PPAR-gamma Fun(gi) With Prostaglandin

In our recent publication, we show for the first time that the fungal pathogen Cryptococcus neoformans is able to manipulate host cells by producing eicosanoids that mimic those found in the host. Using complementary in vivo zebrafish and in vitro macrophage cell culture models of Cryptococcus infection, we found that these eicosanoids manipulate host innate immune cells by activating the host receptor PPAR-gamma which is an important regulator of macrophage inflammatory phenotypes. We initially identified PGE2 as the eicosanoid species responsible for this effect; however, we later found that a derivative of PGE2—15-keto-PGE2—was ultimately responsible and that this eicosanoid acted as a partial agonist to PPAR-gamma. In this commentary, we will discuss some of the concepts and conclusions in our original publication and expand on their implications and future directions.

Using Chromatin-Nuclear Receptor Interactions to Quantitate Endocrine, Paracrine, and Autocrine Signaling

Hormone-activated nuclear receptors (NRs) control myriad cellular processes. The classical paradigm for hormone delivery is secretion from endocrine organs and blood-borne distribution to responding cells. However, many hormones can also be synthesized in the same tissues in which responding cells are found (paracrine signaling). In both endocrine and paracrine signaling, numerous factors affect hormone availability to target cell NRs, including hormone access to and sequestration by carrier proteins, transport across cell membranes, metabolism, and receptor availability. These factors can differ dramatically during development, between anatomical locations, and across cell types, and may cause highly variable responses to the same hormone signal. This has been difficult to study because current approaches are unable to quantify cell-intrinsic exposure to NR hormone ligands, precluding assessment of cell-specific hormone access and signaling. We have used the ligand-dependent interaction of the endogenous glucocorticoid (GC) receptor with chromatin as a biosensor that quantifies systemic access of GCs to cells within tissues at the single cell level, showing that tissues are buffered against circulating GCs. This approach also showed highly targeted paracrine GC signaling within the thymus, where GCs promote the positive selection of thymocytes with moderate affinity for self-antigens and the development of a safe and effective T-cell repertoire. We believe that this and complementary biosensor approaches will be useful to identify endocrine and paracrine target cells in situ and quantify their exposure to hormones regardless of the mode of delivery.

GR Utilizes a Co-Chaperone Cytoplasmic CAR Retention Protein to Form an N/C Interaction

The N-terminal domain (NTD) of nuclear receptor superfamily members has been recently reported to regulate functions of the receptor through the interaction between the NTD and the C-terminal ligand binding domain (LBD), so-called an N/C interaction. Although this N/C interaction has been demonstrated in various nuclear receptors, eg, androgen receptor, this concept has not been observed in glucocorticoid receptor (GR). We hypothesized that GR requires its co-chaperone CCRP (cytoplasmic constitutive active/androstane receptor retention protein) to form a stable N/C interaction. This hypothesis was examined by co-immunoprecipitation assays using GR fragments overexpressing COS-1 cell lysate. Here, we demonstrated that GR undergoes the N/C interaction between the 26VMDFY30 motif in the NTD and the LBD. More importantly, co-chaperone CCRP is now found to induce this interaction. By the fact that a negative charge at Y30 disrupts this interaction, this residue, a potential phosphorylation site, was indicated to regulate the GR N/C interaction critically. Utilizing Y30F and Y30E mutants as N/C interacting and noninteracting forms of GR, respectively, a 2-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to examine whether or not the N/C interaction regulated formation of GR complexes. A cDNA microarray analysis was performed with COS-1 cells expressing Y30F or Y30E. We will present experimental data to demonstrate that CCRP is essential for GR to form the N/C interaction and will discuss its implications in GR functions.

C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors

Nuclear hormone receptors (NRs) are evolutionarily conserved ligand-dependent transcription factors. They are essential for human life, mediating the actions of lipophilic molecules, such as steroid hormones and metabolites of fatty acid, cholesterol, and external toxic compounds. The C2H2-type zinc finger proteins (ZNFs) form the largest family of the transcription factors in humans and are characterized by multiple, tandemly arranged zinc fingers. Many of the C2H2-type ZNFs are conserved throughout evolution, suggesting their involvement in preserved biological activities, such as general transcriptional regulation and development/differentiation of organs/tissues observed in the early embryonic phase. However, some C2H2-type ZNFs, such as those with the Krüppel-associated box (KRAB) domain, appeared relatively late in evolution and have significantly increased family members in mammals including humans, possibly modulating their complicated transcriptional network and/or supporting the morphological development/functions specific to them. Such evolutional characteristics of the C2H2-type ZNFs indicate that these molecules influence the NR functions conserved through evolution, whereas some also adjust them to meet with specific needs of higher organisms. We review the interaction between NRs and C2H2-type ZNFs by focusing on some of the latter molecules.

LXRs, SHP, and FXR in Prostate Cancer: Enemies or Ménage à Quatre With AR?

Androgens and androgen receptor (AR, NR3C4) clearly play a crucial role in prostate cancer progression. Besides, the link between metabolic disorders and the risk of developing a prostate cancer has been emerging these last years. Interestingly, “lipid” nuclear receptors such as LXRα/NR1H3 and LXRβ/NR1H2 (as well as FXRα/NR1H4 and SHP/NR0B2) have been described to decrease the lipid metabolism, while AR increases it. Moreover, these former orphan nuclear receptors can regulate androgen levels and modulate AR activity. Thus, it is not surprising to find such receptors involved in the physiology of prostate. This review is focused on the roles of liver X receptors (LXRs), farnesoid X receptor (FXR), and small heterodimeric partner (SHP) in prostate physiology and their capabilities to interfere with the androgen-regulated pathways by modulating the levels of active androgen within the prostate. By the use of prostate cancer cell lines, mice deficient for these nuclear receptors and human tissue libraries, several authors have pointed out the putative possibility to pharmacologically target these receptors. These data open a new field of research for the development of new drugs that could overcome the castration resistance in prostate cancer, a usual phenomenon in patients.

Regulation of Steroid Hormone Receptors and Coregulators during the Cell Cycle Highlights Potential Novel Function in Addition to Roles as Transcription Factors

Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M.

Estrogen-related Receptor β (ERRβ) – Renaissance Receptor or Receptor Renaissance?

Estrogen-related receptors (ERRs) are founding members of the orphan nuclear receptor (ONR) subgroup of the nuclear receptor superfamily. Twenty-seven years of study have yet to identify cognate ligands for the ERRs, though they have firmly placed ERRα (ESRRA) and ERRγ (ESRRG) at the intersection of cellular metabolism and oncogenesis. The pace of discovery for novel functions of ERRβ (ESRRB), however, has until recently been somewhat slower than that of its family members. ERRβ has also been largely ignored in summaries and perspectives of the ONR literature. Here, we provide an overview of established and emerging knowledge of ERRβ in mouse, man, and other species, highlighting unique aspects of ERRβ biology that set it apart from the other two estrogen-related receptors, with a focus on the impact of alternative splicing on the structure and function of this receptor.

Molecular Targets of Androgen Signaling that Characterize Skeletal Muscle Recovery and Regeneration

The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration. Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR/Nr3c1). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identifed temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.

Identification of Chemical Modulators of the Constitutive Activated Receptor (CAR) in a Gene Expression Compendium

The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher's algorithm (p-value < 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ∼1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of inflammation including acetaminophen, concanavalin A, lipopolysaccharide, and 300 nm silica particles. In conclusion, we have shown that a CAR biomarker signature coupled with a rank-based similarity method accurately predicts CAR activation. This analytical approach, when applied to a gene expression compendium, increased the universe of known chemicals that directly or indirectly activate CAR, highlighting the promiscuous nature of CAR activation and signaling through activation of other xenobiotic-activated receptors.

Mechanisms of Peroxisome Proliferator Activated Receptor γ Regulation by Non-steroidal Anti-inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the β-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.