Post translational modifications of the GR can alter

Post-translational modifications of the GR can alter the transcriptional effects of GR activation [31], and may provide a mechanism for the interactions between cell signaling pathways and GR-mediated transcriptional activity. Dex-BSA treatment reportedly increased p38 MAPK activation in CD14-positive monocytes [19], and both pharmacological and siRNA-mediated inhibition of p38 MAPK increased GR nuclear localization [32], presenting the possibility that Dex-BSA-induced p38 activation could inhibit GR nuclear localization Consistent with this, GR phosphorylated at serine 232, a putative p38 MAPK site, was less transcriptionally active in rat primary cortical neuron cultures [33]. However, in leukemia cell lines [34] and in osteosarcoma and lung cancer cell lines [35], p38 MAPK phosphorylation of the GR at serine 211 (pGR S211, which is orthologous to serine 232 in the rat) increased GRE-mediated transcription, suggesting that the effects of phosphorylation are cell-type specific [36]. We found that Akt activation inhibited the nuclear translocation of GR induced by Dex-BSA, but not by Dex. Akt is part of the PI3 kinase signaling pathway, which can be activated at the plasma membrane by receptor tyrosine kinases and G-protein coupled receptors that respond to growth factors and cytokines [37]. Rapid Akt phosphorylation in response to glucocorticoids has been reported in human epithelial AICAR phosphate in the presence of a transcription inhibitor [38]. We were not able to detect any change in Akt phosphorylation in response to Dex or Dex-BSA treatment or an effect of Akt activation on Dex-mediated GR nuclear localization, which suggests that membrane glucocorticoid signaling may be regulated by the intracellular environment. Under conditions in which the PI3K pathway is active, such as in response to insulin exposure [39], hypothalamic neurons may respond differently to glucocorticoids. The identity of the membrane receptor responsible for signaling to the GR is not known. GR immunoreactivity has been detected in the membrane in lymphoma [40] and breast cancer (MCF-7) cell lines [41], in transfected HEK 293T cells [19], in pituitary cell lines [26], and in native hypothalamic neuroendocrine cells [42], suggesting that the GR itself, or an isoform of the GR, may be the membrane receptor. The GR has also been detected in neuronal membranes in some studies using immunocytochemistry and electron microscopy [43], [44], while other studies have suggested that standard immunocytochemical methods are not sensitive enough to detect low levels of GR in the plasma membrane [41]. Glucocorticoids bind to neuronal membranes with ∼10-fold lower affinity than in the cytosol, suggesting that the membrane receptor is significantly lower in abundance than the cytosolic receptor [42], [45], which may account for the difficulty of detection of the GR in the membrane. On the other hand, rapid actions via a membrane receptor have been shown in several, though not all, studies to have a different pharmacology than the intracellular GR (i.e., they are insensitive to the GR antagonist RU486) [24], [27] and to be blocked by inhibiting G protein activity [25], which suggests that the membrane receptor may be distinct from the classical GR. In conclusion, membrane-restricted Dex-BSA induced the nuclear translocation of GR in hypothalamic neurons to a degree that was comparable to that elicited by Dex, albeit with slower kinetics. Because free Dex does not dissociate from the Dex-BSA [46], this suggests that glucocorticoid signaling from the membrane is capable of inducing the nuclear translocation of the unliganded GR. The molecular mechanism for this membrane-initiated trafficking of the unliganded GR is not known, although it appears to be independent of PKA, Akt, MEK, p38 MAPK, Src and calcium signaling, and it is blocked by Akt activation. Nuclear translocation is part of the classical GR signaling pathway, and is necessary for GRE regulation of transcription; however, we demonstrate here that non-classical glucocorticoid signaling via a membrane GR induces the nuclear translocation of an unliganded GR and subsequent transcriptional regulation that appears to be independent of activation of the GRE. This divergence in the transcriptional signaling of the unliganded and liganded GR may represent a mechanism for finely controlling transcriptional output by glucocorticoids under different physiological and/or cellular conditions.