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De Novo Synthesis of Glucocorticoids ..
AB - Glucocorticoid hormones induce apoptosis in lymphoid cells. This process is transcriptionally regulated and requires de novo RNA/protein synthesis. However, the full spectrum of glucocorticoid-regulated genes mediating this cell death process is unknown. Through gene expression profiling we discovered that the expression of thioredoxin-intereacting protein (txnip) mRNA is significantly induced by the glucocorticoid hormone dexamethasone not only in the murine T-cell lymphoma line WEHI7.2, but also in normal mouse thymocytes. This result was confirmed by Northern blot analysis in multiple models of dexamethasone-induced apoptosis. The induction of txnip mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Deletion and mutation analysis of the txnip promoter identified a functional glucocorticoid response element in the txnip promoter. Reporter assays demonstrated that this glucocorticoid response element was necessary and sufficient for induction of txnip by dexamethasone. Expression of a GFP-TXNIP fusion protein was sufficient to induce apoptosis in WEHI7.2 cells, and repression of endogenous txnip by RNA interference inhibited dexamethasone-induced apoptosis in WEHI7.2 cells. Together, these findings indicate that txnip is a novel glucocorticoid-induced primary target gene involved in mediating glucocorticoid-induced apoptosis.
Glucocorticoid regulation of the hypothalamic-pituitary-adrenal (HPA) axis is believed to depend on multiple actions operative within discrete time domains. However, the underlying cellular and molecular mechanism for those glucocorticoid actions remain undetermined. Moreover, there is an absence of in vivo studies examining whether there are multiple glucocorticoid effects on HPA axis related function within an intermediate feedback time-frame (1–3 h after glucocorticoid elevation), and whether those effects depend on de novo protein synthesis. We examined in rats the effects of protein synthesis inhibition on HPA axis response to restraint (15min) after 1 and 3 h phasic corticosterone (CORT) pretreatment. We measured HPA axis hormones (ACTH and CORT) and gene expression in the paraventricular nucleus (c-fos and crh genes), as well as gene expression in the anterior and intermediate pituitary (c-fos and pomc genes). Both CORT pretreatment intervals produced inhibition of stress-induced ACTH secretion, but no inhibition was seen in the presence of protein synthesis inhibition. CORT pretreatment produced inhibitory effects on stress-induced gene expression that varied for each gene depending on the anatomical site, pretreatment time and protein synthesis dependency. Taken together, the ACTH and gene expression patterns support the presence of multiple independent glucocorticoid actions initiated during the intermediate glucocorticoid negative feedback phase. Moreover, we conclude that those effects are exerted predominantly on the intrinsic anatomical elements of the HPA axis, and some of those effects depend on CORT induction of the expression of one or more regulatory gene products.
De novo synthesis of steroids, ..
The goal of this study was to determine whether a phasic increase in corticosterone (CORT) comparable to a moderate stressor, would produce over the course of several hours (1–3 h) multiple glucocorticoid negative feedback effects. Specifically, we sought to determine if separate glucocorticoid effects could be identified by anatomical site of action, time-course and dependence on de novo protein synthesis. The key strategy that we adopted for these studies was to not only monitor effects of corticosterone (CORT) on stress-induced HPA axis hormone secretion, but also to monitor CORT effects on stress-induced gene expression. We and others have found that a number of genes are rapidly induced (within 15 min) by acute stress within the cellular elements of the HPA axis (; ; ). Some of those genes (e.g. c-fos) function as immediate early genes in a wide range of neuronal and endocrine cell populations. Other genes (e.g. crh gene and pro-opiomelanocortin, pomc, gene) are rapidly induced within a restricted population of cells. Rapid induction of the crh and pomc genes in the PVN and anterior pituitary, respectively, can be observed when measuring levels of their primary transcript (hnRNA) (; ; ).
Surprisingly, there has been very little advance in the subsequent 25 years in determination of the underlying molecular mechanisms by which glucocorticoids produce negative feedback regulation of the HPA axis. For example, it remains undetermined whether each of those phases of feedback function are operative at both the hypothalamic paraventricular nucleus (PVN) and anterior pituitary elements of the HPA axis (intrinsic negative feedback). Glucocorticoid negative feedback also appears to depend on glucocorticoid alteration of neural input to the PVN (extrinsic negative feedback), however the time-course for that influence has not been explored. Although the mechanism of slow feedback is believed to be largely due to glucocorticoid inhibition of corticotrophin releasing hormone (CRH) and adrenocorticotropin hormone (ACTH) production, the molecular mechanism(s) of intermediate feedback remain unknown (). Moreover, it remains to be determined whether intermediate feedback depends on a single or multiple glucocorticoid effects. The prospect of multiple glucocorticoid effects within the intermediate feedback time frame has been questioned based on in vitro study of corticotrophs (). There is also an absence of in vivo studies that clearly illustrate within the same study and experimental conditions the presence of separate dissociable glucocorticoid intermediate negative feedback effects. In addition, in vivo studies have not examined whether de novo protein synthesis is required for glucocorticoid inhibition within the intermediate feedback time frame. Because, intermediate glucocorticoid inhibitory effects are evident within 1 h after treatment (; ), establishing the requirement for de novo protein synthesis is warranted. Determination of the mechanisms of glucocorticoid negative feedback has clinical importance. There is considerable evidence for altered negative feedback function associated with a wide range of clinical disorders (e.g. depression, post traumatic stress disorder, type II diabetes, chronic fatigue syndrome, fibromyalgia and chronic facial pain) and associated precursor conditions (e.g. obesity and systemic hypertension) (; ; ; ; ; ; ; ).
Dysfunctional Skin-Derived Glucocorticoid Synthesis Is …
However, research during the last two decades has revealed that besides the adrenal cortex, other organs also possess the capability of de novo synthesis of biologically active GCs from the precursor substance cholesterol, and recent results suggest that these locally produced GCs mainly act in a paracrine or autocrine mode.
In this study we examined the effect of CORT pretreatment on stress-induced c-fos, crh and pomc gene expression, under normal conditions and in the presence of systemic protein synthesis blockade. Both crh and pomc genes are believed to be direct targets for glucocorticoid receptor (GR) mediated CORT repression (). On the other hand, the c-fos gene appears to not be directly repressed by CORT (; ), although its expression can be inhibited by long-term glucocorticoid treatment (). In previous studies we have found that 1 h glucocorticoid pretreatment is not sufficient to suppress subsequent restraint-induced c-fos mRNA in the PVN or anterior pituitary (; ). It appears then that crh, pomc and c-fos gene expression all reflect aspects of recent stress-induced HPA axis cellular excitation. However, c-fos gene expression may only reflect glucocorticoid actions as they alter stress-induced intercellular and intracellular signals that converge on the c-fos gene promoter. Thus, we may be able to determine the influence of phasic CORT on stress-induced excitatory input to the PVN (i.e. extrinsic feedback) by examining c-fos mRNA. In contrast, the expression of crh and pomc genes appear to integrate information about both the direct presence of activated glucocorticoid receptors (GR) and upstream signaling events.
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is maintained primarily by controlling the level of de novo synthesis
N2 - Glucocorticoid hormones induce apoptosis in lymphoid cells. This process is transcriptionally regulated and requires de novo RNA/protein synthesis. However, the full spectrum of glucocorticoid-regulated genes mediating this cell death process is unknown. Through gene expression profiling we discovered that the expression of thioredoxin-intereacting protein (txnip) mRNA is significantly induced by the glucocorticoid hormone dexamethasone not only in the murine T-cell lymphoma line WEHI7.2, but also in normal mouse thymocytes. This result was confirmed by Northern blot analysis in multiple models of dexamethasone-induced apoptosis. The induction of txnip mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Deletion and mutation analysis of the txnip promoter identified a functional glucocorticoid response element in the txnip promoter. Reporter assays demonstrated that this glucocorticoid response element was necessary and sufficient for induction of txnip by dexamethasone. Expression of a GFP-TXNIP fusion protein was sufficient to induce apoptosis in WEHI7.2 cells, and repression of endogenous txnip by RNA interference inhibited dexamethasone-induced apoptosis in WEHI7.2 cells. Together, these findings indicate that txnip is a novel glucocorticoid-induced primary target gene involved in mediating glucocorticoid-induced apoptosis.
de novo synthesis of cortisol during ..
AB - The glucocorticoid analogue, dexamethasone, stimulated RNA synthesis more than two-fold in rat L6 myoblasts, without affecting the rate of cell proliferation. Treatment of myoblasts for 24 h with 10-7 M dexamethasone resulted in a 30% increase in the cellular RNA level. More than a two-fold stimulation of pre-rRNA gene transcription by dexamethasone, as measured in isolated nuclei and by cell-free transcription, was accompanied by a corresponding increase in pre-rRNA levels. Co-incubation of myoblasts with cycloheximide and dexamethasone did not affect the enhanced pre-rRNA gene transcription demonstrating that de novo protein synthesis was unnecessary to manifest the dexamethasone effect on rDNA transcription. Support for this conclusion is provided by the finding that the levels of UBF1 and UBF2, rDNA upstream binding transcription factors, remain unchanged. The glucocorticoid antagonist RU38486 [11 β-(4-dimethylaminophenyl)17/gb-hydroxy-17α-(prop-1 -ynyl)estra-4,9-dien-3-one]inhibited the dexamethasone-stimulated rRNA gene transcription suggesting that the glucocorticoid receptor is involved in the response mechanism.
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