Cross-talk between the glucocorticoid receptor and MyoD family inhibitor domain-containing protein provides a new mechanism for generating tissue-specific responses to glucocorticoids.

Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic. The molecular factors that govern tissue specificity of glucocorticoids, however, are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast two-hybrid assay. The MyoD family inhibitor domain-containing protein (MDFIC) was identified as a binding partner for GR. MDFIC associated with GR in the cytoplasm of cells, and treatment with glucocorticoids resulted in the dissociation of the GR-MDFIC complex. To investigate the function of the GR-MDFIC interaction, we performed a genome-wide microarray in intact and MDFIC-deficient A549 cells that were treated with glucocorticoids. A large cohort of genes was differentially regulated by GR depending on the presence or absence of MDFIC. These gene changes were strongly associated with inflammation, and glucocorticoid regulation of the inflammatory response was altered in MDFIC-deficient cells. At a molecular level, the interaction of MDFIC with GR altered the phosphorylation status of the receptor. We demonstrate in COS-1 cells that changes in receptor phosphorylation underlie the ability of MDFIC to regulate the transcriptional activity of GR. Finally, we show that GR directly represses the MDFIC gene, revealing a negative feedback loop by which glucocorticoids limit MDFIC activity. These findings identify a new binding partner for cytoplasmic GR that modulates the receptor transcriptome and contributes to the tissue-specific actions of glucocorticoids.

Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gßγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gßγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.

G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of ß-Arrestin.

Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gαißγ heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKCα, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of ß-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.

Glucocorticoids sensitize the innate immune system through regulation of the NLRP3 inflammasome.

Glucocorticoids have long been recognized as powerful anti-inflammatory compounds that are one of the most widely prescribed classes of drugs in the world. However, their role in the regulation of innate immunity is not well understood. We sought to examine the effects of glucocorticoids on the NOD-like receptors (NLRs), a central component of the inflammasome and innate immunity. Surprisingly, we show that glucocorticoids induce both NLRP3 messenger RNA and protein, which is a critical component of the inflammasome. The glucocorticoid-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of proinflammatory molecules, including mature IL-1ß, TNF-α, and IL-6. This effect was specific for glucocorticoids and dependent on the glucocorticoid receptor. These studies demonstrate a novel role for glucocorticoids in sensitizing the initial inflammatory response by the innate immune system.

Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms.

We have used RNA interference previously to demonstrate that G protein-coupled receptor kinase 2 (GRK2) regulates endogenously expressed H1 histamine receptor in human embryonic kidney 293 cells. In this report, we investigate the regulation of endogenously expressed M(3) muscarinic acetylcholine receptor (M(3) mAChR). We show that knockdown of GRK2, GRK3, or GRK6, but not GRK5, significantly increased carbachol-mediated calcium mobilization. Stable expression of wild-type GRK2 or a kinase-dead mutant (GRK2-K220R) reduced calcium mobilization after receptor activation, whereas GRK2 mutants defective in Galpha(q) binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates G(q) after M(3) mAChR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 and the arrestins also significantly enhanced carbachol-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), whereas prolonged ERK1/2 activation was only observed with GRK2 or arrestin-3 knockdown. We also investigated the role of casein kinase-1alpha (CK1alpha) and found that knockdown of CK1alpha increased calcium mobilization but not ERK activation. In summary, our data suggest that multiple proteins dynamically regulate M(3) mAChR-mediated calcium signaling, whereas GRK2 and arrestin-3 play the primary role in regulating ERK activation.

Regulation of CXCR4 signaling.

The chemokine receptor CXCR4 belongs to the large superfamily of G protein-coupled receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immune response. Recent evidence has highlighted the role of CXCR4 in a variety of diseases including HIV, cancer, and WHIM syndrome. Importantly, the involvement of CXCR4 in cancer metastasis and WHIM syndrome appears to be due to dysregulation of the receptor leading to enhanced signaling. Herein we review what is currently known regarding the regulation of CXCR4 and how dysregulation contributes to disease progression.

The five Rs of glucocorticoid action during inflammation: ready, reinforce, repress, resolve, and restore.

Glucocorticoids are essential for maintaining homeostasis and regulate a wide variety of physiological processes. Therapeutically, synthetic glucocorticoids are widely prescribed for the treatment of inflammation, autoimmune disorders, and malignancies of lymphoid origin. In this review we examine emerging evidence highlighting both proinflammatory and anti-inflammatory actions of glucocorticoids on both the innate and adaptive immune systems. We incorporate these findings into the more traditional anti-inflammatory role attributed to glucocorticoids, and propose how the two seemingly disparate processes seamlessly work together to resolve cellular responses to inflammatory stimuli. These ideas provide a framework by which glucocorticoids ready and reinforce the innate immune system, and repress the adaptive immune system, to help to resolve inflammation and restore homeostasis.