Uneven modulation of the annexin 1 system in osteoblast-like cells by dexamethasone

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.     

Glucocorticoid-mediated anti-inflammatory effect through NFκB is preserved in the absence of Dexras1

Glucocorticoids effectively mediate the resolution of inflammation, but long-term use of glucocorticoids inevitably causes metabolic side effects. However, it is unknown if metabolic effectors such as Dexras1, a dexamethasone-stimulated protein, play a role in the anti-inflammatory outcome of dexamethasone. Here, we demonstrate that Dexras1 is required for the dexamethasone-induced upregulation of annexin A1 expression, but is not involved in the reduction of inflammation as evidenced by decreased pro-inflammatory parameters. In the absence of Dexras1, lipopolysaccharide (LPS)-induced interleukin-6 expression was suppressed when murine macrophage RAW264.7 cells were treated with dexamethasone. Similar observations were made in the blood of Dexras1 knockout mice. Furthermore, dexamethasone suppressed the LPS-stimulated increase of NFκB-p65 in both control and Dexras1-absent RAW264.7 cells. Interestingly, depletion of Dexras1 resulted in the loss of pERK production. These results suggest that Dexras1 is involved primarily in the metabolic side effects and its inhibition preserves the anti-inflammatory action of glucocorticoids. Thus, the inhibition of Dexras1 will be an excellent target for reducing steroid-induced side effects.     

Dexamethasone down-regulates the expression of endothelin receptors in vascular smooth muscle cells.

Steroid hormones have been shown to modulate a number of physiological processes in addition to their potent antiinflammatory effects. Endothelin (ET) is a newly discovered vasoconstrictor that is synthesized and released by endothelial cells and acts on adjacent vascular smooth muscle cells by interacting with specific cell surface receptors. Proinflammatory agents such as thrombin and transforming growth factor beta have been shown to up-regulate ET gene expression in vascular endothelial cells. We wondered whether the anti-inflammatory steroids might have any regulatory effect on the ET receptors present in the vascular smooth muscle cells. Rat vascular smooth muscle cells (A-10 cell line, ATCC.CRL 1476) were used as a model system to study the effects of glucocorticoids on ET receptor expression and function. These cells display high density and high affinity ET receptors that belong to the ETA subtype. Pretreatment of these cells with dexamethasone reduced the number of ET receptors by 50-60% without changing the affinity. Of the steroids tested, dexamethasone was most effective followed by prednisolone and hydrocortisone. Aldosterone, a mineralocorticoid, was 5000-fold less potent than dexamethasone. This effect of dexamethasone was dependent on the time of pretreatment and concentration of the steroid used. This down-regulation of ET receptors was also accompanied by an attenuated response to ET-1 in dexamethasone-pretreated cells. The inhibitory effect of dexamethasone was selective for ET receptors because the vasopressin-mediated response was unaffected. In addition, dexamethasone pretreatment of these cells resulted in 50-60% reduction in the steady-state level of ETA receptor mRNA as revealed by Northern analysis. These results suggest that glucocorticoid pretreatment of smooth muscle cells resulted in the down-regulation of the ETA receptor at the mRNA level.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Dexamethasone up-regulates type II IL-1 receptor in mouse primary activated astrocytes

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL-1 receptors including the type I IL-1 receptor (IL-1RI) that transduces IL-1 signals in cooperation with the IL-1 receptor accessory protein (IL-1RAcP) and the type II IL-1 receptor (IL-1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL-1 receptors expression. IL-1beta-activated mouse primary astrocytes were treated with 10(-6) M dexamethasone, and IL-1 receptors were studied at the mRNA and protein levels. Using RT-PCR, IL-1RI and IL-1RII but not IL-1RAcP mRNAs were found to be up-regulated by dexamethasone in a time-dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL-1RI but strongly increased IL-1RII mRNA expression. Binding studies revealed an increase in the number of IL-1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL-1.     

Cell-type specific regulation of galectin-3 expression by glucocorticoids in lung Clara cells and macrophages

Bronchiolar Clara cells are integral components of lung homeostasis, predominantly distributed in distal airways. In addition to the 16 kDa Clara cell protein, a major secretory product with anti-inflammatory effects, rat Clara cells express the glycan-binding protein galectin-3 and secrete it into the airways. Given the essential role of galectin-3 in the control of inflammation and the well-established function of glucocorticoids (GCs) in lung physiology, here we investigated whether galectin-3 is a target of the regulatory effects of GCs. Adult male rats were subjected to bilateral adrenalectomy and the lungs were processed for light and transmission electron microscopy, immunoelectron microscopy and Western blot analysis. Profound changes in bronchiolar Clara cells and macrophage morphology could be observed by electron microscopy after adrenalectomy. While specific galectin-3 staining was detected in the nucleus and cytoplasm of Clara cells and macrophages from control animals, cytoplasmic galectin-3 expression was dramatically reduced after adrenalectomy in both cell types. This effect was cell-specific as it did not affect expression of this lectin in ciliated cells. After dexamethasone treatment, galectin-3 expression increased significantly in the nucleus and cytoplasm of macrophages and Clara cells. Western blot analysis showed a clear decrease in galectin-3 expression in ADX animals, which was recovered after a 7-day treatment with dexamethasone. In peritoneal macrophages, galectin-3 expression was also dependent on the effects of GCs both in vivo and in vitro. Our results identify a cell type-specific control of galectin-3 synthesis by GCs in lung bronchiolar Clara cells and interstitial macrophages, which may provide an alternative mechanism by which GCs contribute to modulate the inflammatory response.     

Formyl peptide-receptor like-1 requires lipid raft and extracellular signal-regulated protein kinase to activate inhibitor-kappa B kinase in human U87 astrocytoma cells

Formyl peptide-receptor like-1 (FPRL-1) may possess critical roles in Alzheimer's diseases, chemotaxis and release of neurotoxins, possibly through its regulation of nuclear factor-κB (NFκB). Here we illustrate that activation of FPRL-1 in human U87 astrocytoma or Chinese hamster ovary cells stably expressing the receptor resulted in the phosphorylations of inhibitor-κB kinase (IKK), an onset kinase for NFκB signaling cascade. FPRL-1 selective hexapeptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM) promoted IKK phosphorylations in time- and dose-dependent manners while pre-treatment of pertussis toxin abrogated the Gα_i/o-dependent stimulations. The FPRL-1-mediated IKK phosphorylation required extracellular signal-regulated protein kinase (ERK), phosphatidylinositol 3-kinase and cellular Src (c-Src), but not c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Despite its ability to mobilize Ca²⁺, WKYMVM did not require Ca²⁺ for the modulation of IKK phosphorylation. Activation of FPRL-1 also induced NFκB-driven luciferase expression. Interestingly, cholesterol depletion from plasma membrane by methyl-β-cyclodextrin abolished the FPRL-1-stimulated IKK phosphorylation, denoting the important role of lipid raft integrity in the FPRL-1 to IKK signaling. Furthermore, we demonstrated that in U87 cells, several signaling intermediates in the FPRL-1-IKK pathway including Gα_i2, c-Src and ERK were constitutively localized at the raft microdomains. WKYMVM administration not only resulted in higher amount of ERK recruitment to the raft region, but also specifically stimulated raft-associated c-Src and ERK phosphorylations. Taken together, these results demonstrate that FPRL-1 is capable of activating NFκB signaling through IKK phosphorylation and this may serve as a useful therapeutical target for FPRL-1-related diseases.     

Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line.

A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.     

Reciprocal regulation of 11��-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression by dexamethasone inhibits human coronary artery smooth muscle cell proliferation in vitro

The actions of glucocorticoids are mediated, in part, by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which amplifies their effects at the pre-receptor level by converting cortisone to cortisol. Glucocorticoids, such as dexamethasone, inhibit vascular smooth muscle cell proliferation; however, the role of 11β-HSD1 in this response remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with dexamethasone (10(-9)-10(-6) mol/l) and found that after 72?h dexamethasone increased 11β-HSD1 expression (14.16?±?1.6-fold, P?相似文献   

The role of MAP kinase phosphatase-1 in the protective mechanism of dexamethasone against endotoxemia

AIMS: We have previously shown that glucocorticoids induce the expression of MAP kinase phosphatase (Mkp)(a)-1 in innate immune cells. Since Mkp-1 is a critical negative regulator of the innate immune response, we hypothesize that Mkp-1 plays a significant role in the anti-inflammatory action of glucocorticoids. The specific aim of the present study is to understand the role of Mkp-1 in the anti-inflammatory function of glucocorticoids. MAIN METHODS: Wild-type and Mkp-1(-/-) mice were treated with different doses of dexamethasone and then challenged with different doses of lipopolysaccharide (LPS). The survival and blood cytokines were assessed. The effects of dexamethasone on cytokine production in wild-type and Mkp-1(-/-) primary macrophages ex vivo were also examined. KEY FINDINGS: We found that dexamethasone induced the expression of Mkp-1 in vivo. Dexamethasone treatment completely protected wild-type mice from the mortality caused by a relatively high dose of LPS. However, dexamethasone treatment offered only a partial protection to Mkp-1(-/-) mice. Dexamethasone attenuated TNF-alpha production in both wild-type and Mkp-1(-/-) mice challenged with LPS, although TNF-alpha production in Mkp-1(-/-) mice was significantly more robust than that in wild-type mice. Dexamethasone pretreatment shortened the duration of p38 and JNK activation in LPS-stimulated wild-type macrophages, but had little effect on p38 or JNK activation in similarly treated Mkp-1(-/-) macrophages. SIGNIFICANCE: Our results indicate that the inhibition of p38 and JNK activities by glucocorticoids is mediated by enhanced Mkp-1 expression. These results demonstrate that dexamethasone exerts its anti-inflammatory effects through both Mkp-1-dependent and Mkp-1-indepent mechanisms.     

Annexin 1 negatively regulates IL-6 expression via effects on p38 MAPK and MAPK phosphatase-1

Annexin 1 (Anx-1) is a mediator of the anti-inflammatory actions of glucocorticoids, but the mechanism of its anti-inflammatory effects is not known. We investigated the role of Anx-1 in the regulation of the proinflammatory cytokine, IL-6. Lung fibroblast cell lines derived from Anx-1(-/-) and wild-type (WT) mice were treated with dexamethasone and/or IL-1. IL-6 mRNA and protein were measured using real-time PCR and ELISA, and MAPK pathway activation was studied. Compared with WT cells, unstimulated Anx-1(-/-) cells exhibited dramatically increased basal IL-6 mRNA and protein expression. In concert with this result, Anx-1 deficiency was associated with increased basal phosphorylated p38, JNK, and ERK1/2 MAPKs. IL-1-inducible phosphorylated p38 was also increased in Anx-1(-/-) cells. The increase in IL-6 release in Anx-1(-/-) cells was inhibited by inhibition of p38 MAPK. Anx-1(-/-) cells were less sensitive to dexamethasone inhibition of IL-6 mRNA expression than WT cells, although inhibition by dexamethasone of IL-6 protein was similar. MAPK phosphatase-1 (MKP-1), a glucocorticoid-induced negative regulator of MAPK activation, was up-regulated by dexamethasone in WT cells, but this effect of dexamethasone was significantly impaired in Anx-1(-/-) cells. Treatment of Anx-1(-/-) cells with Anx-1 N-terminal peptide restored MKP-1 expression and inhibited p38 MAPK activity. These data demonstrate that Anx-1 is an endogenous inhibitory regulator of MAPK activation and IL-6 expression, and that Anx-1 is required for glucocorticoid up-regulation of MKP-1. Therapeutic manipulation of Anx-1 could provide glucocorticoid-mimicking effects in inflammatory disease.     

15-Hydroxyprostaglandin dehydrogenase can be induced by dexamethasone and other glucocorticoids at the therapeutic level in A549 human lung adenocarcinoma cells

Dexamethasone induced the expression of 15-PGDH in a time- and concentration-dependent manner in A549 human lung adenocarcinoma cells. Maximal induction was observed at 10nM. Induction of 15-PGDH expression was also achieved by other synthetic glucocorticoids. Induction was inhibited by the addition of pro-inflammatory cytokines and phorbol ester. These pro-inflammatory agents were also shown to induce COX-2 expression. PMA was found to be the most effective stimulator of COX-2 expression and the most potent inhibitor of dexamethasone-induced 15-PGDH expression. Attenuation of dexamethasone-induced 15-PGDH expression by PMA was, in part, due to a protein kinase C-mediated mechanism. The induction of 15-PGDH expression by dexamethasone was blocked by a glucocorticoid receptor antagonist RU 486 and by a nuclear translocation inhibitor geldanamycin, indicating that the induction is a genetic mechanism. The induction of 15-PGDH expression by dexamethasone and other glucocorticoids at the therapeutic level provides an additional biochemical mechanism for the anti-inflammatory action of these glucocorticoids.     

Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line.

The third component of C, C3, is the key opsonin of the C cascade and is produced locally within the lung by pulmonary epithelial cells, macrophages, and fibroblasts. Because glucocorticoids regulate the maturation and expression of several physiologically important genes in pulmonary epithelial cells, we examined the effects of glucocorticoids on C3 mRNA expression and C3 synthesis by the human pulmonary epithelial cell line, A549. Treatment with dexamethasone enhanced C3 production in a time- and dose-dependent fashion such that concentrations of dexamethasone greater than or equal to 0.001 microM significantly increased C3 production on day 3 of culture. Natural glucocorticoids, corticosterone, cortisol, and 11-deoxycortisol also increased C3 concentrations in A549 supernatants. Both cycloheximide and the glucocorticoid receptor antagonist, RU486, individually inhibited the effect of dexamethasone on C3 production. Northern analysis demonstrated that the steady state 5.2-kb C3 message increased in A549 cells within 10 h of treatment with dexamethasone. RU486 inhibited the effect of dexamethasone on C3 mRNA expression. The integrity of the C3 thiolester bond, as measured by [3H]iodoacetic acid titration and hemolytic assay, was not disrupted by dexamethasone. We conclude that glucocorticoids such as dexamethasone enhance the expression of C3 mRNA and increase the production of functionally active C3 by A549 cells by a mechanism that is mediated by the intracellular glucocorticoid receptor.     

Annexin A1 regulates intestinal mucosal injury, inflammation, and repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.