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1.  Nuclear receptors in inflammation control: repression by GR and beyond 
Inflammation is a protective response of organisms to pathogens, irritation or injury. Primary inflammatory sensors activate an array of signaling pathways that ultimately converge upon a few transcription factors such as AP1, NFκB and STATs that in turn stimulate expression of inflammatory genes to ultimately eradicate infection and repair the damage. A disturbed balance between activation and inhibition of inflammatory pathways can set the stage for chronic inflammation which is increasingly recognized as a key pathogenic component of autoimmune, metabolic, cardiovascular and neurodegenerative disorders. Nuclear Receptors (NRs) are a large family of transcription factors many of which are known for their potent anti-inflammatory actions. Activated by small lipophilic ligands, NRs interact with a wide range of transcription factors, cofactors and chromatin-modifying enzymes, assembling numerous cell- and tissue-specific DNA-protein transcriptional regulatory complexes with diverse activities. Here we discuss established and emerging roles and mechanisms by which NRs and, in particular, the glucocorticoid receptor (GR) repress genes encoding cytokines, chemokines and other pro-inflammatory mediators.
PMCID: PMC3787948  PMID: 23623868
Inflammation; transcriptional repression; nuclear receptors; glucocorticoid receptor
2.  Glucocorticoid receptor coordinates transcription factor-dominated regulatory network in macrophages 
BMC Genomics  2014;15(1):656.
Inflammation triggered by infection or injury is tightly controlled by glucocorticoid hormones which signal via a dedicated transcription factor, the Glucocorticoid Receptor (GR), to regulate hundreds of genes. However, the hierarchy of transcriptional responses to GR activation and the molecular basis of their oftentimes non-linear dynamics are not understood.
We investigated early glucocorticoid-driven transcriptional events in macrophages, a cell type highly responsive to both pro- and anti-inflammatory stimuli. Using whole transcriptome analyses in resting and acutely lipopolysaccharide (LPS)-stimulated macrophages, we show that early GR target genes form dense networks with the majority of control nodes represented by transcription factors. The expression dynamics of several glucocorticoid-responsive genes are consistent with feed forward loops (FFL) and coincide with rapid GR recruitment. Notably, GR binding sites in genes encoding members of the KLF transcription factor family colocalize with KLF binding sites. Moreover, our gene expression, transcription factor binding and computational data are consistent with the existence of the GR-KLF9-KLF2 incoherent FFL. Analysis of LPS-downregulated genes revealed striking enrichment in multimerized Zn-fingers- and KRAB domain-containing proteins known to bind nucleic acids and repress transcription by propagating heterochromatin. This raises an intriguing possibility that an increase in chromatin accessibility in inflammatory macrophages results from broad downregulation of negative chromatin remodelers.
Pro- and anti-inflammatory stimuli alter the expression of a vast array of transcription factors and chromatin remodelers. By regulating multiple transcription factors, which propagate the initial hormonal signal, GR acts as a coordinating hub in anti-inflammatory responses. As several KLFs promote the anti-inflammatory program in macrophages, we propose that GR and KLFs functionally cooperate to curb inflammation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-656) contains supplementary material, which is available to authorized users.
PMCID: PMC4133603  PMID: 25099603
Transcriptional regulation; Glucocorticoid receptor; Inflammation; Feed forward loops; Gene regulatory network; KLF transcription factors
3.  Glucocorticoid-Dependent Phosphorylation of the Transcriptional Coregulator GRIP1 
Molecular and Cellular Biology  2012;32(4):730-739.
Much of the regulatory diversity in eukaryotic transcription is provided by coregulators, which are recruited by DNA-binding factors to propagate signaling to basal machinery or chromatin. p160 family members, including the glucocorticoid receptor (GR)-interacting protein 1 (GRIP1), function as coactivators for GR, a ligand-dependent transcription factor of the nuclear receptor superfamily. Unlike other p160s, GRIP1 also potentiates GR-mediated repression of AP1 and NF-κB targets and, surprisingly, transcriptional activation by interferon regulatory factors. What enables GRIP1 activating or repressing properties or discrimination between physiologically antagonistic pathways is unknown. We found that endogenous GRIP1 in mammalian cells undergoes glucocorticoid-induced, GR interaction-dependent phosphorylation and identified one constitutive and six inducible phosphorylation sites and two putative GRIP1 kinases, casein kinase 2 and cyclin-dependent kinase 9. We raised phosphospecific antibodies to the four closely spaced sites in a previously uncharacterized part of GRIP1 which, combined with mutagenesis, revealed the conservation of GRIP1 phosphorylation across several cell types and species and its functional relevance to GR-activated transcription and to response element-specific recruitment of phospho-GRIP1 to native GR targets. We propose that cofactor engagement by GR is neither passive nor stochastic; rather, GR actively imparts modifications that dictate GRIP1 function in a subset of complexes, adding a layer of specificity to GR transcriptional control.
PMCID: PMC3272970  PMID: 22158970
4.  The Type I Interferon Signaling Pathway Is a Target for Glucocorticoid Inhibition ▿  
Molecular and Cellular Biology  2010;30(19):4564-4574.
Type I interferon (IFN) is essential for host defenses against viruses; however, dysregulated IFN signaling is causally linked to autoimmunity, particularly systemic lupus erythematosus. Autoimmune disease treatments rely on glucocorticoids (GCs), which act via the GC receptor (GR) to repress proinflammatory cytokine gene transcription. Conversely, cytokine signaling through cognate Jak/STAT pathways is reportedly unaffected or even stimulated by GR. Unexpectedly, we found that GR dramatically inhibited IFN-stimulated gene (ISG) expression in macrophages. The target of inhibition, the heterotrimeric STAT1-STAT2-IRF9 (ISGF3) transcription complex, utilized the GR cofactor GRIP1/TIF2 as a coactivator. Consequently, GRIP1 knockdown, genetic ablation, or depletion by GC-activated GR attenuated ISGF3 promoter occupancy, preinitiation complex assembly, and ISG expression. Furthermore, this regulatory loop was restricted to cell types such as macrophages expressing the GRIP1 protein at extremely low levels, and pharmacological disruption of the GR-GRIP1 interaction or transient introduction of GRIP1 restored RNA polymerase recruitment to target ISGs and the subsequent IFN response. Thus, type I IFN is a cytokine uniquely controlled by GR at the levels of not only production but also signaling through antagonism with the ISGF3 effector function, revealing a novel facet of the immunosuppressive properties of GCs.
PMCID: PMC2950533  PMID: 20679482
5.  Glucocorticoid Receptor Interacting Protein-1 Restores Glucocorticoid Responsiveness in Steroid-Resistant Airway Structural Cells 
Glucocorticoid (GC) insensitivity represents a profound challenge in managing patients with asthma. The mutual inhibition of transcriptional activity between GC receptor (GR) and other regulators is one of the mechanisms contributing to GC resistance in asthma. We recently reported that interferon regulatory factor (IRF)-1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle (ASM) cells by interfering with GR signaling (Tliba et al., Am J Respir Cell Mol Biol 2008;38:463–472). Here, we sought to determine whether the inhibition of GR function by IRF-1 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 (GRIP-1), a known GR transcriptional co-activator. We here found that siRNA-mediated GRIP-1 depletion attenuated IRF-1–dependent transcription of the luciferase reporter construct and the mRNA expression of an IRF-1–dependent gene, CD38. In parallel experiments, GRIP-1 silencing significantly reduced GR-mediated transactivation activities. Co-immunoprecipitation and GST pull-down assays showed that GRIP-1, through its repression domain, physically interacts with IRF-1 identifying GRIP-1 as a bona fide transcriptional co-activator for IRF-1. Interestingly, the previously reported inhibition of GR-mediated transactivation activities by either TNF-α and IFN-γ treatment or IRF-1 overexpression was fully reversed by increasing cellular levels of GRIP-1. Together, these data suggest that the cellular accumulation of IRF-1 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of GRIP-1 from the GR transcriptional regulatory complexes.
PMCID: PMC2809222  PMID: 19805480
glucocorticoid; cytokine; airway smooth muscle; IRF-1; GRIP-1
6.  Phosphorylation of Liver X Receptor α Selectively Regulates Target Gene Expression in Macrophages▿ † 
Molecular and Cellular Biology  2008;28(8):2626-2636.
Dysregulation of liver X receptor α (LXRα) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRα target gene selectivity is achieved by modulation of LXRα phosphorylation. Under basal conditions, LXRα is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRα S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRα S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRα-responsive genes.
PMCID: PMC2293109  PMID: 18250151
7.  Estradiol and Selective Estrogen Receptor Modulators Differentially Regulate Target Genes with Estrogen Receptors α and βD⃞ 
Molecular Biology of the Cell  2004;15(3):1262-1272.
Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) α and β to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERα or ERβ regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERα or ERβ. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERα cells synthesized only ERα and that U2OS-ERβ cells expressed exclusively ERβ. U2OS-ERα and U2OS-ERβ cells were treated either with 17β-estradiol (E2), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERα and U2OS-ERβ cells by E2, raloxifene, and tamoxifen, respectively. Most genes regulated in ERα cells in response to E2, raloxifene, and tamoxifen were distinct from those regulated in ERβ cells. Only 38 of the 228 (17%) genes were regulated by E2 in both U2OS-ERα and U2OS-ERβ cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERα and ERβ cells. A subset of genes involved in bone-related activities regulated by E2, raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERα are distinct from those regulated by ERβ in response to E2 and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERα and ERβ
PMCID: PMC363122  PMID: 14699072
8.  Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus 
Molecular Biology of the Cell  2002;13(2):670-682.
The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.
PMCID: PMC65658  PMID: 11854421
9.  Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids 
Molecular and Cellular Biology  1999;19(7):5036-5049.
Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids.
PMCID: PMC84339  PMID: 10373553

Results 1-9 (9)