The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities.

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

Nuclear receptors: Lipid and hormone sensors with essential roles in the control of cancer development.

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that act as biological sensors and use a combination of mechanisms to modulate positively and negatively gene expression in a spatial and temporal manner. The highly orchestrated biological actions of several NRs influence the proliferation, differentiation, and apoptosis of many different cell types. Synthetic ligands for several NRs have been the focus of extensive drug discovery efforts for cancer intervention. This review summarizes the roles in tumour growth and metastasis of several relevant NR family members, namely androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor (TR), retinoic acid receptors (RARs), retinoid X receptors (RXRs), peroxisome proliferator-activated receptors (PPARs), and liver X receptors (LXRs). These studies are key to develop improved therapeutic agents based on novel modes of action with reduced side effects and overcoming resistance.

Glucocorticoid Resistance: Interference between the Glucocorticoid Receptor and the MAPK Signalling Pathways.

Endogenous glucocorticoids (GCs) are steroid hormones that signal in virtually all cell types to modulate tissue homeostasis throughout life. Also, synthetic GC derivatives (pharmacological GCs) constitute the first-line treatment in many chronic inflammatory conditions with unquestionable therapeutic benefits despite the associated adverse effects. GC actions are principally mediated through the GC receptor (GR), a ligand-dependent transcription factor. Despite the ubiquitous expression of GR, imbalances in GC signalling affect tissues differently, and with variable degrees of severity through mechanisms that are not completely deciphered. Congenital or acquired GC hypersensitivity or resistance syndromes can impact responsiveness to endogenous or pharmacological GCs, causing disease or inadequate therapeutic outcomes, respectively. Acquired GC resistance is defined as loss of efficacy or desensitization over time, and arises as a consequence of chronic inflammation, affecting around 30% of GC-treated patients. It represents an important limitation in the management of chronic inflammatory diseases and cancer, and can be due to impairment of multiple mechanisms along the GC signalling pathway. Among them, activation of the mitogen-activated protein kinases (MAPKs) and/or alterations in expression of their regulators, the dual-specific phosphatases (DUSPs), have been identified as common mechanisms of GC resistance. While many of the anti-inflammatory actions of GCs rely on GR-mediated inhibition of MAPKs and/or induction of DUSPs, the GC anti-inflammatory capacity is decreased or lost in conditions of excessive MAPK activation, contributing to disease susceptibility in tissue- and disease- specific manners. Here, we discuss potential strategies to modulate GC responsiveness, with the dual goal of overcoming GC resistance and minimizing the onset and severity of unwanted adverse effects while maintaining therapeutic potential.

The histone demethylase PHF8 is a molecular safeguard of the IFNγ response.

A precise immune response is essential for cellular homeostasis and animal survival. The paramount importance of its control is reflected by the fact that its non-specific activation leads to inflammatory events that ultimately contribute to the appearance of many chronic diseases. However, the molecular mechanisms preventing non-specific activation and allowing a quick response upon signal activation are not yet fully understood. In this paper we uncover a new function of PHF8 blocking signal independent activation of immune gene promoters. Affinity purifications coupled with mass spectrometry analysis identified SIN3A and HDAC1 corepressors as new PHF8 interacting partners. Further molecular analysis demonstrated that prior to interferon gamma (IFNγ) stimulation, PHF8 is bound to a subset of IFNγ-responsive promoters. Through the association with HDAC1 and SIN3A, PHF8 keeps the promoters in a silent state, maintaining low levels of H4K20me1. Upon IFNγ treatment, PHF8 is phosphorylated by ERK2 and evicted from the promoters, correlating with an increase in H4K20me1 and transcriptional activation. Our data strongly indicate that in addition to its well-characterized function as a coactivator, PHF8 safeguards transcription to allow an accurate immune response.

Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.

The NIMA-family kinases Nek9/Nercc1, Nek6 and Nek7 form a signalling module required for mitotic spindle assembly. Nek9, the upstream kinase, is activated during prophase at centrosomes although the details of this have remained elusive. We now identify Plk1 as Nek9 direct activator and propose a two-step activation mechanism that involves Nek9 sequential phosphorylation by CDK1 and Plk1. Furthermore, we show that Plk1 controls prophase centrosome separation through the activation of Nek9 and ultimately the phosphorylation of the mitotic kinesin Eg5 at Ser1033, a Nek6/7 site that together with the CDK1 site Thr926 we establish contributes to the accumulation of Eg5 at centrosomes and is necessary for subsequent centrosome separation and timely mitosis. Our results provide a basis to understand signalling downstream of Plk1 and shed light on the role of Eg5, Plk1 and the NIMA-family kinases in the control of centrosome separation and normal mitotic progression.

Over-expression of muscle glycogen synthase in human diabetic nephropathy.

Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.

Involvement of the cellular phosphatase DUSP1 in vaccinia virus infection.

Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection.

Upon Wnt stimulation, Rac1 activation requires Rac1 and Vav2 binding to p120-catenin.

A role for Rac1 GTPase in canonical Wnt signaling has recently been demonstrated, showing that it is required for ß-catenin translocation to the nucleus. In this study, we investigated the mechanism of Rac1 stimulation by Wnt. Upregulation of Rac1 activity by Wnt3a temporally correlated with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin was modulated by phosphorylation of this protein, which was stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induced the release of p120-catenin from E-cadherin, enabled the interaction of p120-catenin with Vav2 and Rac1, and facilitated Rac1 activation by Vav2. Given that p120-catenin depletion disrupts gastrulation in Xenopus, we analyzed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants that were deficient in the release from E-cadherin or in Vav2 or Rac1 binding failed to rescue p120-catenin depletion. Collectively, these results indicate that binding of p120-catenin to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signaling.

Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney.

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down-regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3ß kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease.

Distinct Responses to IL4 in Macrophages Mediated by JNK.

IL(Interleukin)-4 is the main macrophage M2-type activator and induces an anti-inflammatory phenotype called alternative activation. The IL-4 signaling pathway involves the activation of STAT (Signal Transducer and Activator of Transcription)-6 and members of the MAPK (Mitogen-activated protein kinase) family. In primary-bone-marrow-derived macrophages, we observed a strong activation of JNK (Jun N-terminal kinase)-1 at early time points of IL-4 stimulation. Using selective inhibitors and a knockout model, we explored the contribution of JNK-1 activation to macrophages' response to IL-4. Our findings indicate that JNK-1 regulates the IL-4-mediated expression of genes typically involved in alternative activation, such as Arginase 1 or Mannose receptor, but not others, such as SOCS (suppressor of cytokine signaling) 1 or p21Waf-1 (cyclin dependent kinase inhibitor 1A). Interestingly, we have observed that after macrophages are stimulated with IL-4, JNK-1 has the capacity to phosphorylate STAT-6 on serine but not on tyrosine. Chromatin immunoprecipitation assays revealed that functional JNK-1 is required for the recruitment of co-activators such as CBP (CREB-binding protein)/p300 on the promoter of Arginase 1 but not on p21Waf-1. Taken together, these data demonstrate the critical role of STAT-6 serine phosphorylation by JNK-1 in distinct macrophage responses to IL-4.

DYNLL/LC8 protein controls signal transduction through the Nek9/Nek6 signaling module by regulating Nek6 binding to Nek9.

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser(944), a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation.

Pharmacologic Activation of LXR Alters the Expression Profile of Tumor-Associated Macrophages and the Abundance of Regulatory T Cells in the Tumor Microenvironment.

Liver X receptors (LXR) are transcription factors from the nuclear receptor family that are activated by oxysterols and synthetic high-affinity agonists. In this study, we assessed the antitumor effects of synthetic LXR agonist TO901317 in a murine model of syngeneic Lewis Lung carcinoma. Treatment with TO901317 inhibited tumor growth in wild-type, but not in LXR-deficient mice, indicating that the antitumor effects of the agonist depends on functional LXR activity in host cells. Pharmacologic activation of the LXR pathway reduced the intratumoral abundance of regulatory T cells (Treg) and the expression of the Treg-attracting chemokine Ccl17 by MHCIIhigh tumor-associated macrophages (TAM). Moreover, gene expression profiling indicated a broad negative impact of the LXR agonist on other mechanisms used by TAM for the maintenance of an immunosuppressive environment. In studies exploring the macrophage response to GM-CSF or IL4, activated LXR repressed IRF4 expression, resulting in subsequent downregulation of IRF4-dependent genes including Ccl17. Taken together, this work reveals the combined actions of the LXR pathway in the control of TAM responses that contribute to the antitumoral effects of pharmacologic LXR activation. Moreover, these data provide new insights for the development of novel therapeutic options for the treatment of cancer. SIGNIFICANCE: This study reveals unrecognized roles of LXR in the transcriptional control of the tumor microenvironment and suggests use of a synthetic LXR agonist as a novel therapeutic strategy to stimulate antitumor activity.

CREB and AP-1 activation regulates MKP-1 induction by LPS or M-CSF and their kinetics correlate with macrophage activation versus proliferation.

MAPK phosphatase-1 (MKP-1) is a protein phosphatase that plays a crucial role in innate immunity. This phosphatase inactivates ERK1/2, which are involved in two opposite functional activities of the macrophage, namely proliferation and activation. Here we found that although macrophage proliferation and activation induce MKP-1 with different kinetics, gene expression is mediated by the proximal promoter sequences localized between -380 and -180 bp. Mutagenesis experiments of the proximal element determined that CRE/AP-1 is required for LPS- or M-CSF-induced activation of the MKP-1 gene. Moreover, the results from gel shift analysis and chromatin immunoprecipitation indicated that c-Jun and CREB bind to the CRE/AP-1 box. The distinct kinetics shown by M-CSF and LPS correlates with the induction of JNK and c-jun, as well as the requirement for Raf-1. The signal transduction pathways that activate the induction of MKP-1 correlate kinetically with induction by M-CSF and LPS.

IFN-{gamma}-mediated inhibition of MAPK phosphatase expression results in prolonged MAPK activity in response to M-CSF and inhibition of proliferation.

Macrophages have the capacity to proliferate in response to specific growth factors, such as macrophage-colony stimulating factor (M-CSF). In the presence of several cytokines and activating factors, macrophages undergo growth arrest, become activated, and participate in the development of an immune response. We have previously observed that activation of extracellularly regulated kinase 1/2 (ERK-1/2) is required for macrophage proliferation in response to growth factors. A short and early pattern of ERK activity correlated with the proliferative response. In contrast, slightly prolonged patterns of activity of these kinases were induced by signals that lead to macrophage activation and growth arrest. IFN-gamma is the main endogenous Th1-type macrophage activator. Here we report that stimulation with IFN-gamma prolongs the pattern of ERK activity induced by M-CSF in macrophages. These effects correlate with IFN-gamma-mediated inhibition of the expression of several members of the MAPK phosphatase family, namely MKP-1, -2, and -4. Moreover, inhibition of MKP-1 expression using siRNA technology or synthetic inhibitors also led to elongated ERK activity and significant blockage of M-CSF-dependent proliferation. These data suggest that subtle changes in the time course of activity of members of the MAPK family contribute to the antiproliferative effects of IFN-gamma in macrophages.

Assaying Activation and Subcellular Localization of ERK in Cells and Tissues.

The extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are the focus of many studies due to their involvement in numerous physiological and pathological processes, such as cell proliferation and differentiation, and oncogenic transformation, respectively. ERK1/2 belong to the mitogen-activated protein kinase (MAPKs) family, which are serine/threonine kinases that participate in signal transduction and are activated by dual phosphorylation in the Thr-X-Tyr motif located in their activation loop. In addition, ERK activation induces its dimerization and translocation into the nucleus. On the basis of this knowledge, different assays and tools have been developed to determine ERK activity or monitor its activation. In this chapter, we describe methods to assay ERK activity based on the ability of ERK immunocomplexes to phosphorylate a substrate, as well as on immunoblot analysis using antibodies that recognize ERK1/2 phosphorylated in the Thr-X-Tyr motif. In addition, we describe an immunocytochemistry procedure to reveal stimuli-induced nuclear translocation of ERK1/2.

The Nuclear Receptor LXR Limits Bacterial Infection of Host Macrophages through a Mechanism that Impacts Cellular NAD Metabolism.

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.

Neuroprotective action of flavopiridol, a cyclin-dependent kinase inhibitor, in colchicine-induced apoptosis.

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimer's disease.

Assays to measure stress-activated MAPK activity.

Mitogen-activated protein kinases (MAPKs) are activated by a wide variety of cellular stimuli and involved in the regulation of most, if not all, cellular processes. Among them, the c-Jun N-terminal kinase and p38 MAPKs are predominantly induced in response to proinflammatory cytokines and most environmental stresses, such as pathogenic insults, heat and osmotic shock, ultraviolet light, protein synthesis inhibitors, DNA-damaging agents, and withdrawal of growth factors; thus, they are also known as stress-activated protein kinases. In this chapter we describe alternative methods designed to measure the activity of these stress-activated protein kinases, c-Jun N-terminal kinase, and p38 MAPKs.

In vivo JNK activation in pancreatic ß-cells leads to glucose intolerance caused by insulin resistance in pancreas.

Insulin resistance is a key condition in the development of type 2 diabetes. It is well established that exacerbated Jun NH2-terminal kinase (JNK) activity is involved in promoting insulin resistance in peripheral insulin-target tissues; however, this involvement is less documented in pancreatic ß-cells. Using a transgenic mouse model, here we show that JNK activation in ß-cells led to glucose intolerance as a result of impaired capacity to increase insulinemia in response to hyperglycemia. Pancreatic islets from these mice showed no obvious morphostructural abnormalities or decreased insulin content. In contrast, these islets failed to secrete insulin in response to glucose or insulin but were competent in succinate-, ketoisocaproate-, 3-isobutyl-1-methylxanthine (IBMX-), KCl-, and tolbutamide-induced insulin secretion. At the molecular level, JNK activation in ß-cells inhibited insulin-induced Akt phosphorylation, pancreatic and duodenal homeobox 1 nucleocytoplasmic shuttling, and transcription of insulin-target genes. Remarkably, rosiglitazone restored insulin secretion in response to hyperglycemia in mice and insulin-induced insulin secretion and signaling in isolated islets. In conclusion, the mere activation of JNK suffices to induce insulin resistance in pancreatic ß-cells by inhibition of insulin signaling in these cells, but it is not sufficient to elicit ß-cell death. In addition, we provide the first evidence that thiazolidinediones exert insulin-sensitizing action directly on pancreatic ß-cells.