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    • The rapid actions of glucocorticoids range from the producti

    2022-08-05

    The rapid actions of glucocorticoids range from the production of endocannabinoid as a retrograde messenger [9], to regulation of ion channels [10], [11], to inhibitory effects on immune apelin receptor [12], [13], [14]. Many different kinase signaling pathways have been implicated in the rapid actions of glucocorticoids mediated by putative membrane glucocorticoid receptors, including ERK-MAPK [9], SRC [15], protein kinase A [9], [16], [17], and protein kinase C [18]. Here, we compared the membrane-impermeant glucocorticoid conjugate dexamethasone-bovine serum albumin (Dex-BSA) with the membrane-permeant synthetic glucocorticoid dexamethasone (Dex) in its capacity to stimulate the nuclear translocation of GR and to induce transcriptional activity in hypothalamic cells in vitro and in vivo. We analyzed the Dex-BSA conjugate for its stability and purity in solution in a companion paper [46].
    Experimental
    Results
    Discussion The main finding of this study is that glucocorticoid signaling from the membrane can induce the nuclear translocation of the GR, which suggests trafficking of an unliganded GR that is activated by glucocorticoid binding to a membrane-associated receptor. Corticosterone also caused GR nuclear translocation, indicating that the synthetic steroids acted in a similar manner as the endogenous steroid. The similar response observed in primary hypothalamic cell cultures in vitro and following steroid injection into the PVN in vivo provided evidence that the Dex- and Dex-BSA-induced nuclear translocation of GR observed in the N42 hypothalamic cell line also occurs in native hypothalamic neurons. The Dex-BSA-induced GR translocation was slower than the Dex-induced GR translocation, which could provide insight into mechanisms of unliganded GR trafficking. For example, time might be required following glucocorticoid binding to the membrane for the GR to move to the membrane [26] to be exposed to the steroid signal. Thus, the GR may need to be trafficked to the membrane in order to receive a membrane-restricted signal, either from the ligand directly or from a nearby membrane receptor. Although not studied directly, we saw no evidence for trafficking of the GR to the membrane following glucocorticoid application. The Dex group of the Dex-BSA conjugate is not long enough to protrude through the plasma membrane lipid bilayer, so it would be necessary for some portion of the GR to enter the membrane in order for Dex-BSA to directly interact with the GR. Alternatively, Dex-BSA could bind to an unidentified membrane receptor, which could then relay a signal to the GR to trigger translocation. Either process may be slower than free Dex diffusing into the cell and binding to the GR. Another possible explanation for the difference in kinetics between the two steroids is that the number of Dex molecules in the Dex-BSA conjugate available to bind to the membrane receptor (the Dex-to-BSA ratio in the Dex-BSA conjugate was 37-to-1) was fewer than in the 1-μM Dex solution, which may have slowed the binding reaction. The trafficking of an unliganded GR by glucocorticoid actions at the membrane implicates the activation of a membrane receptor-initiated signaling mechanism. We found previously in ex-vivo brain slices that rapid glucocorticoid actions in hypothalamic neurons and neurons of the basolateral amygdala are dependent on G protein-coupled receptor and protein kinase activation [24], [25], [9], [27]. Other studies have demonstrated that glucocorticoids can rapidly activate multiple kinase pathways in cultured cells, including both serine/threonine kinase and tyrosine kinase signaling pathways [28]. For example, glucocorticoids activate ERK 1/2 within 1 h in an anterior pituitary cell line [29], and rapidly inhibit calcium influx into HT4 and PC12 cells via PKA and PKC activation, respectively [16]. In primary hippocampal neurons, glucocorticoids activate JNK and p38 MAP kinases [18], and inhibition of GR results in the reduction of Akt and ERK phosphorylation within 30 min [30]. In contrast to studies of other forms of rapid glucocorticoid signaling that showed kinase dependence (23–28), we did not find that the GR nuclear translocation induced by membrane glucocorticoid signaling was dependent on, or was activated by, any of a battery of kinases. It is possible that we missed inhibitory or activational effects of drugs tested at a single concentration, however, the concentrations of the kinase activators and inhibitors that we employed in these experiments have been demonstrated to be effective in other studies. The lack of kinase involvement in GR nuclear translocation suggests that either the membrane receptor or the signaling cascades engaged by the membrane receptor may be different for distinct rapid glucocorticoid actions.