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，外源生物不会直接与CAR结合来激活它。我们在破译这种间接激活机制方面取得了重大突破，这在过去10年里一直是一个谜。根据先前发现的蛋白磷酸酶2A抑制剂冈田酸抑制苯巴比妥激活CAR的结果，我们一直在研究CAR通过磷酸化失活的假设。人CAR的苏氨酸38(小鼠CAR中的苏氨酸48)现在被认为是PKC介导的磷酸化的主要部位，它使CAR失活并将其隔离在细胞质中。使用抗磷肽抗体的免疫组织化学显示，苯巴比妥使苏氨酸38去磷酸化以激活CAR并将其移位到细胞核中。因此，苏氨酸38的去磷酸化似乎是CAR间接激活的主要机制。这一发现目前正在进行修订，准备发表在《生物化学杂志》上。 异源生物信号串扰机制：在被外源生物激活后，CAR对基因的调控是不同的，赋予了CAR调控的基因表达的特异性。CAR通过与这些细胞信号的串扰获得这种特异性：早期反应、生长和压力信号。我们已经确定早期生长反应1(Egr1)是CAR激活CYP2B基因的关键因子。EGR L与多个位点结合，使CYP2B6启动子循环，使远端的CAR与近端的HNF4相互作用。我们还将早期反应因子GADD45b(生长停滞和DNA损伤诱导的45bw)表征为一种新的CAR共激活剂。 CAR介导性疾病的靶基因：已知激活CAR和/或PXR的药物(如利福平、苯巴比妥钠和苯妥英)的慢性治疗会导致骨骼疾病。研究发现，CAR和PXR都能与CyP24A启动子内的SMRT-VDR-VDRE相互作用，防止共抑制物SMRT从VDR上解离，抑制VDR依赖的激活。然而，在小鼠中，骨骼疾病与CYP24A基因的PXR调节之间没有这种联系。相反，我们发现PXR-/-小鼠出现低磷血症和骨密度丧失。Na/PI协同转运蛋白SLC34A2在PXR-/-小鼠的肠道中表达严重下调，可能导致低磷酸盐血症。这项低磷酸盐酶研究目前正在修订，将在《药物遗传学》和《基因组学》上发表。

CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We have made the major breakthrough into deciphering this indirect activation mechanism, which has been a mystery for the past 10 years. Taking the previous finding that the protein phosphatase 2A inhibitor okadaic acid represses CAR activation by phenobarbital, we have been working on the hypothesis that CAR is inactivated by phosphorylation. Threonine 38 of human CAR (threonine 48 in mouse CAR) has now been identified as the primary site of PKC-mediated phosphorylation that inactivates CAR and sequesters it in the cytoplasm. Immuohistochemistry using an anti-phosphopeptide antibody showed that phenobarbital dephosphorylates threonine 38 to activate CAR and translocate it into the nucleus. Dephosphorylation of threonine 38, therefore, appears to be the primary mechanism for the indirect activation of CAR. This finding is currently revised for publication in Journal of Biological Chemistry. 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 these cellular signals: early response, growth and stress signals. We have identified early growth response 1 (EGR1) as the essential factor for CAR to activate the CYP2B gene. EGR l binds to multiple sites, looping the CYP2B6 promoter and enabling the distal CAR to interact with the proximal HNF4. We have also characterized the early response factor GADD45b (growth arrest and DNA-damage inducible 45bw as a new CAR co-activator. Target genes for CAR-mediated diseases: Chronic treatment with drugs (e.g. rifampicin, phenobarbtal and phenytoin) that activate CAR and/or PXR is known to cause bone diseases. Both CAR and PXR are found to interact with SMRT-VDR-VDRE within the CYP24A promoter, preventing dissociation of the co-repressor SMRT from the VDR and repressing VDR-dependent activation. In mouse, however, there is no such association between bone disease to the PXR-regulation of the CYP24A gene. Instead, we found that Pxr-/- mice develop hypophosphatemia and loss of bone mineral density. The Na/Pi-cotransporter SLC34A2 is severely down-regulated in the intestine of Pxr-/- mice, possibly causing hypophosphatemina. This hypophosphatemina study is currently revised for publication in Pharmacogenetics and Genomics.