The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation.

Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.

Glucocorticoid receptor coordinates transcription factor-dominated regulatory network in macrophages.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Long glucocorticoid-induced leucine zipper (L-GILZ) protein interacts with ras protein pathway and contributes to spermatogenesis control.

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.

Glucocorticoid-induced leucine zipper is protective in Th1-mediated models of colitis.

BACKGROUND & AIMS: Inflammatory bowel diseases are relatively common diseases of the gastrointestinal tract. The relative therapeutic efficacy of glucocorticoids used in inflammatory bowel diseases resides in part in their capability to inhibit activity of nuclear factor kappaB (NF-kappaB), a transcription factor central to the inflammatory process, and the consequent production of T-helper 1 (Th1)-type cytokines. Previous studies indicate that increased expression in transgenic mice of glucocorticoid-induced leucine zipper (GILZ), a gene rapidly induced by glucocorticoids, inhibits NF-kappaB and Th1 activity. METHODS: We performed experiments with the aim to test the susceptibility of GILZ transgenic (GILZ-TG) mice to dinitrobenzene sulfonic acid-induced colitis. RESULTS: Consistent with a decreased Th1 response, GILZ-TG mice were less susceptible to colitis induction as compared with wild-type littermates, while they were more susceptible to Th2-mediated colitis. The inhibition was comparable to that obtained with dexamethasone treatment. Moreover, diminished intestinal tissue damage, associated with inhibition of NF-kappaB nuclear translocation, interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 production in CD4+ T lymphocytes of the lamina propria, was evident in GILZ-TG as compared with wild-type mice. In addition, inhibition of colitis development was also evident when GILZ fusion protein was delivered in vivo in dinitrobenzene sulfonic acid-treated WT animals as well as in interleukin-10 knockout mice. CONCLUSIONS: Together these results demonstrate that GILZ mimics the effects of glucocorticoids, suggesting a contribution of this protein to the anti-inflammatory activity of glucocorticoids in Th1-induced colitis.

Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages.

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery.

Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages.

The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.

The transcriptional coregulator GRIP1 controls macrophage polarization and metabolic homeostasis.

Diet-induced obesity causes chronic macrophage-driven inflammation in white adipose tissue (WAT) leading to insulin resistance. WAT macrophages, however, differ in their origin, gene expression and activities: unlike infiltrating monocyte-derived inflammatory macrophages, WAT-resident macrophages counteract inflammation and insulin resistance, yet, the mechanisms underlying their transcriptional programming remain poorly understood. We recently reported that a nuclear receptor cofactor-glucocorticoid receptor (GR)-interacting protein (GRIP)1-cooperates with GR to repress inflammatory genes. Here, we show that GRIP1 facilitates macrophage programming in response to IL4 via a GR-independent pathway by serving as a coactivator for Kruppel-like factor (KLF)4-a driver of tissue-resident macrophage differentiation. Moreover, obese mice conditionally lacking GRIP1 in macrophages develop massive macrophage infiltration and inflammation in metabolic tissues, fatty livers, hyperglycaemia and insulin resistance recapitulating metabolic disease. Thus, GRIP1 is a critical regulator of immunometabolism, which engages distinct transcriptional mechanisms to coordinate the balance between macrophage populations and ultimately promote metabolic homeostasis.

Minireview: nuclear receptor coregulators of the p160 family: insights into inflammation and metabolism.

Nuclear receptor coactivators (NCOAs) are multifunctional transcriptional coregulators for a growing number of signal-activated transcription factors. The members of the p160 family (NCOA1/2/3) are increasingly recognized as essential and nonredundant players in a number of physiological processes. In particular, accumulating evidence points to the pivotal roles that these coregulators play in inflammatory and metabolic pathways, both under homeostasis and in disease. Given that chronic inflammation of metabolic tissues ("metainflammation") is a driving force for the widespread epidemic of obesity, insulin resistance, cardiovascular disease, and associated comorbidities, deciphering the role of NCOAs in "normal" vs "pathological" inflammation and in metabolic processes is indeed a subject of extreme biomedical importance. Here, we review the evolving and, at times, contradictory, literature on the pleiotropic functions of NCOA1/2/3 in inflammation and metabolism as related to nuclear receptor actions and beyond. We then briefly discuss the potential utility of NCOAs as predictive markers for disease and/or possible therapeutic targets once a better understanding of their molecular and physiological actions is achieved.

GILZ promotes production of peripherally induced Treg cells and mediates the crosstalk between glucocorticoids and TGF-ß signaling.

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) control immune responses and prevent autoimmunity. Treatment with glucocorticoids (GCs) has been shown to increase Treg cell frequency, but the mechanisms of their action on Treg cell induction are largely unknown. Here, we report that glucocorticoid-induced leucine zipper (GILZ), a protein induced by GCs, promotes Treg cell production. In mice, GILZ overexpression causes an increase in Treg cell number, whereas GILZ deficiency results in impaired generation of peripheral Treg cells (pTreg), associated with increased spontaneous and experimental intestinal inflammation. Mechanistically, we found that GILZ is required for GCs to cooperate with TGF-ß in FoxP3 induction, while it enhances TGF-ß signaling by binding to and promoting Smad2 phosphorylation and activation of FoxP3 expression. Thus, our results establish an essential GILZ-mediated link between the anti-inflammatory action of GCs and the regulation of TGF-ß-dependent pTreg production.

Glucocorticoid-induced leucine zipper (GILZ) over-expression in T lymphocytes inhibits inflammation and tissue damage in spinal cord injury.

Spinal cord injury (SCI) is a traumatic event that causes a secondary and extended inflammation characterized by infiltration of immune cells, including T lymphocytes, release of pro-inflammatory mediators in the lesion site, and tissue degeneration. Current therapeutic approaches for SCI are limited to glucocorticoids (GC) due to their potent anti-inflammatory activity. GC efficacy resides, in part, in the capability to inhibit NF-κB, T lymphocyte activation, and the consequent cytokine production. In this study, we performed experiments aimed to test the susceptibility of glucocorticoid-induced leucine zipper (GILZ) transgenic (GILZ(TG)) mice, in which GILZ is selectively over-expressed in T lymphocytes, to SCI induction. Consistent with a decreased inflammatory response, GILZ(TG) were less susceptible to SCI as compared to wild-type littermates. Notably, inhibition of NF-κB activation and nuclear translocation, diminished T lymphocytes activation and tissue infiltration, as well as decreased release of cytokines were evident in GILZ(TG) as compared to wild-type mice. Moreover, GILZ(TG) showed a reduced tumor necrosis factor-α, IL-1ß, Inductible nitric oxide synthase (iNOS) and nytrotyrosine production, apoptosis, and neuronal tissue damage. Together these results indicate that GILZ mimics the anti-inflammatory effect of GC and represents a potential pharmacological target for modulation of T lymphocyte-mediated immune response in inflammatory disorders, such as SCI.