Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ4-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency.
cancer; bile-acids; hydroxysteroid dehydrogenase; inherited mutations; neurosteroids; synthetic steroids
Androgens play a pivotal role in the regulation of body fat distribution. Adipogenesis is a process whereby multipotent adipose stem cells (ASCs) initially become preadipocytes (ASC commitment to preadipocytes) before differentiating into adipocytes. Androgens inhibit human (h) subcutaneous (SC) abdominal preadipocyte differentiation in both sexes, but their effects on hASC commitment to preadipocyte formation is unknown. We therefore examined whether androgen exposure to human (h) ASCs, isolated from SC abdominal adipose of nonobese women, impairs their commitment to preadipocyte formation and/or subsequent differentiation into adipocytes. For this, isolated hASCs from SC abdominal lipoaspirate were cultured in adipogenesis-inducing medium for 0.5–14 days in the presence of testosterone (T, 0–100 nM) or dihydrotestosterone (DHT, 0–50 nM). Adipogenesis was determined by immunofluorescence microscopy and by quantification of adipogenically relevant transcriptional factors, PPARγ, C/EBPα and C/EBPβ. We found that a 3-day exposure of hASCs to T (50 nM) or DHT (5 nM) in adipogenesis-inducing medium impaired lipid acquisition and decreased PPARγ, C/EBPα and C/EBPβ gene expression. The inhibitory effects of T and DHT at this early-stage of adipocyte differentiation, were partially and completely reversed by flutamide (F, 100 nM), respectively. The effect of androgens on hASC commitment to a preadipocyte phenotype was examined via activation of BMP4 signaling. T (50 nM) and DHT (5nM) significantly inhibited the stimulatory effect of BMP4-induced ASC commitment to the preadipocyte phenotype, as regards PPARγ and C/EBPα gene expression. Our findings indicate that androgens, in part through androgen receptor action, impair BMP4-induced commitment of SC hASCs to preadipocytes and also reduce early-stage adipocyte differentiation, perhaps limiting adipocyte numbers and fat storage in SC abdominal adipose.
testosterone; dihydrotestosterone; adipogenesis; cell commitment
The etiology of the polycystic ovary syndrome (PCOS) remains unclear, despite its high prevalence among infertility disorders in women of reproductive age. Although there is evidence for a genetic component of the disorder, other causes, such as prenatal insults are considered among the potential factors that may contribute to the development of the syndrome. Over the past few decades, several animal models have been developed in an attempt to understand the potential contribution of exposure to excess steroids on the development of this syndrome. The current review summarizes the phenotypes of current animal models exposed to excess steroid during the prenatal and early postnatal period and how they compare with the phenotype seen in women with PCOS.
Infertility; PCOS; fetal programming; androgens; estrogens
Surgically menopausal women incur a 2–5 fold increased risk for dementia and mortality from neurological diseases, but the mechanisms underlying these increased risks remain unclear. Previously, we demonstrated that after global cerebral ischemia (GCI), 17β-estradiol (E2 or estrogen) suppresses hippocampal elevation of the Wnt antagonist Dickkopf-1 (Dkk1), a neurodegenerative factor. We, thus, hypothesized that prolonged loss of E2 may lead to dysregulation of neural Dkk1 and Wnt/β-Catenin signaling, which could contribute to an increased risk of neurodegeneration. To test this hypothesis, we examined the effect of shortterm (1 week - STED) and long-term E2 deprivation (10 weeks - LTED) via ovariectomy upon basal and E2-regulated Dkk1 levels and Wnt/β-Catenin signaling in the hippocampal CA1 region following GCI. In STED rats, E2 exerted robust neuroprotection against GCI, suppressed postischemic elevation of Dkk1, and enhanced pro-survival Wnt/β-Catenin signaling, effects that were lost in LTED rats. Intriguingly, LTED rats displayed modest basal changes in Dkk1 and survivin expression. Further work showed that c-Jun N-Terminal Kinase (JNK) mediated GCI-induced changes in Dkk1 and survivin, and JNK inhibition afforded neuroprotection in LTED rats. Finally, we extended our findings to natural aging, as 24-month-old, reproductively senescent female rats also displayed a modest increase in basal Dkk1 in the CA1, which consistently co-localized with the apoptotic marker TUNEL after GCI and coincided with a loss of E2 neuroprotection. As a whole, this study supports the “critical period hypothesis” and further suggests that perimenopausal estradiol replacement may prevent neurodegenerative changes in the hippocampus by maintaining favorable Wnt/β-Catenin signaling.
Estrogen; Dkk1; Hippocampus; Long-Term Estrogen Deprivation; Menopause; Neuroprotection
Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ERα appears to be the critical isoform. The role of ERα in reproduction has been found to involve a nuclear ERα that induces a genomic mechanism of action. More recent a plasma membrane ERα has been shown to trigger signaling pathways involved in reproduction. Mechanisms underlying membrane-initiated estradiol signaling are emerging, including evidence that activation of plasma membrane ERα involves receptor trafficking. The present study examined the insertion of ERα into the plasma membrane of N-38 neurons, an immortalized murine hypothalamic cell line. We identified, using western blotting and PCR that N-38 neurons express full-length 66 kDa ERα and a 52 kDa ERα spliced variant missing the fourth exon - ERαΔ4. Using surface biotinylation we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ERα protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ERα was blocked by the ER antagonist ICI 182,780 or with the protein kinase C (PKC) pathway inhibitor bisindolylmaleimide (BIS). Downstream membrane-initiated signaling was confirmed by estradiol activation of PKC-theta (PKCθ) and the release of intracellular calcium. These results indicate that membrane ERα levels in N-38 neurons are dynamically autoregulated by estradiol.
N-38 neurons; membrane-initiated estradiol signaling; receptor trafficking; ERα slice variant; surface biotinylation
The steroid hormone, 17β-estradiol (E2) has been reported to enhance executive functions that are known to be mediated by the prefrontal cortex (PFC), although the underlying mechanisms remain unclear. To shed light on the potential mechanisms, we examined the effect of E2 in vivo upon spine density in the rat PFC and the somatosensory cortex (SSC), which has been implicated to be a transient storage site for information that can also contribute to working memory. The results revealed that E2 significantly enhanced the number of dendritic spines in both the SSC and PFC, as well as the expression of spinophilin. In vitro studies revealed further mechanistic insights by demonstrating that E2 enhanced AMPA GluR1 receptor expression and excitatory glutamatergic synapse formation in rat cortical neurons, without an effect upon inhibitory GABAergic synapse formation. Furthermore, E2 rapidly enhanced ERK and Akt activation in cortical neurons, and inhibitors of ERK and Akt activation significantly attenuated E2 induction of excitatory glutamatergic synapses. Administration of E2-BSA likewise significantly enhanced excitatory glutamatergic synapses in cortical neurons, and administration of an ER antagonist, ICI182,780 and a non-NMDA receptor antagonist (NBQX) significantly attenuated the effect of E2 upon enhancement of excitatory glutamatergic synapses, suggesting mediation by extranuclear estrogen receptors and involvement of non-NMDA receptor activation and signaling. As a whole, the studies demonstrate that E2 enhances spine density in both the PFC and SSC, and that E2 enhances excitatory glutamatergic synapse formation in cortical neurons via a rapid extranuclear ER-mediated signaling mechanism that involves up-regulation of GluR1 and mediation by Akt and ERK signaling pathways.
Estradiol; nongenomic; synapse; synaptic plasticity; kinase
Recent studies have suggested that progestins play a role in the etiology of breast cancer; however, the mechanisms by which progestins promote tumor formation/progression have not been defined. Progestin action, in target tissues such as the breast, is mediated by the progesterone receptor (PR). PR signaling is complex and PR regulates transcription of target genes through a variety of mechanisms. Many cell signaling pathways are activated inappropriately in breast cancer cells and these pathways can regulate PR activity. For example, the p42/p44 MAPK pathway can regulate PR function by altering phosphorylation of PR, as well as its coregulators. We found that inhibition of the p42/p44 MAPK signaling pathway with a MEK inhibitor (U0126) impairs PR-mediated gene induction, but not gene repression. In addition, the effects of U0126 on PR-mediated gene transcription are much greater with long-term versus short-term inhibition and are gene-specific. Finally, treatment with U0126 delays phosphorylation of Ser294, but does not block phosphorylation completely, suggesting that p42/p44 MAPK kinase is not the dominant kinase responsible for phosphorylating this site. Collectively, these studies suggest that in addition to the p42/p44 MAPK pathway, other signaling pathways are also important for PR transcriptional activity in breast cancer cells. The integration of PR transcriptional effects and cell signaling pathways has implications for the initiation or progression of breast cancer. Understanding how these pathways interact may aid in the development of prevention and/or treatment strategies for the disease.
Progesterone receptor; breast cancer; p42/p44 MAPK; phosphorylation; U0126; T47D
Androgen ablation therapy is the most common treatment for advanced prostate cancer (PCa), but most patients will develop castration-resistant prostate cancer (CRPC), which has no cure. CRPC is androgen-depletion resistant but androgen receptor (AR) dependent. AR is a nuclear receptor whose transcriptional activity is regulated by hormone binding to the ligand-binding domain (LBD). Constitutively active AR splice variants that lack LBDs often are expressed in CRPC. The expression of these variants indicates that methods to inhibit AR activity that do not rely on inactivating the LBD are needed. Heat shock protein 90 (Hsp90), a potential therapeutic target in PCa, is an AR chaperone crucial for proper folding, hormone binding and transcriptional activity of AR. We generated LNCaP cell lines with regulated expression of the AR-V7 variant as well as a cell line expressing artificially truncated AR (termed AR-NTD) to characterize splice variant function. Using an Hsp90 inhibitor, Geldanamycin (GA), and an AR-Hsp90-FKBP52 specific inhibitor, MJC13, we sought to determine if the AR variants also require Hsp90 and associated co-chaperone, FKBP52, for their activity. GA inhibits AR transcriptional activity but has little effect on AR-V7 activity. Moreover, GA decreases the stability of AR protein, with no effect on AR-V7 levels. Full-length AR activity is strongly inhibited by MJC13 while AR-V7 is unaffected. Thus, the variants are resistant to inhibitors of the Hsp90-AR heterocomplex. Although Hsp90 inhibitors will continue to inhibit growth promoting kinases and signaling through activated full-length AR in CRPC, AR signaling through variants will be retained.
Androgen Receptor (AR); Prostate Cancer; Splice Variants; Hsp90; FKBP52
Androgen receptor (AR) signaling is essential for the initial development and progression of prostate cancer (PCa) as well as the growth and survival of castration-resistant tumors. However, AR action may be opposed by estrogen receptor beta (ERβ) that responds to androgen metabolites produced in the prostate. The balance between the activity of these two receptors is not only influenced by the steroidogenic capacity of the prostatic microenvironment but also by its redox status and local paracrine signals such as transforming growth factor- beta (TGF-β). In this review, we highlight the studies that revealed select roles for AR and ERβ in distinct compartments of the prostate cancer microenvironment. We also discuss new work that identified stromal-epithelial crosstalk through TGF-β1 signaling that drives the production of reactive oxygen species in stromal cells thereby selectively limiting the antitumor activity of ERβ in cancer cells. Therefore, any new therapeutic approaches that seek to limit AR but enhance ERβ activity in PCa, must take into account potential adaptive changes in the tumor microenvironment that utilize paracrine signals and altered redox balance to divert local androgen metabolites towards AR at the expense of ERβ.
androgen receptor; estrogen receptor-beta; prostate cancer; reactive oxygen species; transforming growth factor-beta; tumor microenvironment
The ovarian hormone 17β-estradiol (E2) exerts profound neuroprotective actions against ischemia-induced brain damage in rodent models of global and focal ischemia. This review focuses on the neuroprotective efficacy of post-ischemic administration of E2 and non-feminizing estrogen analogs in the aging brain, with an emphasis on studies in animals subjected to a long-term loss of circulating E2. Clinical findings from the Women’s Health Initiative study as well as data from animal studies that used long-term, physiological levels of E2 treatment are discussed in this context. We summarize major published findings that highlight the effective doses and timing of E2 treatment relative to onset of ischemia. We then discuss recent findings from our laboratory showing that under some conditions the aging hippocampus remains responsive to E2 and some neuroprotective non-feminizing estrogen analogs even after prolonged periods of hormone withdrawal. Possible membrane-initiated signaling mechanisms that may underlie the neuroprotective actions of acutely administered E2 are also discussed. Based on these findings, we suggest that post-ischemic treatment with high doses of E2 or certain non-feminizing estrogen analogs may have great therapeutic potential for treatment of brain damage and neurodegeneration associated with ischemia.
Estrogens; Hippocampus; Global ischemia; Stroke; Neuroprotection; GPR 30
Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity . Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ERα a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ERα and mutants ERα S212A, ERα S212D, ERα S236A and ERα S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ERα. ERα S212D, but not ERα S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ERβ; the ERβ S176D mutant retained its trans-activation activity, but the ERβ S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ERα S212A or ERα S212D, revealed that phosphorylation of serine 212 enabled ERα to regulate a unique set of genes and cellular functions.
Estrogen receptor; Phosphorylation; DNA binding domain; Gene expression; Nuclear receptor CAR
Progesterone is an ovarian steroid hormone that is essential for normal breast development. The actions of progesterone are largely mediated through binding to its cognate steroid hormone receptor, the progesterone receptor (PR). PR isoforms exist in the nucleus and transcriptionally activate genes necessary for proliferation and survival (classical role). Cytoplasmic or membrane-associated PR exists in the cytoplasm where it participates in protein complexes with signaling molecules and other steroid hormone receptors capable of rapid activation of cytoplasmic protein kinase cascades. This review details the extra nuclear scaffolding actions of PR with c-Src and MEK1, the upstream components of MAP kinase modules.
Progesterone receptor; CD domain; Scaffold; Rapid kinase activation; c-Src; MEK1
We have devised an efficient procedure for the synthesis of 5α-dihydrotestosterone (DHT) (1) starting from 3β-hydroxy-5α-androstan-17-one, providing the product in unprecedented 82% yield. A reported method of using toxic Jones reagent is replaced by milder oxidizing agent (NMO/TPAP) in the synthesis of a key intermediate 17β-[(tert-butyldimethylsilyl)oxy]-5α-androstan-3-one (18). This new procedure is simple, does not require special apparatus/precautions or chromatographic purification in most of the steps.
Androgen receptor (AR); 5α-dihydrotestosterone; (DHT); improved synthetic procedure; prostate cancer (PC); steroids
Glucocorticoids are important regulators of metabolism and immune function. Synthetic glucocorticoids are extensively used for immunosuppression/anti-inflammatory therapy. Since the glucocorticoid receptor (GR) is central to most hormone effects; its in vivo regulation will influence hormone/drug action. An alternative splice variant, GRβ, is present in humans and may function as a dominant negative regulator of GR transcriptional activity. Recently, a similar splice variant was reported in mouse, although the mechanism of alternative splicing differs from that in humans. We present evidence that a splice variant of GR with an alternative C-terminus also occurs in the rat by a mechanism of intron inclusion. A highly quantitative qRT-PCR assay for the simultaneous measurement of both splice variants in a single sample was developed in order to accurately measure their regulation. We used this assay to assess the tissue specific expression of both mRNAs, and demonstrate that GRα is predominant in all tissues. In addition, the regulation of both GRα and GRβ mRNA by various physiological factors in rat liver was assessed. GRα showed a robust circadian rhythm, which was entrained with the circadian oscillation of the endogenous hormone. Time series experiments showed that both corticosteroids and LPS but not insulin dosing resulted in the transient down-regulation of GRα mRNA. LPS treatment also resulted in down-regulation of GRβ expression. A modest up-regulation in GRβ expression was observed only in animals having chronically elevated plasma insulin concentrations. However the expression of GRβ was significantly lower than that of GRα in all cases.
glucocorticoids; glucocorticoid receptor; GRβ; qRTPCR
The translocator protein (18-kDa) TSPO is an ubiquitous high affinity cholesterol-binding protein reported to be present in the endothelial and smooth muscle cells of the blood vessels; its expression dramatically increased in macrophages found in atherosclerotic plaques. A domain in the carboxy-terminus of TSPO was identified and characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide could be used as an hypocholesterolemic, with potential anti-atherogenic properties, agent. We report herein the therapeutic benefit that resulted for the administration of the VLNYYVWR human CRAC sequence to guinea pigs fed with a high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. CRAC treatment (3 and 30 mg/kg once daily for 6 weeks) resulted in reduced circulating cholesterol levels in guinea pigs fed with 2% high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. In high cholesterol fed guinea pigs, CRAC treatment administered once daily induced an increase in circulating HDL, decreased total, free and LDL cholesterol, and removed atheroma deposits in the aorta in a dose-dependent manner. The treatment also prevented the high cholesterol diet-induced increase in serum creatine kinase, total and isoforms, markers of neurological, cardiac and muscular damage. No toxicity was observed. Taken together these results support a role of TSPO in lipid homeostasis and atherosclerosis and indicate that CRAC may constitute a novel and safe treatment of hypercholesterolemia and atherosclerosis.
TSPO; cholesterol; CRAC domain; LDL; atheroma; Drug development
Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβ in vitro, modulating estrogen target genes in vivo.
estrogen receptors; DHEA; androstendione; androstendiol; transcription
Polycystic ovary syndrome (PCOS) is mainly defined by hyperandrogenemia, from ovarian and adrenal origin, and is characterized by insulin resistance (IR). Studies found that raising in vivo non-esterified fatty acid (NEFA) levels, which induces lipotoxicity, increases androgen levels and IR. The aim of this study was therefore to determine the effects of in vitro over-exposure to NEFA on androgen synthesis in a bovine adrenocortical cell model.
Bovine fasciculata/reticularis cells were cultured for 2 days in the absence or presence of ACTH (10 nmol/L) or Forskolin (fsk, 10 μmol/L), alone or in combination with the saturated fatty acid (FA) palmitate (100 μmol/L). Steroid production was measured in medium and corrected for initial cell seeding count. CYP17 protein expression and ERK1/2 phosphorylation were assessed by Western blotting.
Under unstimulated conditions, dehydroepiandrosterone (DHEA) levels were barely detected and no difference was observed after palmitate exposure, which was also the case for CYP17 expression and ERK1/2 phosphorylation. Under stimulation, palmitate exposure increased DHEA production by 38% and 69%, for ACTH and fsk, respectively, as compared to untreated conditions (Ps ≤ 0.05). In palmitate-treated vs untreated cells, fsk-stimulated ERK1/2 phosphorylation was reduced by 46% (P = 0.0047), but stimulated CYP17 expression was not significantly affected.
In a model of androgen-producing cells, under stimulated conditions, overexposure to saturated FAs significantly increases androgen production and reduces MEK/ERK activation. Therefore, this study is the first to demonstrate that lipotoxicity can directly trigger androgen overproduction in vitro, in addition to its well-described impact on IR, which strongly supports a central role of lipotoxicity in PCOS pathophysiology.
PMID: 22245830 CAMSID: cams3748
Polycystic ovary syndrome; Androgens; Adrenal glands; Fatty acids; Mitogen-activated protein kinase; Steroid 17α-hydroxylase
Phytoecdysteroids, structurally similar to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. In skeletal muscle cells, phytoecdysteroids increase protein synthesis. In this study we show that in a mouse skeletal muscle cell line, C2C12, 20-hydroxyecdysone (20HE), a common phytoecdysteroid in both insects and plants, elicited a rapid elevation in intracellular calcium, followed by sustained Akt activation and increased protein synthesis. The effect was inhibited by a G-protein Coupled Receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, and a phosphoinositide kinase-3 (PI3K) inhibitor.
ecdysteroid; skeletal muscle; Akt; GPCR; calcium; C2C12
Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.
C-5(6) sterol desaturase; Tetrahymena; endoplasmic reticulum; complementation; Saccharomyces cerevisiae; ergosterol
Androgens may provide protective effects in the vasculature under pathophysiological conditions. Our past studies have shown that dihydrotestosterone (DHT) decreases expression of cyclooxygenase-2 (COX-2) during cytokine, endotoxin, or hypoxic stimulation in human vascular smooth muscle cells, in an androgen receptor (AR)-independent fashion. Classically DHT is regarded as a pure AR agonist; however, it can be endogenously metabolized to 5α-androstane-3β, 17β-diol (3β-diol), which has recently been shown to be a selective estrogen receptor (ERβ) agonist. Therefore, we hypothesized that DHT’s anti-inflammatory properties following cytokine stimulation are mediated through ERβ. Using primary human brain vascular smooth muscle cells (HBVSMC), we tested whether DHT’s effect on IL-1β induced COX-2 expression was mediated via AR or ERβ. The metabolism of DHT to 3β-diol is a viable pathway in HBVSMC since mRNA for enzymes necessary for the synthesis and metabolism of 3β-diol [3alpha-hydroxysteroid dehydrogenase (HSD), 3β-HSD, 17β-HSD, CYP7B1] was detected. In addition, the expression of AR, ERα, and ERβ mRNA was detected. When applied to HBVSMC, DHT (10 nM; 18 h) attenuated IL-1β-induced increases in COX-2 protein expression. The AR antagonist bicalutamide did not block DHT’s ability to reduce COX-2. Both the non-selective estrogen receptor antagonist ICI 182,780 (1 μM) and the selective ERβ antagonist PHTPP (1 μM) inhibited the effect of DHT, suggesting that DHT actions are ERβ-mediated. In HBVSMC and in rat mesenteric arteries, 3β-diol, similar to DHT, reduced cytokine-induced COX-2 levels. In conclusion, DHT appears to be protective against the progression of vascular inflammation through metabolism to 3β-diol and activation of ERβ.
Interleukin-1 beta; Cyclooxygenase-2; Androgen; Vasculature; Vascular smooth muscle
A facile six-step synthesis (15.2% yield) of ent-17β-estradiol from readily accessible precursors is described. The preparation of analogues with 2-alkyl substitutents, double bond unsaturation in the C-ring, a cis C, D-ring fusion and modified substituents at C17 is also reported.
ent-17β-estradiol; enantiomer; neuroprotectants; antioxidants; estradiol analogues
Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active form of vitamin D, inhibits the growth of many malignant cells including breast cancer (BCa) cells. The mechanisms of calcitriol anticancer actions include cell cycle arrest, stimulation of apoptosis and inhibition of invasion, metastasis and angiogenesis. In addition we have discovered new pathways of calcitriol action that are especially relevant in inhibiting the growth of estrogen receptor positive (ER+) BCa cells. Calcitriol suppresses COX-2 expression and increases that of 15-PGDH thereby reducing the levels of inflammatory prostaglandins (PGs). Our in vitro and in vivo studies show that calcitriol decreases the expression of aromatase, the enzyme that catalyzes estrogen synthesis selectively in BCa cells and in the mammary adipose tissue surrounding BCa, by a direct repression of aromatase transcription via promoter II as well as an indirect effect due to the reduction in the levels of PGs, which are major stimulator of aromatase transcription through promoter II. Calcitriol down-regulates the expression of ERα and thereby attenuates estrogen signaling in BCa cells including the proliferative stimulus provided by estrogens. Thus the inhibition of estrogen synthesis and signaling by calcitriol and its anti-inflammatory actions will play an important role in inhibiting ER+ BCa. We hypothesize that dietary vitamin D would exhibit similar anticancer activity due to the presence of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) in breast cells ensuring conversion of circulating 25-hydroxyvitamin D to calcitriol locally within the breast micro-environment where it can act in a paracrine manner to inhibit BCa growth. Cell culture and in vivo data in mice strongly suggest that calcitriol and dietary vitamin D would play a beneficial role in the prevention and/or treatment of ER+ BCa in women.
Calcitriol; breast cancer; anti-proliferative effects; anti-inflammatory effects; aromatase; prostaglandins; aromatase inhibitors; estrogen receptor; dietary vitamin D
Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the granulosa and luteal cells of rodent and primate ovaries. Interestingly, its molecular weight as assessed by Western blot is dependent on its cellular localization with a ≈ 27 kDa form being detected in the cytoplasm and higher molecular weight forms being detected in the nucleus. The higher molecular weight forms of PGRMC1 are sumoylated suggesting that they are involved in regulating gene transcription, since sumoylation of nuclear proteins often is associated with regulation of transcriptional activity of the sumoylated protein.
In order to identify a set of candidate genes that are regulated by PGRMC1, a human granulosa/luteal cell line (hGL5 cells) was treated with PGRMC1 siRNA and changes in gene expression monitored by microarray analysis. The microarray analysis revealed that PGRMC1 generally functioned as a repressor of transcription, since depletion of PGRMC1 resulted in a disproportionate increase in the number of transcripts. Moreover, a pathway analysis implicated PGRMC1 in the regulation of apoptosis, which is consistent with PGRMC1’s known biological action. More importantly these results support the concept that PGRMC1 influences gene transcription. Additional studies reveal that progesterone (P4) acting through a PGRMC1-dependent mechanism suppresses the activity of the transcription factor, Tcf/Lef, thereby identifying one molecular pathway through which P4-PGRMC1 can regulate gene transcription and ultimately apoptosis.
Progesterone; PGRMC1; Ovary; Gene Expression; Tcf/Lef Transcription Factor Activity; Sumoylation; Apoptosis
Hypothesizing that rapid estrogen signaling could be modulated from different estrogen receptors with unique localization patterns, a number of groups have attempted to design drug conjugates that target or restrict compounds to specific subcellular compartments. This article will briefly discuss the history of using conjugates to dissect rapid estrogen signaling and different strategies to attempt to target estrogens and antiestrogens to different locations. It will also detail some of the potential issues that can arise with different types of conjugates, using examples drawn from the authors’ own work.
Rapid Estrogen Signaling; Drug Conjugates; E2-BSA; 4-hydroxytamoxifen; membrane estrogen receptors
The role of metabolic disturbance in polycystic ovary syndrome (PCOS) has been well established, with insulin resistance and the resulting compensatory hyperinsulinemia thought to promote hyperandrogenemia. Genome-wide association studies (GWAS) have established a large number of loci for metabolic conditions such as type 2 diabetes and obesity. A subset of these loci has been investigated for a role in PCOS; these studies generally have not revealed a confirmed role for these loci in PCOS risk. However, a large scale investigation of genes related to these pathways has not previously been performed. We conducted a two stage case control association study of 121,715 single nucleotide polymorphisms (SNPs) selected to represent susceptibility loci associated with traits such as type 2 diabetes, obesity measures, lipid levels and cardiovascular function using the Cardio-Metabochip in 847 PCOS cases and 845 controls. Several hypothesis-generating associations with PCOS were observed (top SNP rs2129107, P = 3.8 × 10−6). We did not find any loci definitively associated with PCOS after strict correction for multiple testing, suggesting that cardio-metabolic loci are not major risk factors underlying the susceptibility to PCOS.
Candidate-wide association study; Polycystic ovary syndrome; Cardio-Metabochip; Single nucleotide polymorphism; Genetic association