Development Of Glucocorticoid Resistance Research Articles

The PMN-MDSC – A key player in glucocorticoid resistance following combined physical and psychosocial trauma

Stress-associated somatic and psychiatric disorders are often linked to non-resolving low-grade inflammation, which is promoted at least in part by glucocorticoid (GC) resistance of distinct immune cell subpopulations. While the monocyte/macrophage compartment was in the focus of many clinical and preclinical studies, the role of myeloid-derived suppressor cells (MDSCs) in stress-associated pathologies and GC resistance is less understood. As GC resistance is a clear risk factor for posttraumatic complications in patients on intensive care, the exact interplay of physical and psychosocial traumatization in the development of GC resistance needs to be further clarified.In the current study we employ the chronic subordinate colony housing (CSC) paradigm, a well-characterized mouse model of chronic psychosocial stress, to study the role of myeloid cells, in particular of MDSCs, in innate immune activation and GC resistance following combined psychosocial and physical (e.g., bite wounds) trauma. Our findings support the hypothesis that stress-induced neutrophils, polymorphonuclear (PMN)-MDSCs and monocytes/monocyte-like (MO)-MDSCs get primed and activated locally in the bone marrow as determined by toll-like receptor (TLR)2 upregulation and increased basal and lipopolysaccharide (LPS)-induced in vitro cell viability. These primed and activated myeloid cells emigrate into the peripheral circulation and subsequently, if CSC is accompanied by significant bite wounding, accumulate in the spleen. Here, PMN-MDSCs and monocytes/MO-MDSCs upregulate TLR4 expression, which exclusively in PMN-MDSCs promotes NF-κB hyperactivation upon LPS-stimulation, thereby exceeding the anti-inflammatory capacities of GCs and resulting in GC resistance.

Development of glucocorticoid resistance over one year among mothers of children newly diagnosed with cancer

Chronic distress associates with peripheral release of cortisol and a parallel upregulation of innate inflammation. Typically, cortisol functions to down-regulate inflammatory processes. However, in the context of chronic stress, it is hypothesized that glucocorticoid receptors within immune cells become less sensitive to the anti-inflammatory effects of cortisol, resulting in increased systemic inflammation. Caring for a child newly diagnosed with cancer is a particularly provocative chronic stressor. Here, we examine evidence for the development of cellular resistance to glucocorticoids among 120 mothers (Aged 18–56 years; 86% Caucasian) across the 12 months following their child’s new diagnosis with cancer. Measures of psychological distress, interleukin (IL)-6, and glucocorticoid resistance (GCR) were assessed 1, 6, and 12 months after the diagnosis. A latent factor for distress was derived from the covariation among symptoms of anxiety, depression, and post-traumatic stress. Latent change score models revealed a significant positive association between change in distress and change in GCR from 0 to 6 months, and 6 months-1 year. This finding provides initial evidence for a longitudinal association between change in maternal distress and change in GCR from the onset of a chronic stressor through one year. Although levels of IL-6 increased during the first six months after the child’s diagnosis, the magnitude of this change was not related to change in distress or change in GCR. Given the possible health consequences of reduced immune sensitivity to glucocorticoids, future work should further explore this stress response and its clinical significance.

Development of glucocorticoid resistance over one year among mothers of children newly diagnosed with cancer

Development of glucocorticoid resistance over one year among mothers of children newly diagnosed with cancer

Histone deacetylases meet microRNA-associated MMP-9 expression regulation in glucocorticoid-sensitive and -resistant cell lines.

Glucocorticoids are largely used in the treatment of inflammatory pathologies and/or hematological malignancies and regulate the expression of a variety of genes involved in inflammation or metastasis such as matrix metalloproteinases(MMP). Long-term exposure to glucocorticoids can result in failure of responsiveness, which is often associated with an unwanted gene expression. Epigenetic mechanisms are involved in gene expression modulated after development of glucocorticoid resistance but how these mechanisms take place must be further studied. The effects of HDAC inhibitors(HDACi) in a context of glucocorticoid resistance are still not well understood and need to be further investigated. We hypothesized that acquired glucocorticoid resistance associated to HDACi could disturbs epigenetic landscape, especially miR expression, leading to a modulation of MMP-9 gene expression and/or protein secretion, described as largely involved in bone remodeling and tumor invasion in multiple myeloma. To this aim, we used sensitive RPMI-8226 cell line and its dexamethasone- and methylprednisolone-resistant derivatives. The resistant cell lines displayed an 'open chromatin' and an MMP-9 overexpression comparatively to the sensitive cell line. HDACi treatment with MS-275 increased even more MMP-9 overexpression not only at an mRNA level but also at the protein level. We showed that MMP-9 expression regulation was not directly linked with HAT/HDAC balance alterations but rather with the deregulation of MMP-9-targeting miRs. Then, we first demonstrated that miR‑149 downregulation was directly involved in the MMP-9 overexpression following a chronic glucocorticoid exposure and that MS-275 could amplify this overexpression by inhibition of miR‑149 expression and miR‑520c overexpression. Taken together, these results indicate that the use of HDACi in a context of acquired glucocorticoid resistance could modify the epigenetic landscape, highlighting the importance of taking the glucocorticoid response status into consideration in treatment with HDACi.

Tissue-specific Glucocorticoid Signaling May Determine the Resistance Against Glucocorticoids in Autoimmune Diseases

Endogenous glucocorticoids exert a diverse array of physiological processes including immune-modulatory or anti-inflammatory responses and play an important role in the pathogenesis of inflammatory and autoimmune diseases. Regulation of inflammatory processes by glucocorticoids is controlled in a cytokine-hypothalamo-pituitary-adrenal axis feedback circuit and on the local, cell-type and context-specific local regulatory system. At the tissue level the sensitivity and response to glucocorticoids are determined by multiple factors: including the local availability to glucocorticoids transported by blood, the locally-formed bioactive glucocorticoids (synthesized and metabolized 11β-hydroxysteroid dehydrogenase enzymes), the number and function of the glucocorticoid receptor (GR) and the GR affinity to its ligands. Numerous molecular factors are known to influence the sensitivity of glucocorticoid response through the GR. Cytokines are one of the major components that can inhibit GR function and can potentiate the resistance against glucocorticoids. GR isoforms, generated by alternative splicing, alternative translation and post-translation modification are further mechanisms which modulate glucocorticoid signaling. Genetic variants within the GR encoding gene are other potential factors that may influence the susceptibility and severity of autoimmune disorders and may play a key role in individual response to medication. In this review our aim was to summarize our knowledge about the connections between the cell type-specific glucocorticoid signaling and the local immune system. Prediction of individual sensitivity to steroids and identification of key players in development of glucocorticoid resistance are essential in individualized therapies. The local, tissue-specific glucocorticoid signaling and its influence by cytokines may be important in determining the magnitude of inflammatory reactions, and may also be related to the success of glucocorticoid-containing therapeutic strategies.

Abstract 3814: Combination of selective modular of the glucocorticoid receptor (SEGRA) with proteasome inhibitors as a novel strategy for chemotherapy of hematologic malignancies

Abstract Glucocorticoids are widely used for the treatment of hematological malignancies; however chronic treatment with steroids results in numerous adverse effects. Glucocorticoid receptor (GR) regulates gene expression via transactivation that requires GR homodimer binding to gene promoters; and transrepression mediated via negative interaction between GR and other transcription factors. GR transactivation is linked to metabolic side effects, while GR transrepression underlies glucocorticoid therapeutic action. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride also called CpdA. CpdA is a synthetic analogue of a aziridine precursor isolated from the Namibian shrub Salsola tuberculatiformis Botschantzev. Using representative human T cell (CEM) and B cell (NCEB) lymphoma cell lines expressing functional GR, and their counterparts with blocked GR expression, we showed that CpdA indeed acted as dissociated GR ligand, and inhibited growth and survival of these cells via GR. Sensitivity to therapeutic effects of GCs directly depends on the amount of functional GR. The 26S protesome controls GR protein stability, and is responsible for desensitization to GCs via hormone-induced GR degradation and following development of glucocorticoid resistance in patients. Thus, the use of proteasome inhibitors represents a pharmacological approach to prevent GR down-regulation and to elevate the level of GR in cells. Proteasome inhibitor Bortezomib (BZ) was approved by FDA for the treatment of patients with multiple myeloma and mantle cell lymphoma. Since proteasome inhibitors stabilize GR, we hypothesized that BZ will augment CpdA effects as a selective GR modulator and enhance its chemotherapeutic activity. We showed that pretreatment of CEM and NCEB cells with proteasome inhibitor Bortezomib resulted in GR accumulation, and enhanced CpdA ligand properties further shifting GR activity towards transrepression evaluated by inhibiton of NF-kB and AP-1 factors. We also revealed remarkable GR-dependent synergy between CpdA and BZ in suppressing growth and survival of T and B lymphoma cells. Overall, our data provide the rationale for novel GR-based therapy for lymphoma and leukemia based on the combination of non-steroidal GR modulators with proteasome inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3814. doi:1538-7445.AM2012-3814

Polymyalgia rheumatica in 2011

Polymyalgia Rheumatica (PMR) is an inflammatory rheumatic disease that commonly affects individuals over 50 years of age, characterised by pain and morning stiffness of the shoulder and pelvic girdle. PMR can present as 'isolated' form or may be associated with giant cell arteritis. The progress of imaging techniques has helped in understanding different clinical patterns: subclinical vasculitis can occur in at least one-third of PMR patients, causing ischaemic complications. It is considered a polygenic disease and environmental factors may play a role in its pathogenesis, such as viral or bacterial triggers, both in the 'wide' form or assembled with adjuvants in vaccines. The response to steroid therapy is generally dramatic and side effects may occur, as well as the development of glucocorticoid resistance. The optimisation of therapy may require steroid-sparing agents as well as modified-release prednisone as 'nighttime' replacement therapy.

Selective Glucocorticoid Receptor Agonists for the Treatment of Inflammatory Bowel Disease: Studies in Mice with Acute Trinitrobenzene Sulfonic Acid Colitis

Despite being a mainstay of inflammatory bowel disease (IBD) therapy, glucocorticoids (GCs) still carry significant risks with respect to unwanted side effects. Alternative drugs with a more favorable risk/benefit ratio than common GCs are thus highly desirable for the management of IBD. New and supposedly selective glucocorticoid receptor (GR) agonists (SEGRAs), with dissociated properties, have been described as promising candidates for circumventing therapeutic problems while still displaying full beneficial anti-inflammatory potency. Here, we report on compound A [CpdA; (2-((4-acetophenyl)-2-chloro-N-methyl)ethylammonium-chloride)] and N-(4-methyl-1-oxo-1H-2,3-benzoxazine-6-yl)-4-(2,3-dihydrobenzofuran-7-yl)-2-hydroxy-2-(trifluoromethyl)-4-methylpentanamide (ZK216348), two GR agonists for the treatment of experimental colitis. Their therapeutic and anti-inflammatory effects were tested in the acute trinitrobenzene sulfonic acid-mediated colitis model in mice against dexamethasone (Dex). In addition to their influence on immunological pathways, a set of possible side effects, including impact on glucose homeostasis, steroid resistance, and induction of apoptosis, was surveyed. Our results showed that, comparable with Dex, treatment with CpdA and ZK216348 reduced the severity of wasting disease, macroscopic and microscopic damage, and colonic inflammation. However, both SEGRAs exhibited no GC-associated diabetogenic effects, hypothalamic pituitary adrenal axis suppression, or development of glucocorticoid resistance. In addition, CpdA and ZK216348 showed fewer transactivating properties and successfully dampened T helper 1 immune response. Unlike ZK216348, the therapeutic benefit of CpdA was lost at higher doses because of toxic apoptotic effects. In conclusion, both SEGRAs acted as potent anti-inflammatory agents with a significantly improved profile compared with classic GCs. Although CpdA revealed a narrow therapeutic window, both GR agonists might be seen as a starting point for a future IBD treatment option.

Variability in stress system regulatory control of inflammation: a critical factor mediating health effects of stress

Chronic stress has been associated with disease but the biological pathways are not completely understood. Stress systems such as the hypothalamus–pituitary–adrenal axis and the autonomic nervous system are prime candidates but alterations in their baseline activity are not consistently found in chronic stress. Evidence suggests that stress-related changes in the sensitivity of inflammatory pathways towards glucocorticoid regulation, that is, the development of glucocorticoid resistance, might help explain inflammatory disinhibition and the subsequent development of disease. Recent data show a similarly important role for sympathetic and parasympathetic modulation of the inflammatory cascade for the maintenance of health. This article argues that variation of target tissue sensitivity towards anti-inflammatory effects of the hypothalamus–pituitary–adrenal axis, as well as sympathetic and parasympathetic signaling, might be involved in the development of low-grade inflammation under chronic psychosocial stress.

Alternative splicing in exon 9 of glucocorticoid receptor pre-mRNA is regulated by SRp40

Increasing evidence indicates that alternative splicing of human glucocorticoid receptor (GR) transcripts is implicated in the development of glucocorticoid resistance but the underlying mechanism was not well known. Serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 play an important role in the spliceosome assembly. In this study, we analyzed the effects of different SR proteins and hnRNP A1 on the alternative splicing of GR pre-mRNA in HeLa and 293T cells using a minigene transfection assay. Our results revealed that only SRp40 could induce a GRalpha to GRbeta shift of pre-mRNA splicing in exon 9 in HeLa cells and this effect induced by SRp40 was further confirmed by small interfering RNA study. However, in 293T cells, SRp40 could not induce this shift. These results indicated that SRp40 may influence the alternative splicing of GR pre-mRNA to regulate the ratio of GRalpha to GRbeta, and this effect is cell-dependent.

Inhibition of NOTCH1 Signaling and Glucocorticoid Therapy in T-ALL

Activating mutations in NOTCH1 are present in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). Consequently, inhibition of NOTCH1 signaling with small molecule γ-secretase inhibitors (GSIs) has been proposed as a targeted therapy for this disease. However, GSIs fail to induce robust apoptosis in T-ALL cells and their clinical application has been limited by the development of severe gastrointestinal toxicity (mucous diarrhea and goblet cell metaplasia) resulting from systemic inhibition of NOTCH signaling. Here we show that combination therapy with GSIs plus glucocorticoids can reverse both glucocorticoid resistance in T-ALL and the occurrence of GSI-induced gut toxicity in vivo. The development of glucocorticoid resistance in T-ALL has been associated with the loss of a positive auto-regulatory transcriptional feedback loop required for glucocorticoid induced apoptosis. Using gene expression profiling and ChIP-on-chip analysis we demonstrated that HES1, a transcriptional repressor controlled by NOTCH1, inhibits specific regulatory elements in the glucocorticoid receptor promoter required for effective glucocorticoid receptor autoupregulation and glucocorticoid induced cell death. Consequently, downregulation of HES1 via inhibition of NOTCH1 signaling with GSIs or HES1 inactivation via shRNA knockdown restores glucocorticoid receptor autoupregulation and reverses glucocorticoid resistance in T-ALL cell lines and primary patient samples. Consistent with these results, combination therapy with dexamethasone and GSIs was highly effective in a mouse xenograft model of glucocorticoid resistant T-ALL. Surprisingly, these studies demonstrated not only improved antileukemic responses in mice treated with dexamethasone plus a GSI compared with dexamethasone or placebo treated controls (P<0.05), but also decreased mortality associated with GSI-induced gut toxicity in this model (P<0.01). These results suggested that glucocorticoids may have an enteroprotective effect against GSI-induced gut toxicity. Detailed histological analysis demonstrated a block of proliferation and increased goblet cell numbers in the intestine of GSI treated animals and a moderate increase in proliferation accompanied by increased Paneth cell numbers in the dexamethasone treated group. Notably, cotreatment with dexamethasone plus a GSI completely reversed the induction of goblet cell metaplasia typically induced by systemic inhibition of NOTCH signaling with GSIs. Likewise, dexamethasone also protected RBPJ-K conditional knockout mice, a genetic model of loss of NOTCH signaling, from developing goblet cell metaplasia in the gut. Interestingly, gene expression profiling analysis showed a marked upregulation of cyclin D2 expression in the gut of glucocorticoid treated mice. Moreover, dexamethasone treatment failed to protect Ccnd2 knockout animals from developing GSI-induced metaplasia, demonstrating a critical role of Ccnd2 upregulation in the protective effects of dexamethasone against GSI-induced gut toxicity. Overall, these results uncover a NOTCH1-HES1-glucocorticoid receptor regulatory axis in glucocorticoid resistant T-ALL and demonstrate an unanticipated protective effect of glucocorticoids against GSI-induced gut toxicity. Our studies warrant the clinical testing of glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

Bombesin attenuates pre-mRNA splicing of glucocorticoid receptor by regulating the expression of serine-arginine protein p30c (SRp30c) in prostate cancer cells

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GRα and GRβ isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GRβ, but not GRα. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GRβ expression and contributing to glucocorticoid resistance in PC cells.

Inhibition of Jun N-Terminal Kinase (JNK) Enhances Glucocorticoid Receptor-Mediated Function in Mouse Hippocampal HT22 Cells

Mitogen-activated protein kinases (MAPKs), including Jun N-terminal kinase (JNK), promote inflammatory and proliferative responses to infection and other environmental stimuli including stress. Relevant to negative regulation of inflammatory pathways by glucocorticoids and the development of glucocorticoid resistance (observed in inflammatory disorders as well as certain neuropsychiatric disorders such as major depression), activation of JNK has been reported to inhibit glucocorticoid receptor (GR) function. In this study, the role of JNK pathways in modulating GR function was further investigated. Treatment of mouse hippocampal (HT22) cells with the selective JNK inhibitor, SP-600125 (0.1-10 microM), resulted in dose-dependent induction of GR-mediated MMTV-luciferase activity. SP-600125 also significantly enhanced dexamethasone-induced MMTV-luciferase activity, while increasing GR binding to the glucocorticoid responsive element, both in the presence and absence of Dex. Similar effects were observed in mouse fibroblast cells (LMCAT), and in HT22 cells treated with a JNK specific antisense oligonucleotide. The induction of GR-mediated function by SP-600125 was not due to altered cytosolic GR binding or GR protein expression or enhancement of GR nuclear translocation as determined by Western blot. Taken together, the data indicate that constitutive expression of JNK plays a tonic inhibitory role in GR function, which is consistent with findings that activation of JNK pathways inhibits GR. The data also identify potential pathways involved in the pathogenesis of the glucocorticoid resistance found in certain chronic immune/inflammatory diseases and subgroups of patients with major depression. Moreover, JNK pathways may represent a therapeutic target for normalization of GR function in these disorders.

Expression of human glucocorticoid receptor in lymphocytes of patients with autoimmune hepatitis.

Background and aim: Alternative splicing of human glucocorticoid receptor (hGR) premessenger RNA (mRNA) generates two highly homogenous isoforms, termed hGRalpha and hGRbeta. hGRalpha is a ligand-activated transcription factor, which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element DNA sequences. In contrast, hGRbeta may be an endogenous inhibitor of glucocorticoid action and transcriptionally inactive. hGRbeta protein has been known to correlate with the development of glucocorticoid resistance. Glucocorticoids can effectively relieve autoimmune hepatitis (AIH), but some patients with this disease are refractory even when glucocorticoids are administered. The aim of this study was to determine the incidence of hGRbeta mRNA in patients with AIH by reverse transcription polymerase chain reaction (RT-PCR), and to compare the clinical characteristics of hGRbeta-positive and -negative patients with AIH. Materials and methods: RNA was obtained from peripheral blood mononuclear cells (PBMCs) of 62 patients, consisting of 26 with AIH, 10 with primary biliary cirrhosis (PBC), seven with chronic viral hepatitis (CVH), 10 with ulcerative colitis (UC), six with pemphigus, and three with systemic lupus erythematosus (SLE), and 10 healthy volunteers. The total RNA obtained was reverse transcribed, the resulting complementary DNA amplified using specific primers for hGRalpha and hGRbeta. Results: The hGRalpha mRNA was detected in RNA from PBMCs of all patients and healthy volunteers. The hGRbeta mRNA was detected in 15 (57.6%) patients with AIH. This incidence was significantly higher than that for patients with PBC (0%) or CVH (28.6%) or for healthy volunteers (20.0%) ( [Formula: see text] ). Of the hGRbeta-positive and -negative groups, serum ALT and total bilirubin (TB) levels were significantly higher in the positive group ( [Formula: see text] ). The total dose of glucocorticoid was higher in the positive group, but the difference was not statistically significant. However, the average monthly dose was significantly higher in the positive group ( [Formula: see text] ). The rate of relapse of AIH was significantly higher in the positive group (60.0%) than in the negative (10.0%) ( [Formula: see text] ). The rate of usage of immunosuppressive drugs was higher in the positive group (33.3%) than in the negative (18.2%), but the difference was not statistically significant. Conclusions: These data show that hGRbeta expression in PBMCs of patients with AIH assessed by RT-PCR is closely associated with resistance to glucocorticoids which affects the outcome of therapy with this drug.

Social stress increases the susceptibility to endotoxic shock

The influence of social disruption stress (SDR) on the susceptibility to endotoxic shock was investigated. SDR was found to increase the mortality of mice when they were challenged with the bacterial endotoxin lipopolysaccharide (LPS). Histological examination of SDR animals after LPS injection revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen, indicating inflammatory organ damage. In situ hybridization histochemical analysis showed that the expression of the glucocorticoid receptor mRNA was down-regulated in the brain and spleen of SDR animals while the ratio of expression of AVP/CRH-the two adrenocorticotropic hormone secretagogue, increased. After LPS injection, the expression of pro-inflammatory cytokines, IL-1β and TNF-α, was found significantly higher in the lung, liver, spleen, and brain of the SDR mice as compared with the LPS-injected home cage control animals. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock and suggest that the development of glucocorticoid resistance and increased production of pro-inflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.