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Cytosolic sulfotransferase (SULT)-mediated sulfation plays an essential role in the detoxification of bile acids and is necessary to avoid pathological conditions, such as cholestasis, liver damage, and colon cancer. In this study, using transgenic mice bearing conditional expression of the activated constitutive androstane receptor (CAR), we demonstrate that activation of CAR is both necessary and sufficient to confer resistance to the hepatotoxicity of lithocholic acid (LCA). Surprisingly, the CARmediated protection is not attributable to the expected and previously characterized CYP3A pathway; rather, it is associated with a robust induction of SULT gene expression and increased LCA sulfation. We have also provided direct evidence that CAR regulates SULT expression by binding to the CAR response elements found within the SULT gene promoters. Interestingly, activation of CAR was also associated with an increased expression of the 3Ј-phosphoadenosine 5Ј-phosphosulfate synthetase 2 (PAPSS2), an enzyme responsible for generating the sulfate donor 3Ј-phosphoadenosine-5Ј-phosphosulfate. Analysis of gene knockout mice revealed that CAR is also indispensable for ligand-dependent activation of SULT and PAPSS2 in vivo. Therefore, we establish an essential and unique role of CAR in controlling the mammalian sulfation system and its implication in the detoxification of bile acids.

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.

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