Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Crohn's disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.

Ligand-independent phosphorylation of the glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling.

Glucocorticoids are stress hormones that maintain homeostasis through gene regulation mediated by nuclear receptors. We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Cells expressing a mutant glucocorticoid receptor incapable of phosphorylation at Ser134 (S134A-GR) had significantly altered hormone-dependent genome-wide transcriptional responses and associated hormone-mediated cellular functions. The phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform of the 14-3-3 class of signaling proteins (14-3-3zeta) on chromatin promoter regions, resulting in a blunted hormone-dependent transcriptional response of select genes. These data argue that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to redirect glucocorticoid-regulated signaling through altered 14-3-3zeta cofactor binding and promoter recruitment. This posttranslational modification allows prior cellular stress signals to dictate the transcriptional response to glucocorticoids.

Glycogen synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles.

Aberrant glycogen synthase kinase 3beta (GSK-3beta) activity is associated with the progression of several pathological conditions such as diabetes, Alzheimer's, and cancer. GSK-3beta regulates cellular processes by directly phosphorylating metabolic enzymes and transcription factors. Here, we discovered a new target for GSK-3beta phosphorylation: the human glucocorticoid receptor (GR). Glucocorticoid signaling is essential for life and regulates diverse biological functions from cell growth to metabolism to apoptosis. Specifically, we found hormone-dependent GR phosphorylation on serine 404 by GSK-3beta. Cells expressing a GR that is incapable of GSK-3beta phosphorylation had a redirection of the global transcriptional response to hormone, including the activation of additional signaling pathways, in part due to the altered ability of unphosphorylatable GR to recruit transcriptional cofactors CBP/p300 and the p65 (RelA) subunit of NF-kappaB. Furthermore, GSK-3beta-mediated GR phosphorylation inhibited glucocorticoid-dependent NF-kappaB transrepression and attenuated the glucocorticoid-dependent cell death of osteoblasts. Collectively, our results describe a novel convergence point of the GSK-3beta and the GR pathways, resulting in altered hormone-regulated signaling. Our results also provide a mechanism by which GSK-3beta activity can dictate how cells will ultimately respond to glucocorticoids.