Showing papers in "Molecular Endocrinology in 2005"

Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes

Abstract: Estrogen receptors (ERs) act by regulating transcriptional processes. The classical mechanism of ER action involves estrogen binding to receptors in the nucleus, after which the receptors dimerize and bind to specific response elements known as estrogen response elements (EREs) located in the promoters of target genes. However, ERs can also regulate gene expression without directly binding to DNA. This occurs through protein-protein interactions with other DNA-binding transcription factors in the nucleus. In addition, membrane-associated ERs mediate nongenomic actions of estrogens, which can lead both to altered functions of proteins in the cytoplasm and to regulation of gene expression. The latter two mechanisms of ER action enable a broader range of genes to be regulated than the range that can be regulated by the classical mechanism of ER action alone. This review surveys our knowledge about the molecular mechanism by which ERs regulate the expression of genes that do not contain EREs, and it gives examples of the ways in which the genomic and nongenomic actions of ERs on target genes converge. Genomic and nongenomic actions of ERs that do not depend on EREs influence the physiology of many target tissues, and thus, increasing our understanding of the molecular mechanisms behind these actions is highly relevant for the development of novel drugs that target specific receptor actions.

Integration of the Extranuclear and Nuclear Actions of Estrogen

Abstract: Estrogen receptors (ERs) are localized to many sites within the cell, potentially contributing to overall estrogen action. In the nucleus, estrogen mainly modulates gene transcription, and the resulting protein products determine the cell biological actions of the sex steroid. In addition, a small pool of ERs localize to the plasma membrane and signal mainly though coupling, directly or indirectly, to G proteins. In response to steroid, signal transduction modulates both nontranscriptional and transcriptional events and impacts both the rapid and more prolonged actions of estrogen. Cross-talk from membrane-localized ERs to nuclear ERs can be mediated through growth factor receptor tyrosine kinases, such as epidermal growth factor receptor and IGF-I receptor. Growth factor receptors enact signal transduction to kinases such as ERK and phosphatidylinositol 3-kinase that phosphorylate and activate nuclear ERs, and this can also occur in the absence of sex steroid. A complex relationship between the membrane and nuclear effects of estrogen also involves membrane-initiated phosphorylation of coactivators, recruiting these proteins to the nuclear transcriptosome. Finally, large pools of cytoplasmic ERs exist, and some are localized to mitochondria. The integration of sex steroid effects at distinct cellular locations of its receptor leads to important cellular physiological outcomes and are manifest in both reproductive and nonreproductive organs.

Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes.

Abstract: 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] regulates calcium homeostasis and controls cellular differentiation and proliferation. The vitamin D receptor (VDR) is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements (VDREs) formed by direct or everted repeats of PuG(G/T)TCA motifs separated by 3 or 6 bp (DR3 or ER6). Here, we have identified direct 1,25(OH)2D3 target genes by combining 35,000+ gene microarrays and genome-wide screens for consensus DR3 and ER6 elements, and DR3 elements containing single nucleotide substitutions. We find that the effect of a nucleotide substitution on VDR binding in vitro does not predict VDRE function in vivo, because substitutions that disrupted binding in vitro were found in several functional elements. Hu133A microarray analyses, performed with RNA from human SCC25 cells treated with 1,25(OH)2D3 and protein synthesis inhibitor cycloheximide, identified more than 900 regulated genes. VDREs lying within −10 to +5 kb of 5′-ends were assi...

Multiple Signaling Pathways Regulating Steroidogenesis and Steroidogenic Acute Regulatory Protein Expression: More Complicated than We Thought

Abstract: Steroid hormone biosynthesis in steroidogenic cells is regulated through trophic hormone activation of protein kinase A (PKA) signaling pathways. However, many examples of the regulation of steroid synthesis via pathways other than the PKA pathway have been documented. In some cases these pathways act independently of PKA activation whereas in other cases, they act synergistically with it. The current understanding of additional signaling pathways and factors, such as the protein kinase C pathway, arachidonic acid metabolites, growth factors, chloride ion, the calcium messenger system, and others capable of regulating/modulating steroid hormone biosynthesis, and in many cases steroidogenic acute regulatory protein expression, are discussed in this review.

Epigenetic Regulation by Histone Methylation and Histone Variants

Abstract: Epigenetics is the study of heritable changes in gene expression that are not mediated at the DNA sequence level. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and chromatin/histone modifications. With the identification of key histone-modifying enzymes, the biological functions of many histone posttranslational modifications are now beginning to be elucidated. Histone methylation, in particular, plays critical roles in many epigenetic phenomena. In this review, we provide an overview of recent findings that shape the current paradigms regarding the roles of histone methylation and histone variants in heterochromatin assembly and the maintenance of the boundaries between heterochromatin and euchromatin. We also highlight some of the enzymes that mediate histone methylation and discuss the stability and inheritance of this modification.

Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor.

Abstract: The constitutive androstane receptor (CAR, NR1I3) is a central regulator of xenobiotic metabolism. CAR activation induces hepatic expression of detoxification enzymes and transporters and increases liver size. Here we show that CAR-mediated hepatomegaly is a transient, adaptive response to acute xenobiotic stress. In contrast, chronic CAR activation results in hepatocarcinogenesis. In both acute and chronic xenobiotic responses, hepatocyte DNA replication is increased and apoptosis is decreased. These effects are absent in CAR null mice, which are completely resistant to tumorigenic effects of chronic xenobiotic stress. In the acute response, direct up-regulation of Mdm2 expression by CAR contributes to both increased DNA replication and inhibition of p53-mediated apoptosis. These results demonstrate an essential role for CAR in regulating both liver homeostasis and tumorigenesis in response to xenobiotic stresses, and they also identify a specific molecular mechanism linking chronic environmental stress and tumor formation.

The Orphan Nuclear Receptor Rev-erbα Recruits the N-CoR/Histone Deacetylase 3 Corepressor to Regulate the Circadian Bmal1 Gene

Abstract: Transcriptional regulation plays a fundamental role in controlling circadian oscillation of clock gene expression. The orphan nuclear receptor Rev-erbalpha has recently been implicated as a major regulator of the circadian clock. Expression of Bmal1, the master regulator of circadian rhythm in mammals, is negatively correlated with Rev-erbalpha mRNA level, but the molecular mechanism underlying this regulation is largely unknown. Here we show that Rev-erbalpha dramatically represses the basal activity of the mouse Bmal1 gene promoter via two monomeric binding sites, both of which are required for repression and are conserved between mouse and human. Rev-erbalpha directly binds to the mouse Bmal1 promoter and recruits the endogenous nuclear receptor corepressor (N-CoR)/histone deacetylase 3 (HDAC3) complex, in association with a decrease in histone acetylation. The endogenous N-CoR/HDAC3 complex is also associated with the endogenous Bmal1 promoter in human HepG2 liver cells, where a reduction in cellular HDAC3 level markedly increases the expression of Bmal1 mRNA. These data demonstrate a new function for the N-CoR/HDAC3 complex in regulating the expression of genes involved in circadian rhythm by functioning as corepressor for Rev-erbalpha.

The nuclear receptor superfamily: a rosetta stone for physiology.

Abstract: In the December 1985 issue of Nature, we described the cloning of the first nuclear receptor cDNA encoding the human glucocorticoid receptor (GR) (1). In the 20 yr since that event, our field has witnessed a quantum leap by the subsequent discovery and functional elaboration of the nuclear receptor superfamily (2)—a family whose history is linked to the evolution of the entire animal kingdom and whose actions, by decoding the genome, span the vast diversity of biological functions from development to physiology, pathology, and treatment. A messenger is an envoy or courier charged with transmitting a communication or message. In one sense, the cloning of that first messenger (the GR) represented the completion of a prediction that began with Elwood Jensen’s characterization of the first steroid receptor protein (3) and continued with the pioneering work of others in the steroid receptor field (including Gorski, O’Malley, Gustafsson, and Yamamoto). Yet, like the discovery of the Rosetta stone in 1799, the revelation of the GR sequence heralded a completely unpredictable demarcation in the field, helping to solve mysteries unearthed nearly 100 yr ago as well as opening a portal to the future. The beginnings of the adventure lie in disciplines such as medicine and nutrition, which gave rise to the emergent field of endocrinology in the first half of the last century. The purification of chemical messengers ultimately known as hormones from organs and vitamins from foods spurred the study of these compounds and their physiologic effects on the body. At about the same time, the field of molecular biology was emerging from the disciplines of chemistry, physics, and their application to biological problems such as the structure of DNA and the molecular events surrounding its replication and transcription. It would not be until the late 1960s and 1970s that endocrinology and molecular biology would begin to intersect as the link between receptors and transcriptional control were being laid down. During this time, the work of Jensen (4) and Gorski (5) identified a high-affinity estrogen receptor (ER) that suggested an action in the nucleus. Gordon Tomkins and his associates (J. Baxter, G. Ringold, E. B. Thompson, H. Samuels, H. Bourne, and others) were one of the most creative forces studying glucocorticoid action (6). Concurrent work by O’Malley, Gustafsson, and Yamamoto provided further, important evidence supporting a link between steroid receptor action and transcription (see accompanying perspective articles in this issue of Molecular Endocrinology). But whereas the steroid hormone field continued to evolve in this direction, it is of interest to note that the mechanism of action of thyroid hormone and retinoids remained clouded and controversial until the eventual cloning of their receptors in the late 1980s. Likewise, no one had foreseen the possibility that other lipophilic molecules (like oxysterols, bile acids, and fatty acids) would also function through a similar mechanism, or that other steroid receptor-like proteins existed that would play an important role in transcriptional regulation of so many diverse pathways. Thus, the GR isolation in 1985 led to the concept of a hidden superfamily of receptors that in a very real way provided the needed molecular code to unravel the puzzle of physiologic homeostasis.

Thyroid Hormone Induces Rapid Activation of Akt/Protein Kinase B-Mammalian Target of Rapamycin-p70S6K Cascade through Phosphatidylinositol 3-Kinase in Human Fibroblasts

Abstract: We have demonstrated that T3 increases the expression of ZAKI-4alpha, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4alpha expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4alpha was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70(S6K), a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Deltap85alpha). Furthermore, the association between thyroid hormone receptor beta1 (TRbeta1) and PI3K-regulatory subunit p85alpha, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70(S6K) cascade by T3 provides a new molecular mechanism for thyroid hormone action.

The mineralocorticoid receptor: a journey exploring its diversity and specificity of action.

Abstract: The mineralocorticoid receptor (MR) integrates hormonal signaling and activates the expression of aldosterone target genes, which control various physiological processes. In recent years, evidence has been provided for an important role of MR not only in the regulation of sodium and water homeostasis but also in cardiovascular function, neuronal fate, and adipocyte differentiation. MR belongs to the steroid receptor family that displays common mechanism of action. As a result, some apparent similarities with the glucocorticoid receptor (GR) have shaded MR's own specificities. The description of its gene structure, messenger isoforms, protein variants, functional domains, and posttranslational modifications (phosphorylation, ubiquitinylation, sumoylation, acetylation) as well as a panel of interactions with coregulators, progressively depicted an original portrait of MR and shed light on its specific mechanism of action. In this review, after an overview of MR characteristics, the multiple levels of MR selectivity over other steroid receptors, in particular GR, will be described as well as the consequences for aldosterone-regulated gene expression.

A Nuclear Receptor Atlas: 3T3-L1 adipogenesis.

Abstract: The differentiation of a preadipocyte into a mature adipocyte represents a fundamental process in biology that requires a scripted program of transcriptional events leading to changes in gene expression. As part of our contribution to the Nuclear Receptor Signaling Atlas (NURSA), we used quantitative real-time PCR to profile the temporal expression of all 49 members of the mouse nuclear receptor superfamily at selected time points during differentiation of 3T3-L1 cells into mature, lipid-bearing adipocytes using two differentiation inducers [DMI (a cocktail of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin) and rosiglitazone]. We also included a comparative analysis of nuclear receptor expression in mouse primary preadipocytes and mature adipocytes. In addition to confirming the expression of receptors known to be required for adipogenesis, this analysis revealed the existence of a tightly regulated transcriptional cascade that appeared in three distinct temporal phases. The first phase began within 4 h of adipogenic initiation with the transient, sequential expression of four previously uncharacterized receptors, followed by biphasic expression of a second subset, and ended with the sequential increase in a third receptor subset over a period of 2 wk after initiation. The discovery that these receptors may serve as adipogenic biomarkers and as potential therapeutic targets in adipose-related diseases highlights the utility of quantitative expression profiling as a method for directing mechanism-based approaches to study complex regulatory pathways.

A Nuclear Receptor Atlas: Macrophage Activation

Abstract: Macrophage activation is an essential cellular process underlying innate immunity, enabling the body to combat bacteria and other pathogens. In addition to host defense, activated macrophages play a central role in atherogenesis, autoimmunity, and a variety of inflammatory diseases. As members of the Nuclear Receptor Signaling Atlas (NURSA) program, we employed quantitative real-time PCR (qPCR) to provide a comprehensive assessment of changes in expression of the 49 members of the murine nuclear receptor superfamily. In this study, we have identified a network of 28 nuclear receptors associated with the activation of bone marrow-derived macrophages by lipopolysaccharide or the prototypic cytokine interferon gamma. More than half of this network is deployed in three intricate and highly scripted temporal phases that are unique for each activator. Thus, early receptors whose expression peaks within 4 h after lipopolysaccharide exposure, such as glucocorticoid receptor, peroxisome proliferator-activated receptor gamma, and neuronal growth factor 1B, are found as late rising markers of the interferon gamma cascade, occurring 16 h or later. The discovery of precise serial expression patterns reveals that macrophage activation is the product of an underlying process that impacts the genome within minutes and identifies a collection of new therapeutic targets for controlling inflammation by disruption of presumptive regulatory cascades.

p38 Mitogen-activated protein kinase (MAPK) is a key mediator in glucocorticoid-induced apoptosis of lymphoid cells: correlation between p38 MAPK activation and site-specific phosphorylation of the human glucocorticoid receptor at serine 211.

Abstract: Glucocorticoids (GCs) induce apoptosis in lymphoid cells through activation of the GC receptor (GR). We have evaluated the role of p38, a MAPK, in lymphoid cell apoptosis upon treatment with the synthetic GCs dexamethasone (Dex) or deacylcortivazol (DAC). The highly conserved phosphoprotein p38 MAPK is activated by specific phosphorylation of its threonine180 and tyrosine182 residues. We show that Dex and DAC stimulate p38 MAPK phosphorylation and increase the mRNA of MAPK kinase 3, a specific immediate upstream activator of p38 MAPK. Enzymatic assays confirmed elevated activity of p38 MAPK. Pharmacological inhibition of p38 MAPK activity was protective against GC-driven apoptosis in human and mouse lymphoid cells. In contrast, inhibition of the MAPKs, ERK and cJun N-terminal kinase, enhanced apoptosis. Activated p38 MAPK phosphorylates specific downstream targets. Because phosphorylation of the GR is affected by MAPKs, we examined its phosphorylation state in our system. We found serine 211 of the human GR to be a substrate for p38 MAPK both in vitro and intracellularly. Mutation of this site to alanine greatly diminished GR-driven gene transcription and apoptosis. Our results clearly demonstrate a role for p38 MAPK signaling in the pathway of GC-induced apoptosis of lymphoid cells.

Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into Leydig cell mitochondria

Abstract: Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.

Sumoylation of the estrogen receptor alpha hinge region regulates its transcriptional activity.

Abstract: The steroid hormone 17beta-estradiol (estrogen) plays a significant role in the normal physiology of the mammary gland and breast cancer development primarily through binding to its receptor, the estrogen receptor alpha (ERalpha). ERalpha is a nuclear transcription factor undergoing different types of posttranslational modifications, i.e. phosphorylation, acetylation, and ubiquitination, which regulate its transcriptional activation and/or stability. Here we identify ERalpha as a new target for small ubiquitin-like modifier (SUMO)-1 modification in intact cells and in vitro. Moreover, ERalpha sumoylation occurs strictly in the presence of hormone. SUMO-1 appears to regulate ERalpha-dependent transcription. Using a series of mutants, we demonstrated that ERalpha is sumoylated at conserved lysine residues within the hinge region. Mutations that prevented SUMO modification impaired ERalpha-induced transcription without influencing ERalpha cellular localization. In addition to identifying protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIAS3 as E3 ligases for ERalpha, we also found that PIAS1 and PIAS3, as well as Ubc9, modulated ERalpha-dependent transcription independently from their SUMO-1 conjugation activity. These findings identify sumoylation as a new mechanism modulating ERalpha-dependent cellular response and provide a link between the SUMO and estrogen pathways.

Physiological Role for the Cochaperone FKBP52 in Androgen Receptor Signaling

Abstract: Molecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-meditated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues. (Molecular Endocrinology 19: 1654–1666, 2005)

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.

Abstract: We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V–X (1–5)–Y–X (1–5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance...

Structural disorder in the complex of human pregnane x receptor and the macrolide antibiotic rifampicin

Abstract: The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 A crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200-210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR's promiscuous response to xenobiotics.

CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-α

Abstract: In estrogen target cells, estrogen receptor-α (ERα) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERα is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERα stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERα protein degradation was investigated. In ER-negative HeLa cells transfected with ERα and CHIP, ERα proteasomal degradation increased, whereas ERα-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in...

Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 by inducing interaction with 14-3-3.

Abstract: Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an aldosterone-regulated early response gene product that regulates the activity of several ion transport proteins, most notably that of the epithelial sodium channel (ENaC). Recent evidence has established that SGK1 phosphorylates and inhibits Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), a ubiquitin ligase that decreases cell surface expression of the channel and possibly stimulates its degradation. The mechanistic basis for this SGK1-induced Nedd4-2 inhibition is currently unknown. In this study we show that SGK1-mediated phosphorylation of Nedd4-2 induces its interaction with members of the 14-3-3 family of regulatory proteins. Through functional characterization of Nedd4-2-mutant proteins, we demonstrate that this interaction is required for SGK1-mediated inhibition of Nedd4-2. The concerted action of SGK1 and 14-3-3 appears to disrupt Nedd4-2-mediated ubiquitination of ENaC, thus providing a mechanism by which SGK1 modulates the ENaC-mediated Na(+) current. Finally, the expression pattern of 14-3-3 is also consistent with a functional role in distal nephron Na(+) transport. These results demonstrate a novel, physiologically significant role for 14-3-3 proteins in modulating ubiquitin ligase-dependent pathways in the control of epithelial ion transport.

Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions.

Abstract: The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inh...

Role of PROP1 in pituitary gland growth.

Abstract: Mutations in the PROP1 transcription factor gene lead to reduced production of thyrotropin, GH, prolactin, and gonadotropins as well as to pituitary hypoplasia in adult humans and mice. Some PROP1-deficient patients initially exhibit pituitary hyperplasia that resolves to hypoplasia. To understand this feature and to explore the mechanism whereby PROP1 regulates anterior pituitary gland growth, we carried out longitudinal studies in normal and Prop1-deficient dwarf mice from early embryogenesis through adulthood, examining the volume of Rathke's pouch and its derivatives, the position and number of dividing cells, the rate of apoptosis, and cell migration by pulse labeling. The results suggest that anterior pituitary progenitors normally leave the perilumenal region of Rathke's pouch and migrate to form the anterior lobe as they differentiate. Some of the cells that seed the anterior lobe during organogenesis have proliferative potential, supporting the expansion of the anterior lobe after birth. Prop1-deficient fetal pituitaries are dysmorphic because mutant cells are retained in the perilumenal area and fail to differentiate. After birth, mutant pituitaries exhibit enhanced apoptosis and reduced proliferation, apparently because the mutant anterior lobe is not seeded with progenitors. These studies suggest a mechanism for Prop1 action and an explanation for some of the clinical findings in human patients.

Release of methyl CpG binding proteins and histone deacetylase 1 from the estrogen receptor α (ER) promoter upon reactivation in ER-negative human breast cancer cells

Abstract: Estrogen receptor alpha (ER) is an epigenetically regulated gene. Inhibitors of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) synergistically activate the methylated ER gene promoter in ER-negative MDA-MB-231 human breast cancer cells. Chromatin immunoprecipitation was used to examine the chromatin status and repressor complex associated with silenced ER and changes in the key regulatory factors during reactivation by inhibitors of DNMT (5-aza-2'-deoxycytidine) and HDAC (trichostatin A). The silencing of ER due to CpG hypermethylation correlates with binding of specific methyl-binding proteins, DNMTs, and HDAC proteins. Inhibition of HDAC activity by trichostatin A results in the accumulation of hyperacetylated core histones. The activation of ER gene expression by 5-aza-2'-deoxycytidine also involves the release of the repressor complex involving various methyl-binding proteins, DNMTs, and HDAC1. HDAC and DNMT inhibitors modulate histone methylation at H3-K9 and H3-K4 to form a more open chromatin structure necessary for reactivation of silenced ER transcription. Together these results impart a better understanding of molecular mechanisms of chromatin remodeling during ER reactivation by DNMT and HDAC inhibitors. These findings will aid in the application of agents targeting epigenetic changes in the treatment of breast cancer.

Feedback Inhibition of Leptin Receptor/Jak2 Signaling via Tyr1138 of the Leptin Receptor and Suppressor of Cytokine Signaling 3

Abstract: Leptin is an adipocyte-derived hormone that communicates the status of body energy stores to the brain to regulate feeding and energy balance. The inability of elevated leptin levels to adequately suppress feeding in obesity suggests attenuation of leptin action under these conditions; the activation of feedback circuits due to high leptin levels could contribute to this leptin resistance. Using cultured cells exogenously expressing the long form of the leptin receptor (LRb) or an erythropoietin receptor/LRb chimera, we show that chronic stimulation results in the attenuation of LRb signaling and the establishment of a state in which the receptor is refractory to reactivation. Mutation of LRb Tyr1138 (the site that recruits signal transducer and activator of transcription 3) alleviated this feedback inhibition, suggesting that signal transducer and activator of transcription 3 mediates the induction of a feedback inhibitor, such as suppressor of cytokine signaling 3 (SOCS3), during chronic LRb stimulation. Indeed, manipulation of the expression or activity of the LRb-binding tyrosine phosphatase, SH2-domain containing phosphatase-2, by overexpression of wild-type and dominant negative isoforms or RNA interference-mediated knockdown did not alter the attenuation of LRb signals. In contrast, SOCS3 overexpression repressed LRb signaling, whereas RNA interference-mediated knockdown of SOCS3 resulted in increased LRb signaling that was not attenuated during chronic ligand stimulation. These data suggest that Tyr1138 of LRb and SOCS3 represent major effector pathways for the feedback inhibition of LRb signaling. Furthermore, we show that mice expressing an LRb isoform mutant for Tyr1138 display increased activity of the leptin-dependent growth and immune axes, suggesting that Tyr1138-mediated feedback inhibition may regulate leptin sensitivity in vivo.

The orphan receptor tyrosine kinase Ror2 modulates canonical Wnt signaling in osteoblastic cells.

Abstract: Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.

Alterations in mitogen-activated protein kinase kinase and extracellular regulated kinase signaling in theca cells contribute to excessive androgen production in polycystic ovary syndrome.

Abstract: We have investigated the involvement of the MAPK signaling pathway in increased androgen biosynthesis and CYP17 gene expression in women with polycystic ovary syndrome (PCOS). A comparison of MAPK kinase (MEK1/2) and ERK1/2 phosphorylation in propagated normal and PCOS theca cells, revealed that MEK1/2 phosphorylation was decreased more than 70%, and ERK1/2 phosphorylation was reduced 50% in PCOS cells as compared with normal cells. Infection with dominant-negative MEK1 increased CYP17 mRNA and dehydroepiandrosterone (DHEA) abundance, whereas constitutively active MEK1 reduced DHEA production and CYP17 mRNA abundance. Similarly, the MEK inhibitor, PD98059, increased CYP17 mRNA accumulation and CYP17 promoter activity to levels observed in PCOS cells. Remarkably, in theca cells maintained in the complete absence of insulin, ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, and CYP17 mRNA and DHEA synthesis were increased in PCOS theca cells. These studies demonst...

Progesterone receptors induce cell cycle progression via activation of mitogen-activated protein kinases.

Abstract: Progestins induce proliferation of breast cancer cells and are implicated in the development of breast cancer. The effects of progestins are mediated by progesterone receptors (PRs), although it is unclear whether proliferative effects are delivered through activities as ligand-activated transcription factors or via activation of cytoplasmic kinases. We report that progestin induces S phase entry of T47D cells stably expressing either wild-type (wt) PR-B or a transcriptionally impaired PR-B harboring a point mutation at Ser294, a ligand-dependent and MAPK consensus phosphorylation site (S294A). Both wt and S294A PR are capable of activating p42/p44 MAPKs and promoting proliferation. However, cells expressing wt, but not S294A PR, exhibited enhanced proliferation in response to combined epidermal growth factor and progestin. S phase progression correlated with up-regulation of cyclin D1. The PR antagonist RU486 also induced MAPK activation, increased cyclin D1 expression, and stimulated S phase entry, which was blocked by inhibition of either p42/p44 or p38 MAPKs, whereas proliferation induced by R5020 was sensitive only to p42/p44 MAPK inhibition. MCF-7 cells stably expressing a mutant PR unable to bind c-Src and activate MAPK failed to support progestin-induced proliferation. These data suggest that PR mediate cell cycle progression primarily through activation of cytoplasmic kinases and independently of direct regulation of transcription, whereas the coordinate regulation of both aspects of PR action are required for enhanced proliferation in response to progestins in the presence of growth factors. Targeting the ability of steroid receptors to activate MAPKs may be beneficial for breast cancer patients.

The glucose-6-phosphatase catalytic subunit gene promoter contains both positive and negative glucocorticoid response elements.

Abstract: Glucose-6-phosphatase catalyzes the final step in the gluconeogenic and glycogenolytic pathways. Glucocorticoids stimulate glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription and studies performed in H4IIE hepatoma cells demonstrate the presence of a glucocorticoid response unit (GRU) in the proximal G6Pase promoter. In vitro deoxyribonuclease I footprinting analyses show that the glucocorticoid receptor binds to three glucocorticoid response elements (GREs) in the -231 to -129 promoter region and transfection results indicate all three contribute to glucocorticoid induction of G6Pase gene transcription. Furthermore, binding sites for hepatocyte nuclear factor-1 and -4, CRE binding factors, and FKHR (FOXO1a) are required for the full glucocorticoid response. Chromatin immunoprecipitation assays show that dexamethasone treatment stimulates glucocorticoid receptor and FKHR binding to the endogenous G6Pase promoter. Surprisingly, although glucocorticoids stimulate G6Pase gene transcription, deoxyribonuclease I footprinting and transfection analyses demonstrate the presence of a negative GRE and an associated negative accessory factor element in the -271 to -225 promoter region, which inhibit the glucocorticoid response. This appears to be the first report of a promoter that contains both positive and negative GREs, which function within the same cellular environment. We hypothesize that targeted signaling to the negative accessory element within the GRU may provide tight regulation of the glucocorticoid stimulation.

Cytosolic action of thyroid hormone leads to induction of hypoxia-inducible factor-1alpha and glycolytic genes.

Abstract: Thyroid hormone (TH) effects are mediated through T(3), which regulates gene expression by binding to the nuclear TH receptors, TRalpha and TRbeta. Using microarrays and real-time PCR we found mRNAs of the following genes increased in response to T(3) in a TRbeta-specific manner: the transcription factor hypoxia-inducible factor (HIF)-1alpha, its target genes glucose transporter (GLUT)1 and platelet-type phosphofructokinase (PFKP), and the monocarboxylate transporter (MCT)4. The products of these genes have important roles in cellular glucose metabolism. HIF-1alpha expression and activity can be regulated through phosphatidylinositol-OH-3-kinase (PI3K) and MAPK signaling; thus the possibility of alternative, nonnuclear pathways of TH action was raised. We examined the involvement of these pathways in mediating TH effects by treating human skin fibroblasts with 2 nm T(3) in the absence or presence of either the PI3K inhibitor LY294002 or the MAPK inhibitor PD98059. T(3) induced HIF-1alpha mRNA by 2.7-fold (+/-0.4; P < 0.013). This increase was completely abrogated by LY294002 (1.1 +/- 0.1; nonsignificant = 0.57), but preserved in the presence of PD98059 (2.2 +/- 0.2; P < 0.009). Western blotting confirmed these results at the protein level, indicating dependency on the PI3K pathway. The same pattern of response was observed for GLUT1, PFKP, and MCT4 expression. To examine whether HIF-1alpha is directly induced, we used the translation inhibitor cycloheximide (CHX). T(3) induction of HIF-1alpha mRNA was not affected by CHX, whereas T(3) effect on GLUT1, PFKP, and MCT4 mRNA was completely abrogated by CHX. These results demonstrate that cytosolic activation of the PI3K signaling pathway has a role in TH-mediated direct (HIF-1alpha) and indirect (GLUT1, PFKP, MCT4) gene expression, and possibly provides a link between TH and cellular glucose metabolism in human fibroblasts.