Progesterone induces meiosis through two obligate co-receptors with PLA2 activity.

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, its detailed signal transduction remains elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/ß hydrolase domain-containing protein 2) as an essential mPRß co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires both P4 and mPRß. This PLA2 activity bifurcates P4 signaling by inducing mPRß clathrin-dependent endocytosis and producing lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing the ABHD2 PLA2 activity that requires both mPRß and ABHD2 as obligate co-receptors.

Lipid Signaling During Gamete Maturation.

Cell lipids are differentially distributed in distinct organelles and within the leaflets of the bilayer. They can further form laterally defined sub-domains within membranes with important signaling functions. This molecular and spatial complexity offers optimal platforms for signaling with the associated challenge of dissecting these pathways especially that lipid metabolism tends to be highly interconnected. Lipid signaling has historically been implicated in gamete function, however the detailed signaling pathways involved remain obscure. In this review we focus on oocyte and sperm maturation in an effort to consolidate current knowledge of the role of lipid signaling and set the stage for future directions.

Correction: Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

[This corrects the article DOI: 10.1371/journal.pbio.3000901.].

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis.

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260-275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

miRNA-dependent regulation of STIM1 expression in breast cancer.

Store-operated Ca2+ entry (SOCE) has been shown to be important for breast cancer metastasis in xenograft mouse models. The ER Ca2+ sensor STIM1 and Orai plasma membrane Ca2+ channels molecularly mediate SOCE. Here we investigate the role of the microRNA machinery in regulating STIM1 expression. We show that STIM1 expression is regulated post-transcriptionally by the miRNA machinery and identify miR-223 and miR-150 as regulators of STIM1 expression in the luminal non-aggressive MCF7 breast cancer cell line. In contrast, STIM1 expression in the more aggressive basal triple-negative MDA-MB-231 cell line is not significantly modulated by a single miRNA species but is rather upregulated due to inhibition of the miRNA machinery through downregulation of Ago2. Consistently, overexpression of Ago2 results in decreased STIM1 protein levels in MDA-MB-231 cells. Clinically, STIM1 and Ago2 expression levels do not correlate with breast cancer progression, however in the basal subtype high STIM1 expression is associated with poorer survival. Our findings show that STIM1 expression is differentially regulated by the miRNA machinery in different cell types and argue for a role for this regulation in breast cancer.

The VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling.

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRß (PAQR8) partner that is required for mPRß plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.

Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling.

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different.

AKT1 has dual actions on the glucocorticoid receptor by cooperating with 14-3-3.

Glucocorticoids are important therapeutic compounds for acute lymphoblastic leukemia (ALL). AKT1 or the protein kinase B is frequently activated in ALL, and contributes to the development of glucocorticoid resistance. We examined impact of AKT1 on glucocorticoid receptor (GR)-induced transcriptional activity in cooperation with phospho-serine/threonine-binding protein 14-3-3. AKT1 has two distinct actions on GR transcriptional activity, one through segregation of GR in the cytoplasm by phosphorylating GR at Ser-134 and subsequent association of 14-3-3, and the other through direct modulation of GR transcriptional activity in the nucleus. For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GR, accompanied by AKT1-dependent p300 phosphorylation, H3S10 phosphorylation and H3K14 acetylation at the DNA site. These two actions of AKT1 regulate distinct sets of glucocorticoid-responsive genes. Our results suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR may be a promising target for treating glucocorticoid resistance observed in ALL.

Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway.

Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest.

Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSßγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest.

Salmonella-mediated tumor regression involves targeting of tumor myeloid suppressor cells causing a shift to M1-like phenotype and reduction in suppressive capacity.

The effectiveness of attenuated Salmonella in inhibiting tumor growth has been demonstrated in many therapeutic models, but the precise mechanisms remain incompletely understood. In this study, we show that the anti-tumor capacity of Salmonella depends on a functional MyD88-TLR pathway and is independent of adaptive immune responses. Since myeloid suppressor cells play a critical role in tumor growth, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics, and functional activation in spleen and tumor tissue of B16.F1 melanoma-bearing mice. Salmonella treatment led to increased accumulation of splenic and intratumoral CD11b(+)Gr-1(+) myeloid cells, exhibiting significantly increased expression of various activation markers such as MHC class II, costimulatory molecules, and Sca-1/Ly6A proteins. Gene expression analysis showed that Salmonella treatment induced expression of iNOS, arginase-1 (ARG1), and IFN-γ in the spleen, but down-regulated IL-4 and TGF-ß. Within the tumor, expression of iNOS, IFN-γ, and S100A9 was markedly increased, but ARG1, IL-4, TGF-ß, and VEGF were inhibited. Functionally, splenic CD11b(+) cells maintained their suppressive capacity following Salmonella treatment, but intratumoral myeloid cells had significantly reduced suppressive capacity. Our findings demonstrate that administration of attenuated Salmonella leads to phenotypic and functional maturation of intratumoral myeloid cells making them less suppressive and hence enhancing the host's anti-tumor immune response. Modalities that inhibit myeloid suppressor cells may be useful adjuncts in cancer immunotherapy.

How to make a good egg!: The need for remodeling of oocyte Ca(2+) signaling to mediate the egg-to-embryo transition.

The egg-to-embryo transition marks the initiation of multicellular organismal development and is mediated by a specialized Ca(2+) transient at fertilization. This explosive Ca(2+) signal has captured the interest and imagination of scientists for many decades, given its cataclysmic nature and necessity for the egg-to-embryo transition. Learning how the egg acquires the competency to generate this Ca(2+) transient at fertilization is essential to our understanding of the mechanisms controlling egg and the transition to embryogenesis. In this review we discuss our current knowledge of how Ca(2+) signaling pathways remodel during oocyte maturation in preparation for fertilization with a special emphasis on the frog oocyte as additional reviews in this issue will touch on this in other species.

Liver x receptors regulate the transcriptional activity of the glucocorticoid receptor: implications for the carbohydrate metabolism.

GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism.

AMPK regulates metabolic actions of glucocorticoids by phosphorylating the glucocorticoid receptor through p38 MAPK.

Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies.

A novel point mutation in helix 10 of the human glucocorticoid receptor causes generalized glucocorticoid resistance by disrupting the structure of the ligand-binding domain.

CONTEXT: Generalized glucocorticoid resistance syndrome is a rare familial or sporadic condition characterized by partial insensitivity to glucocorticoids, caused by mutations in the glucocorticoid receptor (GR) gene. Most of the reported cases are adults, demonstrating symptoms associated with mineralocorticoid and/or adrenal androgen excess caused by compensatively increased secretion of the adrenocorticotropic hormone. PATIENT: We identified a new 2-yr-old female case of generalized glucocorticoid resistance syndrome. The patient (TJ) presented with a generalized seizure associated with hypoglycemia and hypokalemia. She also had hypertension and premature pubarche, whereas dexamethasone effectively suppressed these clinical manifestations. RESULTS: The patient's GR gene had a heterozygotic mutation (G-->A) at nucleotide position 2141 (exon 8), which resulted in substitution of arginine by glutamine at amino acid position 714 in the ligand-binding domain (LBD) of the GR alpha. Molecular analysis revealed that the mutant receptor had significantly impaired transactivation activity with a 2-fold reduction in affinity to ligand. It showed attenuated transactivation of the activation function (AF)-2 and reduced binding to a p160 nuclear receptor coactivator. Computer-based structural analysis revealed that replacement of arginine by glutamine at position 714 transmitted a conformational change to the LBD and the AF-2 transactivation surface, resulting in a decreased binding affinity to ligand and to the LXXLL coactivator motif. CONCLUSIONS: Dexamethasone treatment is effective in controlling the premature pubarche, hypoglycemia, hypertension, and hypokalemia in this child case, wherein arginine 714 plays a key role in the proper formation of the ligand-binding pocket and the AF-2 surface of the GR alpha LBD.

Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor.

The availability of nutrients influences cellular growth and survival by affecting gene transcription. Glucocorticoids also influence gene transcription and have diverse activities on cell growth, energy expenditure, and survival. We found that the growth arrest-specific 5 (Gas5) noncoding RNA, which is abundant in cells whose growth has been arrested because of lack of nutrients or growth factors, sensitized cells to apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including the one encoding cellular inhibitor of apoptosis 2. Gas5 bound to the DNA-binding domain of the glucocorticoid receptor (GR) by acting as a decoy glucocorticoid response element (GRE), thus competing with DNA GREs for binding to the GR. We conclude that Gas5 is a "riborepressor" of the GR, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR.

Interactions of the circadian CLOCK system and the HPA axis.

Organisms have developed concurrent behavioral and physiological adaptations to the strong influence of day/night cycles, as well as to unforeseen, random stress stimuli. These circadian and stress-related responses are achieved by two highly conserved and interrelated regulatory networks, the circadian CLOCK and stress systems, which respectively consist of oscillating molecular pacemakers, the Clock/Bmal1 transcription factors, and the hypothalamic-pituitary-adrenal (HPA) axis and its end-effector, the glucocorticoid receptor. These systems communicate with one another at different signaling levels and dysregulation of either system can lead to development of pathologic conditions. In this review, we summarize the mutual physiologic interactions between the circadian CLOCK system and the HPA axis, and discuss their clinical implications.

Sex hormone-binding globulin gene expression in the liver: drugs and the metabolic syndrome.

Sex hormone-binding globulin (SHBG) is the main transport binding protein for sex steroid hormones in plasma and regulates their accessibility to target cells. Plasma SHBG is secreted by the liver under the control of hormones and nutritional factors. In the human hepatoma cell line (HepG2), thyroid and estrogenic hormones, and a variety of drugs including the antioestrogen tamoxifen, the phytoestrogen, genistein and mitotane (Op'DDD) increase SHBG production and SHBG gene promoter activity. In contrast, monosaccharides (glucose or fructose) effectively decrease SHBG expression by inducing lipogenesis, which reduces hepatic HNF-4alpha levels, a transcription factor that play a critical role in controlling the SHBG promoter. Interestingly, diminishing hepatic lipogenesis and free fatty acid liver biosynthesis also appear to be associated with the positive effects of thyroid hormones and PPARgamma antagonists on SHBG expression. This mechanism provides a biological explanation for why SHBG is a sensitive biomarker of insulin resistance and the metabolic syndrome, and why low plasma SHBG levels are a risk factor for developing hyperglycemia and type 2 diabetes, especially in women. These important advances in our knowledge of the regulation of SHBG expression in the liver open new approaches for identifying and preventing metabolic disorder-associated diseases early in life.

Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications.

Glucocorticoids, end products of the hypothalamic-pituitary-adrenal axis, influence functions of virtually all organs and tissues through the glucocorticoid receptor (GR). Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms in both the central nervous system and periphery. We found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT-activity- dependent fashion. In serum-shock-synchronized cells, transactivational activity of GR, accessed by mRNA expression of an endogenous-responsive gene, fluctuated spontaneously in a circadian fashion in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically, and CLOCK suppressed binding of GR to its DNA recognition sequences by acetylating multiple lysine residues located in its hinge region. These findings indicate that CLOCK/BMAL1 functions as a reverse-phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing biological actions of diurnally fluctuating circulating glucocorticoids. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences the functions of every organ and tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids.