BackgroundIt has been suggested that stress provokes neuropathological changes and may thus contribute to the precipitation of affective disorders such as depression. Likewise, the pharmacological therapy of depression requires chronic treatment and is thought to induce a positive neuronal adaptation, presumably based on changes in gene transcription. The transcription factor cAMP-responsive element binding protein (CREB) and its binding site (CRE) have been suggested to play a major role in both the development of depression and antidepressive therapy.Methodology/Principle FindingsTo investigate the impact of stress and antidepressant treatment on CRE/CREB transcriptional activity, we generated a transgenic mouse line in which expression of the luciferase reporter gene is controlled by four copies of CRE. In this transgene, luciferase enzyme activity and protein were detected throughout the brain, e.g., in the hippocampal formation. Chronic social stress significantly increased (by 45 to 120%) CRE/CREB-driven gene expression measured as luciferase activity in several brain regions. This was also reflected by increased CREB-phosphorylation determined by immunoblotting. Treatment of the stressed mice with the antidepressant imipramine normalized luciferase expression to control levels in all brain regions and likewise reduced CREB-phosphorylation. In non-stressed animals, chronic (21 d) but not acute (24 h) treatment with imipramine (2×10 mg/kg/d) reduced luciferase expression in the hippocampus by 40–50%.Conclusions/SignificanceOur results emphasize a role of CREB in stress-regulated gene expression and support the view that the therapeutic actions of antidepressants are mediated via CRE/CREB-directed transcription.

The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3beta (glycogen-synthase-kinase 3beta) were not involved as specific GSK3beta inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium.

The molecular mechanism of action of the mood stabilizer lithium is assumed to involve changes in gene expression leading to neuronal adaptation. The transcription factor CREB (cAMP-responsive element binding protein) regulates the expression of many genes and has been implicated in important brain functions and the action of psychogenic agents. We here investigated the effect of lithium on cAMPresponsive element (CRE)/CREB-mediated gene transcription in the brain, using transgenic reporter mice that express the luciferase reporter gene under the control of four copies of the rat somatostatin gene promoter CRE. Chronic (21 days) but not acute (24 h) treatment with lithium (7.5 mmol/kg) significantly decreased CRE/CREB-directed gene expression in hippocampus, cortex, hypothalamus, and striatum to 60-70%, and likewise reduced CREB phosphorylation. As bipolar disorder is also considered as a stress-related disorder, the effect of lithium was determined in mice submitted to a paradigm for chronic psychosocial stress. As shown before, stress for 25 days significantly increased CRE/CREB-directed gene expression in several brain regions by 100-150%. Treatment of stressed mice with lithium decreased stress-induced CRE/CREB-directed gene expression to control levels in nearly all brain regions and likewise reduced CREB phosphorylation. Chronic lithium treatment induced b-catenin accumulation and decreased cAMP levels, indicating an inhibitory effect of lithium on glycogen synthase kinase 3 and the adenylate cyclase/protein kinase A signalling cascade, which are known to modulate CREB activity. We here for the first time show that lithium regulates CRE/CREB-directed gene transcription in vivo and suggest CREB as a putative mediator of the neuronal adaptation after chronic lithium treatment.

Prostaglandin E 2 receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G q , EP2 and EP4 to G s , and EP3 to G i . The EP4-R, in contrast to the EP3␤-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G i -coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E 2 , or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAGepitope-tagged rat EP3␤-R (rEP3␤-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3␤-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E 2 stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time-and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAGrEP3␤-R. Agonist-induced phosphorylation of the FLAGhEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.Prostaglandin E 2 receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domains (Negishi, 1994). There are four subtypes of E-prostaglandin receptors (EP-Rs) that differ in their affinity to synthetic ligands and their G protein coupling specificity. EP1-Rs are linked to G q and increase inositol trisphosphate (InsP 3 ) and, hence, cytosolic Ca 2ϩ concentration. EP2-Rs and EP4-Rs are coupled to G s and increase intracellular cAMP. EP3-Rs are coupled to G i and decrease hormone-stimulated cyclic AMP (cAMP) formation ( Fig. 1) (Coleman, 1994). These receptors display an overall sequence homology of about 50%; the putative transmembrane domains are the most conserved (Coleman, 1994).