Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.     

Cyclooxygenase 2 (COX-2) inhibition increases the inflammatory response in the brain during systemic immune stimuli

Non-steroidal anti-inflammatory drugs (NSAIDs) and inhibitors of the cyclooxygenase (COX) pathways are currently recommended for the prevention and treatment of several inflammatory diseases, including neurodegenerative disorders. However non-selective blockade of COX was found to have pro-inflammatory properties, because they have the ability to alter the plasma glucocorticoid levels that play a critical role in the control of the innate immune response. The present study investigated the role of non-selective (ketorolac or indomethacin) or specific inhibitors of COX-1 (SC-560) and COX-2 (NS-398) in these effects. Mice challenged systemically with the endotoxin lipopolysaccharide (LPS) exhibited a robust hybridization signal for numerous inflammatory genes in vascular-associated cells of the brain and microglia across the cerebral tissue. Ketorolac, indomethacin and NS-398 significantly increased the ability of LPS to trigger such an innate immune response at time 3 h post challenge, whereas SC-560 failed to change gene expression in the brain of animals treated with the endotoxin. These data together with the crucial role of COX-2-derived prostaglandin E2 (PGE2) in the increase of glucocorticoids during systemic immune stimuli provide evidence that inhibition of this pathway results in an exacerbated early innate immune reaction. This may have a major impact on the use of these drugs in diseases where inflammation is believed to be a contributing and detrimental factor.     

Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3epsilon Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3epsilon-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.     

Disruption of glucocorticoid action by environmental chemicals: potential mechanisms and relevance

Glucocorticoids play an essential role in the regulation of key physiological processes, including immunomodulation, brain function, energy metabolism, electrolyte balance and blood pressure. Exposure to naturally occurring compounds or industrial chemicals that impair glucocorticoid action may contribute to the increasing incidence of cognitive deficits, immune disorders and metabolic diseases. Potentially, “glucocorticoid disruptors” can interfere with various steps of hormone action, e.g. hormone synthesis, binding to plasma proteins, delivery to target cells, pre-receptor regulation of the ratio of active versus inactive hormones, glucocorticoid receptor (GR) function, or export and degradation of glucocorticoids. Several recent studies indicate that such chemicals exist and that some of them can cause multiple toxic effects by interfering with different steps of hormone action. For example, increasing evidence suggests that organotins disturb glucocorticoid action by altering the function of factors that regulate the expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) pre-receptor enzymes, by direct inhibition of 11β-HSD2-dependent inactivation of glucocorticoids, and by blocking GR activation. These observations emphasize on the complexity of the toxic effects caused by such compounds and on the need of suitable test systems to assess their effects on each relevant step.     

Glucocorticoid-induced leucine zipper (GILZ) mediates glucocorticoid action and inhibits inflammatory cytokine-induced COX-2 expression

Cyclooxygenase-2 (COX-2) plays an important role in rheumatoid arthritis and therefore, has been a major target for anti-arthritis therapies. The expression of COX-2 is induced by inflammatory cytokines such as TNF-alpha and IL-1beta, and inhibited by glucocorticoids. However, the molecular mechanisms underlying the anti-inflammatory and immune suppressive actions of glucocorticoids are not well defined. Here we report that glucocorticoid-induced leucine zipper (GILZ) mimics glucocorticoid action and inhibits inflammatory cytokine-induced COX-2 expression in bone marrow mesenchymal stem cells, the cells that have been recently implicated in the pathogenesis and progression of rheumatoid arthritis. Using a retrovirus-mediated gene expression approach we demonstrate that overexpression of GILZ inhibits TNF-alpha and IL-1beta-induced COX-2 mRNA and protein expression, and knockdown of GILZ by shRNA reduces glucocorticoid inhibition of cytokine-induced COX-2 expression. Consistent to these results, overexpression of GILZ also inhibits NF-kappaB-mediated COX-2 promoter activity. Finally, we show that GILZ inhibits COX-2 expression by blocking NF-kappaB nuclear translocation. Our results suggest that GILZ is a key glucocorticoid effect mediator and that GILZ may have therapeutic value for novel anti-inflammation therapies.     

Red ginseng represses hypoxia-induced cyclooxygenase-2 through sirtuin1 activation

BackgroundKorean red ginseng (KRG) is a traditional herbal medicine made by steaming and drying the fresh ginseng, leading to chemical transformation of some components by heat. It ameliorates various inflammatory diseases and strengthens the endocrine, immune, and central nervous systems. The cyclooxygenase-2 (COX-2)/prostaglandin E2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumorigenesis.PurposeIn this study we examined the effects and the mechanism underlying Korean red ginseng water extract (KRG-WE) inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells.Study designThe effect of the KRG on suppression of hypoxia-induced COX-2 in A549 cells were determined by Western blot and/or qRT-PCR. The anti-invasive effect of KRG-WE was evaluated on A549 cells using matrigel invasion assay. The activation of glucocorticoid receptor (GR) and sirtuin1 (Sirt1) was examined by using specific inhibitors.ResultsWe first observed that hypoxia induced COX-2 protein and mRNA levels and promoter activity were suppressed by KRG-WE. Second, we observed that hypoxia-induced cell migration is dramatically reduced by KRG-WE. Third, we found that the effect of KRG-WE was not antagonized by the GR antagonist RU486 implying that the effect is mediated other than GR pathway. Finally, we demonstrated that inhibition of Sirt1 abolished the effect of KRG-WE on hypoxia-induced COX-2 suppression and cell-invasion indicating that the suppression is mediated by Sirt1.ConclusionTaken together, KRG-WE inhibits the hypoxic induction of COX-2 expression and cell invasion through Sirt1 activation. Our results imply that KRG-WE could be effective for suppression of inflammation under hypoxia.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.     

Regulation of IL-1 and TNF receptor expression and function by endogenous macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.     

TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis

Immune homeostasis is essential for the normal functioning of the immune system, and its breakdown leads to fatal inflammatory diseases. We report here the identification of a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, designated TIPE2, that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid tissues, and its deletion in mice leads to multiorgan inflammation, splenomegaly, and premature death. TIPE2-deficient animals are hypersensitive to septic shock, and TIPE2-deficient cells are hyper-responsive to Toll-like receptor (TLR) and T cell receptor (TCR) activation. Importantly, TIPE2 binds to caspase-8 and inhibits activating protein-1 and nuclear factor-kappaB activation while promoting Fas-induced apoptosis. Inhibiting caspase-8 significantly blocks the hyper-responsiveness of TIPE2-deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR function, and its selective expression in the immune system prevents hyperresponsiveness and maintains immune homeostasis.     

A single regulatory module of the carbamoylphosphate synthetase I gene executes its hepatic program of expression

A 469-base pair (bp) upstream regulatory fragment (URF) and the proximal promoter of the carbamoylphosphate synthetase I (CPS) gene were analyzed for their role in the regulation of spatial, developmental, and hormone-induced expression in vivo. The URF is essential and sufficient for hepatocyte-specific expression, periportal localization, perinatal activation and induction by glucocorticoids, and cAMP in transgenic mice. Before birth, the transgene is silent but can be induced by cAMP and glucocorticoids, indicating that these compounds are responsible for the activation of expression at birth. A 102-bp glucocorticoid response unit within the URF, containing binding sites for HNF3, C/EBP, and the glucocorticoid receptor, is the main determinant of the hepatocyte-specific and hormone-controlled activity. Additional sequences are required for a productive interaction between this minimal response unit and the core CPS promoter. These results show that the 469-bp URF, and probably only the 102-bp glucocorticoid response unit, functions as a regulatory module, in that it autonomously executes a correct spatial, developmental and hormonal program of CPS expression in the liver.     

Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.     

Regulation of lung fibroblast activation by annexin A1

Annexin‐A1 (AnxA1) is a glucocorticoid‐induced protein with multiple actions in the regulation of inflammatory cell activation. The contribution of AnxA1 to human cell biology is not well understood. We investigated the contribution of AnxA1 and its receptor, formyl‐peptide receptor 2 (FPR2), to the regulation of inflammatory responses in human normal lung fibroblasts (NLF). Silencing constitutive AnxA1 expression in NLF using small interfering RNA (siRNA) was associated with moderate but significant increases in tumor necrosis factor (TNF)‐induced proliferation and interleukin (IL)‐6 production, accompanied by reduction of ERK and NF‐κB activity. AnxA1 regulation of ERK and NF‐κB activation was associated with effects on proliferation. Blocking FPR2 using the specific antagonist WRW4 mimicked the effects of AnxA1 silencing on TNF‐induced proliferation, IL‐6, ERK, and NF‐κB activation. AnxA1 silencing also impaired inhibitory effects of glucocorticoid on IL‐6 production and on the expression of glucocorticoid‐induced leucine zipper (GILZ), but blocking FPR2 failed to mimic these effects of AnxA1 silencing. These data suggest that AnxA1 regulates TNF‐induced proliferation and inflammatory responses in lung fibroblasts, via effects on the ERK and NF‐κB pathways, which depend on FPR2. AnxA1 also mediates effects of glucocorticoids and GILZ expression, but these effects appear independent of FPR2. These findings suggest that mimicking AnxA1 actions might have therapeutic potential in chronic inflammatory lung diseases. J. Cell. Physiol. 228: 476–484, 2013. © 2012 Wiley Periodicals, Inc.     

Glucocorticoids sensitize the innate immune system through regulation of the NLRP3 inflammasome

Glucocorticoids have long been recognized as powerful anti-inflammatory compounds that are one of the most widely prescribed classes of drugs in the world. However, their role in the regulation of innate immunity is not well understood. We sought to examine the effects of glucocorticoids on the NOD-like receptors (NLRs), a central component of the inflammasome and innate immunity. Surprisingly, we show that glucocorticoids induce both NLRP3 messenger RNA and protein, which is a critical component of the inflammasome. The glucocorticoid-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of proinflammatory molecules, including mature IL-1β, TNF-α, and IL-6. This effect was specific for glucocorticoids and dependent on the glucocorticoid receptor. These studies demonstrate a novel role for glucocorticoids in sensitizing the initial inflammatory response by the innate immune system.     

Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action.

Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory mediator with the unique ability to counter-regulate the inhibitory effects of glucocorticoids on immune cell activation. MIF is released from cells in response to glucocorticoids, certain pro-inflammatory stimuli, and mitogens and acts to regulate glucocorticoid action on the ensuing inflammatory response. To gain insight into the molecular mechanism of MIF action, we have examined the role of MIF in the proliferation and intracellular signaling events of the well characterized, NIH/3T3 fibroblast cell line. Both endogenously secreted and exogenously added MIFs stimulate the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). The MIF-induced activation of these kinases was sustained for a period of at least 24 h and was dependent upon protein kinase A activity. We further show that MIF regulates cytosolic phospholipase A2 activity via a protein kinase A and ERK dependent pathway and that the glucocorticoid suppression of cytokine-induced cytoplasmic phospholipase A2 activity and arachidonic acid release can be reversed by the addition of recombinant MIF. These studies indicate that the sustained activation of p44/p42 MAP kinase and subsequent arachidonate release by cytoplasmic phospholipase A2 are important features of the immunoregulatory and intracellular signaling events initiated by MIF and provide the first insight into the mechanisms that underlie the pro-proliferative and inflammatory properties of this mediator.     

Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms.

Interleukin 12 (IL-12) is a crucial cytokine in the regulation of T helper 1 vs. T helper 2 immune responses. In the present study, we investigated the effect of the endogenous purine nucleoside adenosine on the production of IL-12. In mouse macrophages, adenosine suppressed IL-12 production. Although the order of potency of adenosine receptor agonists suggested the involvement of A2a receptors, data obtained with A2a receptor-deficient mice showed that the adenosine suppression of IL-12 and even TNF-alpha production is only partly mediated by A2a receptor ligation. Studies with adenosine receptor antagonists or the adenosine uptake blocker dipyridamole showed that adenosine released endogenously also decreases IL-12. Although adenosine increases IL-10 production, the inhibition of IL-12 production is independent of the increased IL-10. The mechanism of action of adenosine was not associated with alterations of the activation of the p38 and p42/p44 mitogen-activated protein kinases or the phosphorylation of the c-Jun terminal kinase. Adenosine failed to affect steady-state levels of either IL-12 p35 or p40 mRNA, but augmented IL-10 mRNA levels. In summary, adenosine inhibits IL-12 production via various adenosine receptors. These results support the notion that adenosine-based therapies might be useful in certain autoimmune and/or inflammatory diseases.     

Critical but overlapping role of FcgammaRIII and FcgammaRIV in activation of murine neutrophils by immobilized immune complexes

Immune complex-induced activation of neutrophils through cell surface FcRs plays a central role in the pathogenesis of autoimmune inflammatory diseases. These diseases are often modeled using genetically modified mice. However, in contrast to the number of studies on human cells, the identity of FcRs involved in immune complex activation of murine neutrophils is at present unknown. Furthermore, little is known about the cellular functions mediated by the recently identified murine FcgammaRIV. In this study, we tested the identity of FcRs involved in the activation of neutrophils by plate-bound immune complexes, using various knockout mouse strains, function-blocking mAbs, or the combination of both approaches. Activation of murine neutrophils by immobilized IgG immune complexes was abrogated in FcR gamma-chain-deficient cells, but not by the single or combined deficiency of the gamma-chain-associated FcgammaRI and FcgammaRIII, or by blocking Abs against either FcgammaRIII or FcgammaRIV alone. However, treatment of FcgammaRIII-deficient neutrophils with FcgammaRIV-blocking Abs or simultaneous blocking of FcgammaRIII and FcgammaRIV in wild-type cells completely inhibited the immune complex-induced cellular responses. In parallel studies, activation of human neutrophils by immobilized immune complexes was abrogated by blocking Abs against either FcgammaRIIA or FcgammaRIIIB alone. Taken together, neutrophil activation by immobilized immune complexes requires the murine FcgammaRIII/FcgammaRIV or the human FcgammaRIIA/FcgammaRIIIB molecules. Although both of the two human receptors are required for this response, the two murine receptors play overlapping, redundant roles. These results promote our understanding of autoimmune diseases and identify an IgG-dependent cellular function of FcgammaRIV.