Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids

Abstract

Inhibition of cytokine gene expression by the hormone-activated glucocorticoid receptor (GR) is the key component of the anti-inflammatory actions of glucocorticoids, yet the underlying molecular mechanisms remain obscure. Here we report that glucocorticoid repression of cytokine genes in primary macrophages is mediated by GR-interacting protein (GRIP)1, a transcriptional coregulator of the p160 family, which is recruited to the p65-occupied genomic NFκB-binding sites in conjunction with liganded GR. We created a mouse strain enabling a conditional hematopoietic cell-restricted deletion of GRIP1 in adult animals. In this model, GRIP1 depletion in macrophages attenuated in a dose-dependent manner repression of NFκB target genes by GR irrespective of the upstream Toll-like receptor pathway responsible for their activation. Furthermore, genome-wide transcriptome analysis revealed a broad derepression of lipopolysaccharide (LPS)-induced glucocorticoid-sensitive targets in GRIP1-depleted macrophages without affecting their activation by LPS. Consistently, conditional GRIP1-deficient mice were sensitized, relative to the wild type, to a systemic inflammatory challenge developing characteristic signs of LPS-induced shock. Thus, by serving as a GR corepressor, GRIP1 facilitates the anti-inflammatory effects of glucocorticoids in vivo.

Inflammation is a major defense mechanism against infection and injury. Acute inflammation at the site of invasion is initiated by resident cells of the innate immune system (neutrophils, macrophages, and dendritic cells) that recognize certain molecules shared by pathogenic agents via pattern-recognition receptors such as Toll-like receptors (TLRs), Nod-like receptors, or retinoic acid-inducible gene I family members. TLRs recognize pathogenic molecular patterns, such as lipopolysaccharide (LPS), peptidoglycans, single- and double-stranded nucleic acids, and common bacterial surface proteins (1). TLR-activated signaling cascades ultimately converge on a few transcription factors of the NFκB/Rel, AP1, and IRF families (2), which, acting in concert, induce the expression of hundreds of genes encoding cytokines, chemokines, and other proinflammatory mediators. These, in turn, further activate and stimulate the migration of monocytes, neutrophils, and leukocytes to injured sites, eventually leading to pathogen clearance and tissue repair. Although normally a protective response, an excessive production of cytokines may result in local or systemic tissue damage, multiple organ failure, and death. Many pathological and autoimmune conditions, such as rheumatoid arthritis or lupus, are associated with chronic inflammation and are characterized by unique “signatures” of overproduced cytokines (3).

Because unabated inflammation imposes an intrinsic danger to the host, numerous mechanisms have evolved to control the expression and activities of proinflammatory mediators in a spatial and temporal manner. Locally, inflammatory stimuli induce the expression of anti-inflammatory cytokines, such as IL10, factors destabilizing cytokine mRNA, and microRNAs targeting various steps in proinflammatory cytokine production and signaling (4). When local responses fail to contain an inflammatory reaction, cytokines produced by immune cells activate the hypothalamic–pituitary–adrenal (HPA) axis stimulating the adrenal cortex to synthesize and release glucocorticoids (GCs), which ultimately suppress proinflammatory gene expression (5). A disruption of the HPA axis, either physical or pharmacological, decreases the level of circulating GCs and results in hypersensitivity to inflammatory stimuli (6, 7). Due to their uniquely potent ability to counter inflammation, for over half a century, GCs have remained among the most cost-effective and widely used anti-inflammatory and immunosuppressive drugs available.

GCs act through the cytoplasmic glucocorticoid receptor (GR), a transcription factor of the nuclear receptor (NR) superfamily (8), which, upon ligand binding, translocates into the nucleus and either binds directly to a palindromic DNA sequence, a glucocorticoid response element (GRE), composed of two hexameric AGAACA half-sites or “tethers” to DNA through protein:protein interactions with other DNA-bound factors such as AP1 or NFκB (9). Although GR can either activate or repress transcription, typically, binding to “conventional” palindromic GREs leads to activation, whereas GR tethering to AP1 or NFκB usually attenuates transcription of target genes. Both activation of anti-inflammatory genes and repression of proinflammatory genes have been implicated in GC-mediated suppression of inflammation (2, 9, 10).

GR elicits transcriptional changes by recruiting multiple cofactors that act upon components of basal machinery or chromatin. From over 100 GR-interacting proteins (GRIP), the members of the p160 family—SRC1/Ncoa1, GRIP1/TIF2/Ncoa2 (hereafter, GRIP1), and SRC3/Ncoa3—are considered primary coactivators by serving as binding platforms for additional cofactors with chromatin-modifying and remodeling activities, e.g., histone acetyltransferases CBP/p300, methyltransferases CARM1, G9a, Suv4-20h1, and a host of secondary adapter proteins (reviewed in ref. 11). All three p160s are recruited to “conventional” palindromic GREs via their conserved NR interaction domain (NID) in a ligand-dependent manner and facilitate transcriptional activation.

GR-mediated “transrepression,” i.e., the antagonism of AP1- and NFκB-dependent transcription by the tethered ligand-activated GR has historically been considered the dominant mechanism of the anti-inflammatory actions of GCs (9). Indeed, GR has long been known to directly bind the p65 subunit of NFκB and the Jun subunit of AP1, which correlates with inhibition of NFκB and AP1 reporters and endogenous targets (12, 13). Furthermore, a recent genome-wide analysis of GR and p65 binding in HeLa cells costimulated with GC and TNFα revealed a substantial number of shared sites (14); yet, the molecular consequences of these interactions are unknown.

In principle, GR recruitment to AP1/NFκB sites is likely to alter the composition or function of associated transcriptional regulatory complexes or the activity of RNA polymerase (Pol)2 itself. For example, in the context of the IL8 and ICAM1 promoters, GR does not interfere with Pol2 recruitment or initiation complex assembly, but, instead, blocks Pol2 CTD phosphorylation and productive elongation (13). In an alternate scenario, GR may engage a context-specific corepressor; however, the identity of such a coregulator remains enigmatic. It has been recently shown that GR bound to atypical “negative” GREs recruits the established NR corepressors NCoR and SMRT (15), yet there is no evidence of their function at the GR tethering sites and, consistently, GR-mediated transrepression of inflammatory cytokine genes was unaffected in NCoR-deficient mice (16). Intriguingly, the recruitment of the p160 cofactor GRIP1, and not other p160s, to an AP1 site in the human MMP13 promoter correlated with glucocorticoid repression (17), perhaps suggesting that GRIP1 may play a role in GC-mediated repression of proinflammatory genes. GRIP1-null mice display multiple metabolic, endocrine, and reproductive defects (18, 19); however, the role of GRIP1 in the context of the immune system or inflammation is unknown. In addition, GRIP1 deficiency disrupts the normal development of the adrenal glands leading to the aberrant regulation of the HPA axis and GC production (20), making these mice poorly suited for assessing immune responses in vivo. To elucidate the function of GRIP1 in a relevant cellular environment in pro- and anti-inflammatory settings, we created a mouse strain enabling a conditional GRIP1 depletion in myeloid cells in adult mice. Here we describe the genome-wide impact of GRIP1 deficiency in macrophages with respect to the inflammatory gene expression program. We further provide evidence for the critical role of GRIP1 in controlling systemic inflammation in vivo.

Results

GR Represses TLR-Induced Cytokine Gene Expression at the Level of Transcription.

To evaluate the effect of GCs on cytokine gene expression, we treated bone marrow-derived macrophages (BMMΦ) from C57B/6 mice with ligands for TLR2 (Pam3Cys), TLR4 (LPS), and TLR7 (CL264) in the absence or presence of a synthetic GC dexamethasone (Dex). As expected, within 1 h, treatment with TLR ligands induced the expression of cytokines IL1α, IL1β, and TNFα and chemokines CCL2, CCL3, and CCL4 and other inflammatory mediators (Fig. 1A). Both synthetic (Dex, Fig. 1A) and natural murine (corticosterone, Fig. S1B) GCs markedly decreased the expression of all TLR-induced genes tested. Although the kinetics and magnitude of inhibition was gene-specific, it was evident as early as 30 min at Dex concentration as low as 1 nM (Fig. S1A). These results indicate that GCs inhibit TLR target genes irrespective of the specific TLR responsible for their activation.

Fig. 1.

GCs inhibit the transcription of TLR-induced genes. (A) Dex inhibits TLR-induced expression of inflammatory mediators. BMMΦ were treated with 10 ng/mL LPS, 100 ng/mL Pam3Cys, or 1 μg/mL CL264 ± 100 nM Dex for 1 h. mRNA levels of proinflammatory cytokines and chemokines were assayed by RT-quantitative PCR (qPCR), normalized to β-actin or GAPDH, and fold induction was calculated relative to the untreated (= 1). Data are represented as mean ± SEM. (B) Dex suppresses LPS-induced gene expression in the absence of new protein synthesis. BMMΦ were pretreated for 30 min with 20 μg/mL CHX, followed by treatment with 10 ng/mL LPS ± 100 nM Dex, as indicated. mRNA levels of target genes were assessed as in A. (C–F) GCs inhibit cytokine gene expression at the level of transcription. BMMΦ were treated with 10 ng/mL LPS ± 100 nM Dex for 30 min, and Pol2 occupancy at the TSS and downstream regions of the indicated genes was assessed by ChIP with anti-Pol2 antibody. For each location, qPCR signals were normalized to those at the control r28S gene and expressed as fold enrichment over normal IgG (= 1). Data are derived from three or more independent experiments and expressed as mean ± SD. P values were calculated using two-tailed Student’s t-test.

GC-activated GR may repress TLR target genes either directly or by inducing the expression of another protein that in turn down-regulates cytokine genes. To discriminate between these possibilities, we preincubated BMMΦ with the protein synthesis inhibitors cycloheximide (CHX) or puromycin (Puro) followed by treatment with LPS ± Dex and monitored RNA levels of GC-sensitive inflammatory cytokines. As seen previously (21), CHX and Puro elevated the baseline expression of most analyzed genes (Fig. 1B and Fig. S1C); nonetheless, incubation with LPS resulted in further cytokine gene induction, which was attenuated by Dex, demonstrating that GC-mediated cytokine down-regulation is direct and does not rely on the prior synthesis of a protein intermediate.

Both Pol2 recruitment and productive elongation are rate-limiting steps for signal-regulated transcription. To assess the effect of GCs on Pol2 dynamics, we evaluated the effect of LPS and Dex on Pol2 occupancy at the transcription start sites (TSS) of cytokine genes and intragenically by chromatin immunoprecipitation (ChIP). In uninduced MΦ, little Pol2 was detected at IL1α, IL1β, and CCL4 (Fig. 1 C, D, and F, respectively), and comparatively high Pol2 occupancy was detected near the TNFα TSS (Fig. 1E), consistent with the presence of stalled Pol2 in the TNFα promoter-proximal region (22). LPS treatment resulted in a robust recruitment of Pol2 to the TSS as well as intragenic regions of IL1α, IL1β, and CCL4 (Fig. 1 C, D, and F). At the TNFα, Pol2 occupancy was strongly induced intragenically, reflecting the appearance of elongating polymerase in the body of the gene (Fig. 1E). This LPS-dependent increase in occupancy was nearly abrogated by Dex, indicating that liganded GR antagonizes transcription initiation (for IL1α, IL1β, and CCL4) or elongation (for TNFα). Further corroborating the transcriptional effects of GR on IL1α, IL1β, TNFα, and CCL4 expression, the dramatic LPS-dependent accumulation of their nascent unprocessed transcripts was abrogated by Dex (Fig. S1D).

Dex-Mediated Repression of LPS-Induced Genes Involves GR and GRIP1 Recruitment to NFκB-Binding Sites.

NFκB is a primary transcriptional effector of TLR signaling. To evaluate the effect of GCs on NFκB recruitment to its targets, we first determined p65 occupancy at the NFκB-binding sites in the regulatory regions of IL1α, IL1β, TNFα, and CCL4 (Fig. 2A). Treatment with LPS induced a dramatic recruitment of p65 to several NFκB-binding sites, including those in the IL1α/β intergenic region and upstream of the TNFα and CCL4 TSS (Fig. 2B). Coadministration of Dex had no effect on p65 occupancy at any of the nine sites analyzed (Fig. 2B), indicating that the loss of Pol2 and repression of transcription are not due to NFκB displacement.

Fig. 2.

Transcription complexes at the NFκB-binding sites at activated and repressed cytokine genes. (A) IL1α/β and TNFα loci maps showing the NFκB-binding peaks derived from the p65 ChIP-Seq data set (23). The raw read data were mapped to the mouse genome and visualized using CLC Genomics Workbench 4.8. The center positions of NFκB sites that we validated by ChIP with p65 antibodies are indicated. Binding sites are shown as red, blue, and green rectangles under the read distributions for NFκB, GR, and GRIP1, respectively. (B) Dex does not affect LPS-dependent p65 recruitment to cytokine genes. BMMΦ were treated with 10 ng/mL LPS ± 100 nM Dex for 1 h, and ChIPs for p65 were performed as in Fig. 1C. Error bars represent mean ± SD for three or more independent experiments. (C and D) GR and GRIP1 are recruited to p65-binding sites in LPS + Dex-dependent manner. ChIPs for GR (C) and GRIP1 (D) were performed in BMMΦ treated as indicated. Amplification of the GILZ GRE is used as a positive control for Dex-dependent GR and GRIP1 recruitment. Fold enrichments over normal IgG are calculated as in Fig. 1C (*P < 0.05).

Interactions between NFκB and GR in vivo and in vitro (13) play a critical role in GC-mediated inhibition of NFκB-driven transcription. To determine whether GR is recruited to NFκB-binding sites, we surveyed GR occupancy by ChIP. In control experiments, GR was recruited to the previously reported GR-binding sites (GBS) at the GILZ promoter following a 1-h treatment with Dex or LPS + Dex (Fig. 2C, Right). Furthermore, Dex + LPS cotreatment, but not Dex alone, also triggered a significant GR loading onto a subset of NFκB-binding sites in BMMΦ (Fig. 2C), suggesting that NFκB activation and p65 binding (Fig. 2B) are necessary for the recruitment of GR.

Although the mechanisms of GR-mediated repression at tethering sites are unknown, the p160 cofactor GRIP1 was shown to be recruited to the AP1 site of the repressed human MMP13 gene in osteosarcoma cells (17). To determine whether GRIP1 was a component of GR repression complexes at tethering GBS of proinflammatory genes in MΦ, we assessed GRIP1 occupancy at the NFκB-binding sites in our candidate cytokine genes. In parallel with GR, GRIP1 was recruited to the above NFκB sites in response to LPS + Dex, but not Dex alone (Fig. 2D). Conversely, treatment with Dex alone was sufficient to facilitate the enrichment of GRIP1 at the GBS of the GILZ promoter (Fig. 2D), which correlated with GR loading (Fig. 2C). Combined, these results indicate that GC-mediated repression of cytokine genes correlates with recruitment of GR and GRIP1 to a set of p65-occupied NFκB-binding sites.

Generation of Conditionally GRIP1-Deficient Mice.

GRIP1 knock-out (null) mice, in addition to their fertility phenotype, display pleiotropic changes in metabolism and disrupted adrenal architecture and function with compensatory changes in the HPA axis (20), which complicates the assessment of GC signaling in vivo. To overcome these issues, we created a mouse strain enabling an inducible deletion of GRIP1 in adult animals preferentially in hematopoietic cells including MΦ. The GRIP1-WT mice (^−/−:GRIP1^FL/FL) were bred to Mx1Cre mice (Mx1Cre-WT) expressing polyinosinic:polycytidylic acid (pIC)-inducible Cre recombinase to ultimately generate GRIP1-KD, GRIP1-HET, and GRIP1-WT mice (see SI Materials and Methods and Fig. S2A for details). This protocol resulted in a deletion of a floxed allele and a 95% depletion of GRIP1 transcript and protein in BMMΦ from GRIP1-KD mice as determined by RT-quantitative PCR (qPCR) and Western blotting, respectively, whereas the expression of GR (Fig. 3A) or the other two p160 family members (Fig. S2B) was unchanged. Notably, pIC injections 2 wk before bone marrow isolation did not trigger nonspecific long-term changes in the expression or chromatin of TLR-regulated genes (Fig. S2 C and D). In addition, “acute” conditional GRIP1 depletion did not affect MΦ differentiation based on the expression of the surface markers CD11b and F4/80 in GRIP1-deficient vs. GRIP1-sufficient BMMΦ (Fig. S2E).

Fig. 3.

GR represses TLR-induced genes in a GRIP1-dependent manner. (A) GRIP1 expression in the BMMΦ of GRIP1-KD mice. GRIP1 mRNA in WT and KD BMMΦ was measured by RT-qPCR, normalized to β-actin, and expressed relative to WT (= 100%). Error bars show mean ± SD; n = 6. GRIP1 and GR protein levels in WT and KD BMMΦ were assessed by immunoblotting with tubulin as a loading control. (B) GRIP1 depletion attenuates GR-mediated repression of TLR-induced genes. BMMΦ from WT and GRIP1-KD littermate mice were treated as in Fig. 2B. The relative amounts of indicated transcripts were measured and expressed as ratios of LPS/LPS + Dex (“fold repression”) for each genotype. Average fold repression in WT vs. KD was compared using a two-tailed Student’s t-test (nWT = 6, nKD = 7). (C) GRIP1 facilitates repression in a dose-dependent manner. BMMΦ from WT (WT/WT), HET (WT/FL), and GRIP1-KD (FL/FL) mice were treated as in B, and the TNFα relative RNA amount was measured and expressed as fold repression for each genotype. The corresponding GRIP1 RNA levels are shown as percentage of WT (= 100). (D) GRIP1 mediates GR-dependent repression of cytokine genes irrespective of the TLR pathway responsible for their activation. WT and GRIP1-KD BMMΦ were treated with LPS, Pam3Cys, or CL264 ± Dex, and fold repression of TNFα and IL1β RNA was derived as in B.

GRIP1 Deletion Attenuates GR-Mediated Repression of LPS-Induced Genes in BMMΦ.

To determine the effect of GRIP1 disruption on GC-mediated repression, we treated WT and GRIP1-KD BMMΦ with LPS ± Dex and determined the mRNA levels of proinflammatory cytokines. As expected, LPS potently induced cytokine RNAs in both MΦ populations (Fig. S2F). Strikingly, Dex-mediated repression of IL1α, IL1β, TNFα, and CCL4 was significantly attenuated in GRIP1-KD BMMΦ (Fig. 3B). The effect of GRIP1 depletion was dose-dependent, as an intermediate level of repression was observed in the GRIP1 haplo-insufficient GRIP1-HET BMMΦ (Fig. 3C). Furthermore, GRIP1 deletion similarly reversed GC repression of cytokine genes induced by ligands to TLR2 and TLR7 (Fig. 3D), suggesting that GR-mediated repression is GRIP1-dependent regardless of the TLR pathway responsible for gene activation.

To determine the genome-wide effect of GRIP1 deletion on the inflammatory gene expression program, we evaluated the MΦ transcriptome by RNA-Seq in WT and GRIP1-KD BMMΦ. The Illumina cDNA libraries were prepared from BMMΦ of both genotypes treated with Dex, LPS, or both for 1 h and untreated control and sequenced. Of 349 genes induced by LPS >2-fold in WT and KD BMMΦ, 152 were repressed ≥1.4-fold in the WT BMMΦ (Fig. 4A). We ranked Dex-repressed genes in the WT according to their “fold repression” and evaluated the corresponding levels of repression in the KD. Strikingly, over 60 LPS-induced Dex-repressed genes were derepressed by >15% in GRIP1 KD relative to WT BMMΦ (Fig. 4B; Fig. S3A; Table S1), suggesting an unexpectedly broad role of GRIP1 in GR-mediated repression. Gene Ontology term enrichment analysis of derepressed genes performed with G:Profile (24) revealed a high prevalence of terms related to regulation of immune and inflammatory responses, cytokine production, and cell death (Table S2). Interestingly, a computational analysis of the 1,000-bp region upstream of the TSS of these genes showed a significant enrichment of the binding sites for the members of the NFκB and AP1 families, but not of the binding sites for GR or other NRs (Table S3).

Fig. 4.

GRIP1 deletion globally attenuates GR-mediated repression of LPS-induced genes in BMMΦ. (A) Transcriptome analysis of WT and GRIP1-KD BMMΦ treated for 1 h with LPS or LPS + Dex. Venn diagram shows gene overlap between treatments and genotypes. (B) A subset of Dex-repressed genes is derepressed in GRIP1-KD BMMΦ. Gene expression levels were determined by RNA-Seq, normalized to gene length, and expressed as reads per kilobase of exon per million mapped reads (SI Materials and Methods). Fold repression by Dex was determined as a ratio of expression levels in LPS− vs. LPS + Dex-treated BMMΦ. LPS-induced (more than twofold) genes repressed by Dex in WT were ranked by the fold repression, and the corresponding ratios for 64 genes derepressed in a KD are plotted. The median fold repression for each population and the results of the Mann–Whitney comparison are shown in the Inset. (C) GRIP1 depletion does not affect gene induction by LPS. Gene expression levels were determined by RNA-Seq, and LPS-induced (more than twofold) genes in WT and GRIP1-KD BMMΦ were compared as in B.

Because we defined fold repression as a ratio of expression in the presence of LPS over LPS + Dex, in principle, it could be skewed due to a hypothetical global effect of GRIP1 deletion on the induction of various genes by LPS. To rule out this potential variable, we compared fold LPS induction of the 349 genes activated by LPS in WT BMMΦ to corresponding values in the KD. Our analysis detected no statistically significant difference in the transcriptome-wide LPS induction between WT and KD cells (Fig. 4C). Because GRIP1 serves as a GR coactivator in the context of palindromic GREs, we assessed whether GRIP1 depletion also attenuates the induction of the GR transcriptional targets with anti-inflammatory activities: DUSP1, IκB, and GILZ. We found no statistically significant difference in the expression of these genes between WT vs. KD BMMΦ in two independent RNA-Seq experiments (Fig. S3B), arguing against their contribution to the observed attenuation of glucocorticoid repression. This finding is consistent with the lack of an effect of the protein synthesis inhibitors on GR-mediated repression (Fig. 1B and Fig. S1C).

GRIP1 Is Protective Against Systemic Inflammatory Responses in Vivo.

The production of endogenous GCs is an important negative feedback mechanism that is initiated in response to inflammatory stimuli and inhibits a heightened inflammatory response by limiting the transcription of cytokines and other proinflammatory mediators. Because GRIP1 depletion in MΦ impaired GR-mediated repression of cytokine genes (Fig. 3B; Fig. 4B; Fig. S3), we speculated that GRIP1 may be involved in the HPA-dependent anti-inflammatory feedback in vivo, thereby conferring protection against an exaggerated inflammatory response. LPS-induced toxic shock is commonly used as a model of unabridged inflammation. We first determined that 20 mg/kg of LPS administered i.p. is the maximum sublethal dose tolerated by WT controls. We then injected age-matched WT and GRIP-KD mice with LPS and monitored them for 96 h for hallmarks of septic shock such as changes in temperature and weight, locomotor activity, and shivering; mice found in a moribund state were euthanized as per the National Institutes of Health and Institutional Animal Care and Use Committee protocols. As shown in Fig. 5A, the mortality rate of GRIP1-KD animals was significantly higher (P < 0.05), which correlated with a greater loss of body weight than that of their WT counterparts (Fig. 5B). These observations suggested that GRIP1-depleted mice may suffer from a “cytokine storm” due to failure to control the production of proinflammatory cytokines. Indeed, the levels of serum TNFα, IL1β, and IFNγ were significantly higher (P < 0.05) in GRIP1-KD compared with the WT mice (Fig. 5C), suggesting that GRIP1 depletion results in the deregulation of endogenous responses curbing inflammation.

Fig. 5.

GRIP1 plays a protective role in a systemic inflammatory response in vivo. (A and B) GRIP1 conditionally depleted mice are sensitized to septic shock. GRIP1-WT and GRIP1-KD mice (n = 6 for each genotype) were injected i.p. with 20 mg/kg LPS and monitored for the indicated time. Survival is shown via the Kaplan–Meier plot (A). Body weight of WT and GRIP1-KD mice 36 h post injection was expressed as a percentage of pre-LPS weight and compared using the Mann–Whitney test (B). (C) GRIP1 deletion results in increased serum cytokine levels in response to systemic LPS challenge. GRIP1-WT and GRIP1-KD mice were injected with LPS, and their serum cytokines were measured at 1.5 h (TNFα) or 5 h (IL1β and IFNγ). P values (n = 5 or 6) were calculated using two-tailed Student’s t-test.

Discussion

The immune system is under constant pressure from a variety of environmental antigens in a state aptly termed “chronic immunological stress” (25). Indeed, from an evolutionary perspective, the proper function of the immune system requires a trade-off between hypo-activation that leads to rampant multiplication and spread of pathogens and hyper-activation that may result in immunopathologies and a breakdown of homeostasis. This delicate equilibrium is maintained by a number of highly redundant mechanisms that gradually escalate immune responses commensurate with the magnitude of injurious signal. The “decision” to proceed to a more vigorous response often hinges on a balance between pro- and anti-inflammatory cytokines and chemokines. Tipping this balance toward proinflammatory mediators triggers a systemic response that, among others, increases the level of serum GCs, which, in turn, suppresses the expression of proinflammatory cytokines, thereby abating inflammation.

The effect of GR on gene expression is remarkably broad. Even a brief exposure of BMMΦ to GCs results in the inhibition of over 100 LPS-induced genes. Others have shown that, following a prolonged treatment, up to 30% of total expressed genes become affected (26, 27), making the mechanistic analysis of GR action a daunting task. Indeed, although the suppressive effect of GCs on cytokine transcription has been studied for almost 3 decades, no unifying mechanism has been proposed, and, in all likelihood, numerous pathways are engaged to suppress excessive inflammatory signaling. It is conceivable that the mechanisms of repression are at least in part determined by the events responsible for cytokine gene induction in response to TLR ligands. Recent studies broadly classified TLR-induced genes into immediate-early (IE) genes, which are activated by the resident transcription factors (NFκB, IRFs, and AP1) within minutes of stimulation and “delayed” genes, whose activation requires protein synthesis and amplification of the initial signal by the IE cytokines (28–30). Furthermore, IE vs. delayed genes are often activated at different stages of the transcription cycle (22, 28). Specifically, IE genes (e.g., TNFα) display features that are characteristic of “stalled” genes activated by Pol2 release from the early elongation block: extensive histone acetylation, hypomethylation, and a high level of Pol2 near the TSS in uninduced state. Conversely, the delayed genes commonly lack detectable Pol2 at the TSS before stimulation and are induced by signal-dependent Pol2 recruitment and transcription initiation. Surprisingly, we found that, in both cases, GR activation imparted a dramatic, protein synthesis-independent repression, as well as the chromatin marks typical of unstimulated cells.

What are the molecular targets of GR for each class of genes? Physical interactions between NFκB or AP1 family members and GR have long been implicated in the direct inhibition of proinflammatory gene transcription. In MΦ, we observed GR recruitment to several NFκB sites in the regulatory regions of TNFα, IL1α, and IL1β that was contingent upon LPS activation and occurred concomitantly with p65 recruitment. Interestingly, a recent genome-wide analysis of GR and p65 binding in HeLa cells cotreated with the GC triamcinolone acetonide and TNFα revealed 1,033 GR binding peaks (12% of the total GR cistrome), more than half of which were also co-occupied by p65 (14). Consistent with our findings, some of these sites are associated with genes encoding proinflammatory mediators whose transcription is inhibited by GCs.

The potential involvement of GRIP1 in GR-mediated repression was suggested by an earlier observation of its recruitment to the AP1:GR tethering complex at the MMP13 promoter in the U2OS-GR stable cell line (17). However, the physiological relevance of this finding to the inflammatory gene expression program or regulation of inflammation in vivo was never assessed. Among the various phenotypes of GRIP1-deficient mice, those in reproduction and energy metabolism were most extensively described, but reflect primarily the multifaceted functions of GRIP1 as a cofactor for other NRs, such as PPARγ and RORα (18, 31). The study of GC signaling in the immune system of GRIP1-null animals is further complicated by their adrenal insufficiency and decreased level of corticosterone in the blood (20). Our conditional GRIP1-KD mice enabled a comprehensive evaluation of the GRIP1 function in shaping the MΦ transcriptome and its physiological implications for the control of systemic inflammation. This genome-wide approach revealed an unexpectedly broad role of GRIP1 in GR-mediated repression of proinflammatory genes without appreciably affecting their induction by LPS. Indeed, derepression of many Dex-sensitive TLR target genes in GRIP1-KD was seen not only upon LPS stimulation, but also at the level of basal expression (not shown). This result is not unexpected, given that many AP1 sites are constitutively occupied by cJun, which may provide a recruitment site for GR. Consistently, the regulatory regions of the derepressed genes were devoid of GREs but enriched for AP1- and NFκB-binding sites.

Taken together, our results identify GRIP1 as a GR corepressor of proinflammatory mediators in BMMΦ; however, the mechanistic basis of corepression remains to be determined. The GRIP1 corepressor activity, at least in vitro, was localized to a unique repression domain (RD) encompassing GRIP1 amino acids 767–1006, which is not shared by other p160 family members (32) and contains no predicted structural motifs or enzymatic activities. Instead, we discovered that RD participates in protein:protein interactions with several transcriptional regulators including members of the IRF family and the histone methyltransferase Suv4-20h1 (33–35). Conceivably, the RD-interacting secondary GR corepressor mediating GRIP1 actions at tethering elements awaits identification. Unexpectedly, much like the effects of GR itself, the loss of GRIP1 affected both stalled and initiation-controlled genes, perhaps suggesting that, in conjunction with GR, GRIP1 has as-yet-unknown functions in elongation control.

GRIP1 was originally described as an NR coactivator (36, 37); therefore, in principle it was possible that GRIP1 deletion would also attenuate GR-dependent activation of anti-inflammatory genes, such as GILZ, DUSP1, or IκB. Indeed, both GILZ and DUSP1 contribute to the inhibition of inflammation in various systems (10). Interestingly, we detected no significant difference in GILZ, DUSP1, and IκB RNA levels upon a 1-h induction by Dex between the WT and KD BMMΦ in the presence or absence of LPS, suggesting either that the expression of these genes does not rely on GRIP1 or that other members of the p160 family expressed in BMMΦ are sufficient.

Recent analyses of transcriptional regulatory networks revealed that a large number of DNA-binding regulators rely on relatively few cofactors to coordinate gene expression in response to a variety of environmental stimuli. Indeed, GRIP1 has a wide-ranging specificity of protein:protein interactions, including those with MEF2C, MyoD, and IRFs (33, 35, 38, 39), regulating processes as diverse as muscle-cell differentiation and innate immune response to double-stranded RNA. What promotes the preferential targeting of GRIP1 to a specific regulatory complex or its ability to inhibit or activate transcription is unknown, but recently discovered GRIP1 posttranslational modifications such as SUMOylation and phosphorylation (40, 41) may serve as such a mechanism. In addition, GRIP1 expression itself is potentially amenable to regulation: it was found to be overexpressed or fused to other regulators, including MOZ, HEY1, and ETV6 in a number of cancers (refs. 42 and 43 and references therein). Here, we established that GRIP1-KD mice are hypersensitive to LPS due to a failure to repress the IE proinflammatory cytokines (e.g., TNFα, IL1α, and IL1β) and a secondary amplification of cytokines such as IFNγ, which are not direct targets for GR:GRIP1. Future studies will reveal whether specific changes in GRIP1 modifications or expression occur in pathological conditions characterized by chronic inflammation when the balance of pro- and anti-inflammatory signaling is disrupted. Intriguingly, a survey of GRIP1 expression in autoimmune diseases using the NextBio database (www.nextbio.com) revealed that in synovial fibroblasts from patients with rheumatoid arthritis GRIP1 was down-regulated by inflammatory stimulation; GRIP1 mRNA levels were also reduced in peripheral monocytes of patients with lupus and in lupus models. Thus, a dysregulation of GRIP1 may provide the molecular basis for the instances of clinical GC resistance when the function of GR itself appears grossly normal—an attractive hypothesis that remains to be tested.

Materials and Methods

Cell Culture and Reagents.

BMMΦ were prepared from 8- to 12-wk-old mice as described (36). Dex, corticosterone, LPS, pIC, and CHX were purchased from Sigma. Pam3Cys, CL264, and Puro were purchased from Invivogen.

RNA Isolation, Real-Time PCR, and RNA-Seq.

Total RNA isolation, random-primed cDNA synthesis, qPCR with ROX containing SYBR-green master mix (Fermentas), and δδCt analysis were performed as described (34). Primers are listed in Table S4. For RNA-Seq, total RNA was isolated from BMMΦ from two independent pairs of GRIP1-WT and KD littermates and subjected to RNA-Seq as described in SI Materials and Methods.

Immunoblotting and ChIP.

See SI Materials and Methods.

Transgenic Mice.

See SI Materials and Methods.

Survival Analysis.

GRIP1-WT and KD mice were injected i.p. with 20 mg/kg of LPS. Mice were weighed before and at 36 h post injection, and the significance of difference between WT and KD groups was determined using the two-tailed Student’s t-test. The recovery of mice following LPS injection was monitored for up to 96 h. The survival curves of the two groups were evaluated by Kaplan–Meyer analysis, and the P value was determined using a log-rank test.

Cytokine Measurement.

Mouse inflammation and IL1β Flex Cytometric Bead Array kits (BD Biosciences) were used for detecting TNFα, IFNγ, and IL1β, and the data were analyzed with FACScan.