Live Cell Imaging Unveils Multiple Domain Requirements for In Vivo Dimerization of the Glucocorticoid Receptor

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Ogara, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Stortz, Martin Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Alvarez, Lautaro Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Pooley, John R.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. University of Bristol; Reino UnidoFil: Schiltz, R. Louis. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Grøntved, Lars. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Johnson, Thomas A.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Mittelstadt, Paul R.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ashwell, Jonathan D.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ganesan, Sundar. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. National Institute of Allergy and Infectious Diseases; Estados UnidosFil: Burton, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Hager, Gordon L.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Gene expression profiling of mucinous ovarian tumors and comparison with upper and lower gastrointestinal tumors identifies markers associated with adverse outcomes.

PURPOSE: Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. EXPERIMENTAL DESIGN: Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). RESULTS: Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04–7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04–1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01–1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). CONCLUSIONS: An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies

Single-Cell Resolution and Quantitation of Targeted Glucocorticoid Delivery in the Thymus

Summary: Glucocorticoids are lipid-soluble hormones that signal via the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Circulating glucocorticoids derive from the adrenals, but it is now apparent that paracrine glucocorticoid signaling occurs in multiple tissues. Effective local glucocorticoid concentrations and whether glucocorticoid delivery can be targeted to specific cell subsets are unknown. We use fluorescence detection of chromatin-associated GRs as biosensors of ligand binding and observe signals corresponding to steroid concentrations over physiological ranges in vitro and in vivo. In the thymus, where thymic epithelial cell (TEC)-synthesized glucocorticoids antagonize negative selection, we find that CD4+CD8+TCRhi cells, a small subset responding to self-antigens and undergoing selection, are specific targets of TEC-derived glucocorticoids and are exposed to 3-fold higher levels than other cells. These results demonstrate and quantitate targeted delivery of paracrine glucocorticoids. This approach may be used to assess in situ nuclear receptor signaling in a variety of physiological and pathological contexts. : Glucocorticoids signal via the GR, a ligand-dependent transcription factor, and paracrine glucocorticoid signaling occurs in the thymus. Taves et al. use chromatin-associated GRs as biosensors to estimate glucocorticoid concentrations in vitro and in vivo. In the thymus, antigen-signaled CD4+8+TCRhi cells are targeted by epithelial cell-synthesized glucocorticoids to promote positive selection. Keywords: Cyp11b1, glucocorticoids, glucocorticoid receptor, lymphocytes, nuclear receptors, paracrine, steroids, steroidogenesis, transcription facto

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis

<div><p>Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR^CD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR^CD11c-cre mice, CD8⁺ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c⁺ cells rescued LPS-induced loss of CD8⁺ DCs but not other DC subsets. Unlike wild-type animals, exposure of GR^CD11c-cre mice to low-dose LPS did not induce CD8⁺ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.</p></div

Elevated proinflammatory cytokines in GR^CD11c-cre mice.

<p>(A–C, E, and F) Kinetics of LPS-induced cytokine levels in WT and GR^CD11c-cre mice. The sera used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.g001" target="_blank">Fig 1C</a> were also used to determine cytokine concentrations at each time point. Data shown are from four to eight animals per strain per time point pooled from multiple independent experiments. (D) Semiquantitative RT-PCR analysis of IFN-α4 transcription in DC subsets from WT and GR^CD11c-cre mice 1 hr after LPS challenge. β-actin was used as internal control. Results shown are one representative of two independent experiments with pooled splenocytes from two WT and two GR^CD11c-cre mice. (G) Splenocytes were prepared from mice 12 hr after LPS injection, and cultured in vitro for 4 hr with Brefeldin A. Intracellular IFN-γ was measured for gated NK and T cells by flow cytometry. One mouse from each genotype is shown out of two independent experiments, each using three mice per group. (H) Mean fluorescence intensity (MFI) and percentage of IFN-γ⁺ cells in WT and GR^CD11c-cre mice 12 hr post LPS challenge (<i>n</i> = 3). (I) GR^CD11c-cre or WT mice, each represented by an individual symbol, were injected with the indicated doses of neutralizing anti-IL-12 antibody 1 hr before challenge with a lethal dose of LPS (10 μg/g body weight). Surface body temperature at 30 hr is plotted. Results shown are pooled from two independent experiments, each with three to four mice per group. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002269#pbio.1002269.s001" target="_blank">S1 Data</a>.</p