Transgenic mice lacking CREB and CREM in noradrenergic and serotonergic neurons respond differently to common antidepressants on tail suspension test.

Evidence exists that chronic antidepressant therapy enhances CREB levels and activity. Nevertheless, the data are not conclusive, as previous analysis of transgenic mouse models has suggested that CREB inactivation in fact contributes to antidepressant-like behavior. The aim of this study was to evaluate the role of CREB in this context by exploiting novel transgenic mouse models, characterized by selective ablation of CREB restricted to noradrenergic (Creb1DBHCre/Crem-/-) or serotonergic (Creb1TPH2CreERT2/Crem-/-) neurons in a CREM-deficient background to avoid possible compensatory effects of CREM. Selective and functional ablation of CREB affected antidepressant-like behavior in a tail suspension test (TST) after antidepressant treatment. Contrary to single Creb1DBHCre mutants, Creb1DBHCre/Crem-/- mice did not respond to acute desipramine administration (20 mg/kg) on the TST. On the other hand, single Creb1TPH2CreERT2 mutants displayed reduced responses to fluoxetine (10 mg/kg) on the TST, while the effects in Creb1TPH2CreERT2/Crem-/- mice differed by gender. Our results provide further evidence for the important role of CREM as a compensatory factor. Additionally, the results indicate that new models based on the functional ablation of CREB in select neuronal populations may represent a valuable tool for investigating the role of CREB in the mechanism of antidepressant therapy.

A negative feedback loop of ICER and NF-κB regulates TLR signaling in innate immune responses.

The NF-κB pathway has important roles in innate immune responses and its regulation is critical to maintain immune homeostasis. Here, we report a newly discovered feedback mechanism for the regulation of this pathway by TLR ligands in macrophages. Lipopolysaccharide (LPS) induced the expression of ICER via p38-mediated activation of CREB in macrophages. ICER, in turn, inhibited the transcriptional activity of NF-κB by direct interaction with the p65 subunit of NF-κB. Deficiency in ICER elevated binding of NF-κB to promoters of pro-inflammatory genes and their subsequent gene expression. Mice deficient in ICER were hypersensitive to LPS-induced endotoxic shock and showed propagated inflammation. Whereas ICER expression in ICER KO bone marrow transplanted mice rescued the ultra-inflammation phenotype, expression of a p65 binding-deficient ICER mutant failed to do so. Our results thus establish p38-CREB-ICER as key components of a negative feedback mechanism necessary to regulate TLR-driven inflammation.

Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis.

Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.

The role of cAMP response element-binding protein in estrogen negative feedback control of gonadotropin-releasing hormone neurons.

The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.

The CREB/CREM transcription factors negatively regulate early synaptogenesis and spontaneous network activity.

The family of CREB (cAMP response element-binding protein) transcription factors are involved in a variety of biological processes including the development and plasticity of the nervous system. In the maturing and adult brain, CREB genes are required for activity-dependent processes, including synaptogenesis, refinement of connections and long-term potentiation. Here, we use CREB1(Nescre)CREM(-/-) (cAMP-responsive element modulator) mutants to investigate the role of these genes in stimulus-independent patterns of neural activity at early stages. We show that lack of CREB/CREM genes specifically in neural tissue leads to increased synaptogenesis and to a dramatic increase in the levels of spontaneous network activity at embryonic stages. Thus, the functions of CREB/CREM genes in neural activity differ in distinct periods of neural development.

CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis.

Induction of specific gene expression patterns in response to activity confers functional plasticity to neurons. A principal role in the regulation of these processes has been ascribed to the cAMP responsive element binding protein (CREB). Using genome-wide expression profiling in mice lacking CREB in the forebrain, accompanied by deletion of the cAMP responsive element modulator gene (CREM), we here show that the role of these proteins in activity-induced gene expression is surprisingly selective and highly context dependent. Thus, only a very restricted subset of activity-induced genes (i.e., Gadd45b or Nr4a2) requires these proteins for their induction in the hippocampus after kainic acid administration, while they are required for most of the cocaine-induced expression changes in the striatum. Interestingly, in the absence of CREB, CREM is able to rescue activity-regulated transcription, which strengthens the notion of overlapping functions of the two proteins. In addition, we show that cholesterol metabolism is dysregulated in the brains of mutant mice, as reflected coordinated expression changes in genes involved in cholesterol synthesis and neuronal accumulation of cholesterol. These findings provide novel insights into the role of CREB and CREM in stimulus-dependent transcription and neuronal homeostasis.

CREM deficiency in mice alters the response of bone to intermittent parathyroid hormone treatment.

CREM belongs to the ATF/CREB family of basic leucine zipper transcription factors. We previously showed that PTH induces ICER (inducible cAMP early repressor) in osteoblasts. ICER proteins, which are transcribed from the P2 promoter of the Crem gene, act as transcriptional attenuators. The objective of this study was to determine whether the Crem gene plays a role in the response of bone to intermittent PTH. Adult Crem knockout (KO) and wild type (WT) male mice were given daily subcutaneous injections of vehicle or hPTH(1-34) (160 mug/kg) for 10 days. Bone mineral content and density (BMC and BMD, respectively) were measured in femur and tibia by dual energy X-ray absorptiometry (DEXA). Bone morphometry was analyzed by X-ray computed microtomography (microCT) and histomorphometry. Serum bone turnover markers were measured. In vitro osteoclast formation assays were performed in bone marrow cultures treated with PTH or the combination of RANKL and M-CSF. KO mice had slightly higher basal bone mass than wild type mice. PTH treatment increased tibial BMC and BMD to a greater extent in WT mice compared to KO mice. PTH increased both cortical area and trabecular bone area in WT but not in KO femurs. PTH increased the bone formation rate and percent osteoblast surface to the same extent in femurs of WT and KO mice but increased osteoclast parameters and calvarial porosity to a greater extent in KO mice. PTH increased serum osteocalcin levels to the same extent in WT and KO mice. PTH-induced osteoclast formation was 2-fold greater in bone marrow cultures from KO mice. Collectively, our data suggest that the CREM deficiency in mice alters the response of bone to intermittent PTH treatment such that osteoclastogenesis is increased. Crem gene may specify the anabolic response to intermittent PTH treatment by restraining PTH-induced osteoclastogenesis.

Transcription factors, cAMP-responsive element modulator (CREM) and Tisp40, act in concert in postmeiotic transcriptional regulation.

We previously isolated 80 TISP (transcript induced in spermiogenesis) genes whose transcription is dramatically induced during spermiogenesis. Our analysis here of the expression of these genes in the testis of the cAMP-responsive element modulator (CREM)-null mouse revealed that 54 TISP genes are under the transcriptional regulation of CREM. One CREM-regulated gene is TISP40, which encodes a basic leucine zipper (bZip)-type transcription factor bearing a transmembrane domain that generates the two proteins Tisp40alpha and Tisp40beta. Both of these proteins function by binding to UPRE (unfolded protein-response element) but do not recognize CRE motifs. We show here that Tisp40alpha mRNA is generated under the direct transcriptional regulation of CREM. CREMtau and Tisp40 form a heterodimer, which functions through CRE but not through UPRE. Furthermore, binding ability of CREM to CRE is dramatically up-regulated by forming a heterodimer with Tisp40alphaDeltaTM, a truncated form of Tisp40alpha that lacks the transmembrane domain. We confirmed that Tisp40 and CREM actually bind to the Tisp40 promoter in vivo by chromatin immunoprecipitation assay. Finally, we demonstrate that the Tisp40DeltaTM-CREMtau heterodimer acts as a recruiter of HIRA, a histone chaperone, to CRE. Taken together, we propose that Tisp40 is an important transcriptional regulator during spermiogenesis.

CREM: a transcriptional master switch during the spermatogenesis differentiation program.

Spermatogenesis is a complex differentiation process under the hormonal control of the hypothalamic-pituitary axis. The CREM gene encodes activators and repressors of cAMP-mediated gene transcription. The transcript corresponding to the activator isoform CREMtau is found at high levels in pachytene spermatocytes onwards. The CREMtau protein, however, is present only in post-meiotic spermatids where it activates the transcription of testis-specific genes, such as the protamines and transition proteins. Mice in which the CREM gene has been inactivated by homologous recombination have been generated. Homozygous male mutant mice are sterile and produce no spermatozoa. Histological analysis of the seminiferous tubules reveal a complete arrest of spermatogenesis at the first step of spermiogenesis. CREM deficiency results in the lack of post-meiotic gene expression and a ten-fold increase in apoptotic germ cells. These results demonstrate the essential role of CREM in spermatogenesis and are reminiscent of some cases of male infertility.