β-arrestin-2 regulation of the cAMP response element binding protein

Previous work demonstrated that cystic fibrosis (CF) cells exhibit an increase in cAMP-mediated signaling as a characteristic response to lost CFTR function. Evidence for increased cAMP-mediated signaling in CF included increased phosphorylation of the cAMP response element binding protein (CREB) and elevated β-arrestin-2 (βarr2) expression. However, subsequent studies reveal that CREB activation in CF cells is independent of protein kinase-A (PKA). The goal of this study is to test the hypothesis that elevated βarr2 expression leads to increased CREB activation in a PKA-independent mechanism. βarr2-GFP expressing tracheal epithelial cells (βarr2-GFP) exhibit an increase of pCREB content and subsequent CRE activation compared to GFP expressing control cells. βarr2 activation of the ERK cascade represents a candidate mechanism leading to CREB activation. ERK exhibits increased activation in βarr2-GFP cells compared to cont-GFP cells, and ERK inhibition diminishes CRE activation in both GFP and βarr2-GFP cells. To test directly whether CREB regulation in CF is βarr2-dependent, nasal epithelium excised from wt mice (Cftr +/+; βarr2 +/+), CF mice (Cftr -/-; βarr2 +/+), and DKO mice (Cftr -/-; βarr2 -/-) were analyzed for pCREB protein content. Removal of βarr2 expression from CF mice reduces both pCREB and pERK content to wt levels. These data indicate that CF-related CREB regulation is mediated directly through βarr2 expression via the ERK pathway.     

Time-Course of Changes in Phosphorylated CREB in Neuroblasts and BDNF in the Mouse Dentate Gyrus at Early Postnatal Stages

Cyclic AMP (cAMP) response element-binding protein (CREB) is involved in memory, learning, and synaptic transmission. In this study, we observed changes of phosphorylated CREB (pCREB) immunoreactivity and its protein levels as well as brain-derived neurotrophic factor (BDNF) levels in the hippocampal dentate gyrus at postnatal (P) 1, 7, 14, and 21 in mice. In addition, we also investigated pCREB expression in doublecortin (DCX, a marker for neuronal progenitors) immunoreactive neuroblasts at P21. pCREB immunoreaction at P1 was detected in most of cells in the dentate gyrus, thereafter pCREB immunoreactivity was decreased in all the layers of the dentate gyrus with time, however, strong pCREB immunoreactivity was shown in cells confined to the subgranular zone of the dentate gyrus at P21. In this group, many pCREB immunoreactive cells were co-localized with DCX immunoreactive neuroblasts. In addition, pCREB protein levels were decreased with age, showing that their levels were very low at P21, while BDNF protein levels were increased with age. These results suggest that pCREB may play important roles in functional maturity of granule cells in mice.     

The effect of ICER on screening methods involving CRE-mediated reporter gene expression

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.     

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)₂cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.