外源性信号串扰机制：在被外源性物质激活后，CAR调节基因彼此不同，赋予CAR调节的基因表达特异性。CAR通过与细胞信号传导的串扰获得这种特异性。我们已经鉴定了多种内源性细胞信号作为CAR激活和功能的重要调节因子：垂体因子、p38信号传导、SGK 2信号传导和早期反应因子GADD 45（生长停滞和DNA损伤诱导45。鉴于这些发现，我们正在研究这些信号调节CAR激活和功能的分子机制。 CAR介导的疾病：已知用药物（例如苯巴比妥）的慢性治疗会激活CAR并在啮齿动物中引起肝细胞癌（HCC）。我们现在已经将GADD 45表征为苯巴比妥促进HCC发展的CAR靶点：CAR与GADD 45蛋白相互作用，这种相互作用抑制JNK 1的磷酸化，从而抑制细胞凋亡并可能促进肿瘤发生。已知3，5-二乙氧羰基-1，4-二氢可力丁（DDC）治疗可引起严重的肝损伤并使出生后肝祖卵圆细胞增殖。利用CAR KO小鼠，我们已经确定DDC激活CAR，并且这种激活对于肝损伤和卵圆细胞增殖的发展至关重要。

CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We previously determined that threonine 48 of endogenous CAR is phosphorylated in mouse primary hepatocytes and that phenobarbital treatment de-phosphorylates this threonine, activating CAR and translocating it into the nucleus. We have now identified protein phosphatase 2A as the enzyme that de-phosphorylates threonine 48 of CAR. Moreover, receptor for activated C-kinase1 (RACK1) was characterized as the essential regulatory subunit that activates the PP2A core enzyme to de-phosphorylate threonine 48 of CAR. We have also defined the growth factor-MEK1/2-ERK1/2 pathway as a cell signaling mechanism that represses de-phosphorylation of threonine 48, thus repressing CAR activation. Activated P-ERK1/2 directly interacts with phosphorylated CAR and prevents PP2A from interacting with CAR and de-phosphorylating threonine 48. Phenobarbital treatment results in inactivation of P-ERK1/2 and enables PP2A to de-phosphorylate threonine 48 in mouse primary hepatocytes. Our investigations have defined this de-phosphorylation as the principle regulator of CAR activation and functions. Xenobiotic-signal crosstalk mechanism: Upon activation by xenobiotics, CAR regulates genes differently from one another, conferring specificity to CAR-regulated gene expression. CAR acquires this specificity via crosstalk with cell signaling. We have identified various endogenous cell signals as the essential regulator of CAR activation and function: pituitary factor, p38 signaling, SGK2 signaling and the early response factor GADD45 (growth arrest and DNA-damage inducible 45. Given these findings, we are investigating the molecular mechanisms by which these signaling regulate CAR activation and functions. CAR-mediated diseases: Chronic treatment with drugs, such as phenobarbital, is known to activate CAR and cause hepatocellular carcinoma (HCC) in rodents. We have now characterized GADD45 as a CAR target for phenobarbital promotion of HCC development: CAR interacts with GADD45 protein and this interaction inhibits phosphorylation of JNK1, thus repressing apoptosis and possibly promoting tumor genesis. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment is known to cause severe liver injury and proliferate postnatal hepatic progenitor oval cells. Utilizing CAR KO mice, we have determined that DDC activates CAR and this activation is essential for the developments of liver injury and oval cell proliferation.