Nongenomic effects of estradiol vs. the birth control estrogen ethinyl estradiol on signaling and cell proliferation in pituitary tumor cells, and differences in the ability of R-equol to neutralize or enhance these effects.

Ethinyl estradiol (EE2, the active component of many birth control formulations) persists in treated waste waters and it has become a concerning endocrine-disrupting contaminant throughout the world. Previous studies have not examined the behavior of EE2 in nongenomic signaling pathways and the subsequent functional responses (either alone or in mixtures) or conducted comparisons with the physiological estrogen estradiol (E2). In this study, mitogen-activated protein kinases (MAPKs), ERK, and JNK were activated in pituitary tumor cells by fM EE2, but p38 activation was insensitive to

Xenoestrogen interference with nongenomic signaling actions of physiological estrogens in endocrine cancer cells.

Rapid nongenomic signaling by estrogens (Es), initiated near the cell membrane, provides new explanations for the potent actions of environmental chemicals that imperfectly mimic physiological Es. These pathways can affect tumor growth, stabilization, or shrinkage via a number of signaling streams such as activation/inactivation of mitogen-activated protein kinases and caspases, generation of second messengers, and phospho-triggering of cyclin instability. Though prostate cancers are better known for their responsiveness to androgen deprivation, ∼17% of late stage tumors regress in response to high dose natural or pharmaceutical Es; however, the mechanisms at the cellular level are not understood. More accurate recent measurements show that estradiol (E2) levels decline in aging men, leading to the hypothesis that maintaining young male levels of E2 may prevent the growth of prostate cancers. Major contributions to reducing prostate cancer cell numbers included low E2 concentrations producing sustained ERK phospho-activation correlated with generation of reactive oxygen species causing cancer cell death, and phospho-activation of cyclin D1 triggering its rapid degradation by interrupting cell cycle progression. These therapeutic actions were stronger in early stage tumor cells (with higher membrane estrogen receptor levels), and E2 was far more effective compared to diethylstilbestrol (the most frequently prescribed E treatment). Xenoestrogens (XEs) exacerbated the growth of prostate cancer cells, and as we know from previous studies in pituitary cancer cells, can interfere with the nongenomic signaling actions of endogenous Es. Therefore, nongenomic actions of physiological levels of E2 may be important deterrents to the growth of prostate cancers, which could be undermined by the actions of XEs.

Multi-well plate immunoassays for measuring signaling protein activations/deactivations and membrane vs. intracellular receptor levels.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

Non-genomic effects of xenoestrogen mixtures.

Xenoestrogens (XEs) are chemicals derived from a variety of natural and anthropogenic sources that can interfere with endogenous estrogens by either mimicking or blocking their responses via non-genomic and/or genomic signaling mechanisms. Disruption of estrogens' actions through the less-studied non-genomic pathway can alter such functional end points as cell proliferation, peptide hormone release, catecholamine transport, and apoptosis, among others. Studies of potentially adverse effects due to mixtures and to low doses of endocrine-disrupting chemicals have recently become more feasible, though few so far have included actions via the non-genomic pathway. Physiologic estrogens and XEs evoke non-monotonic dose responses, with different compounds having different patterns of actions dependent on concentration and time, making mixture assessments all the more challenging. In order to understand the spectrum of toxicities and their mechanisms, future work should focus on carefully studying individual and mixture components across a range of concentrations and cellular pathways in a variety of tissue types.

Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I.

UNLABELLED: Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERß and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, ß, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/ß antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. CONCLUSIONS: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.

Effects of progesterone treatment on expression of genes involved in uterine quiescence.

An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. The causes of preterm labor are not well understood, so progesterone action on the myometrium can provide clues about the processes that keep the uterus from contracting prematurely. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone appears to inhibit uterine excitability by a number of mechanisms, including increased expression of calcium and voltage-operated K(+) channels, which dampens the electrical activity of the myometrial cell, downregulation of agents, and receptors involved in myometrial contraction, reduction in cell signal components that lead to increased intracellular Ca(2+) concentrations in response to contractile stimuli, and downregulation of proteins involved in the cross-linking of actin and myosin filaments to produce uterine contractions.

Endocrine disruption via estrogen receptors that participate in nongenomic signaling pathways.

When inappropriate (non-physiologic) estrogens affect organisms at critical times of estrogen sensitivity, disruption of normal endocrine functions can result. Non-physiologic estrogen mimetics (environmental, dietary, and pharmaceutical) can signal rapidly and potently via the membrane versions of estrogen receptors, as can physiologic estrogens. Both physiologic and non-physiologic estrogens activate multiple signaling pathways, leading to altered cellular functions (e.g. peptide release, cell proliferation or death, transport). Xenoestrogens' mimicry of physiologic estrogens is imperfect. When superimposed, xenoestrogens can alter endogenous estrogens' signaling and thereby disrupt normal signaling pathways, leading to malfunctions in many tissue types. Though these xenoestrogen actions occur rapidly via nongenomic signaling pathways, they can be sustained with continuing ligand stimulation, combinations of ligands, and signaling that perpetuates downstream, eventually also impinging on genomic regulation by controlling the activation state of transcription factors. Because via these pathways estrogens and xenoestrogens cause nonmonotonic stimulation patterns, they must be carefully tested for activity and toxicity over wide dose ranges. Nongenomic actions of xenoestrogens in combination with each other, and with physiologic estrogens, are still largely unexplored from these mechanistic perspectives.

Combinations of physiologic estrogens with xenoestrogens alter ERK phosphorylation profiles in rat pituitary cells.

BACKGROUND: Estrogens are potent nongenomic phospho-activators of extracellular-signal-regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously. OBJECTIVES: We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E2), estrone (E1), and estriol (E3). METHODS: We quantified hormone/mimetic-induced ERK phosphorylations in the GH3/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands. RESULTS: Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated. CONCLUSIONS: XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause).

Estrogens of multiple classes and their role in mental health disease mechanisms.

Gender and sex hormones can influence a variety of mental health states, including mood, cognitive development and function, and vulnerability to neurodegenerative diseases and brain damage. Functions of neuronal cells may be altered by estrogens depending upon the availability of different physiological estrogenic ligands; these ligands and their effects vary with life stages, the genetic or postgenetic regulation of receptor levels in specific tissues, or the intercession of competing nonphysiological ligands (either intentional or unintentional, beneficial to health or not). Here we review evidence for how different estrogens (physiological and environmental/dietary), acting via different estrogen receptor subtypes residing in alternative subcellular locations, influence brain functions and behavior. We also discuss the families of receptors and transporters for monoamine neurotransmitters and how they may interact with the estrogenic signaling pathways.

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells.

BACKGROUND: Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens. METHODS: We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERß, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively). RESULTS: All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses. CONCLUSIONS: Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.

Subchronic exposure to phytoestrogens alone and in combination with diethylstilbestrol - pituitary tumor induction in Fischer 344 rats.

BACKGROUND: Subchronic administration of the potent pharmaceutical estrogen diethylstilbestrol (DES) to female Fischer 344 (F344) rats induces growth of large, hemorrhagic pituitaries that progress to tumors. Phytoestrogens (dietary plant estrogens) are hypothesized to be potential tumor inhibitors in tissues prone to estrogen-induced cancers, and have been suggested as "safer" estrogen replacements. However, it is unknown if they might themselves establish or exacerbate the growth of estrogen-responsive cancers, such as in pituitary. METHODS: We implanted rats with silastic capsules containing 5 mg of four different phytoestrogens - either coumestrol, daidzein, genistein, or trans-resveratrol, in the presence or absence of DES. We examined pituitary and other organ weights, blood levels of prolactin (PRL) and growth hormone (GH), body weights, and pituitary tissue histology. RESULTS: Blood level measurements of the administered phytoestrogens confirmed successful exposure of the animals to high levels of these compounds. By themselves, no phytoestrogen increased pituitary weights or serum PRL levels after 10 weeks of treatment. DES, genistein, and resveratrol increased GH levels during this time. Phytoestrogens neither changed any wet organ weight (uterus, ovary, cervix, liver, and kidney) after 10 weeks of treatment, nor reversed the adverse effects of DES on pituitaries, GH and PRL levels, or body weight gain after 8 weeks of co-treatment. However, they did reverse the DES-induced weight increase on the ovary and cervix. Morphometric examination of pituitaries revealed that treatment with DES, either alone or in combination with phytoestrogens, caused gross structural changes that included decreases in tissue cell density, increases in vascularity, and multiple hemorrhagic areas. DES, especially in combination with phytoestrogens, caused the development of larger and more heterogeneous nuclear sizes in pituitary. CONCLUSIONS: High levels of phytoestrogens by themselves did not cause pituitary precancerous growth or change weights of other estrogen-sensitive organs, though when combined with DES, they counteracted the growth effects of DES on reproductive organs. In the pituitary, phytoestrogens did not reverse the effects of DES, but they did increase the sizes and size heterogeneity of nuclei. Therefore, phytoestrogens may oppose some but not all estrogen-responsive tissue abnormalities caused by DES overstimulation, and appear to exacerbate DES-induced nuclear changes.

Nongenomic signaling pathways of estrogen toxicity.

Xenoestrogens can affect the healthy functioning of a variety of tissues by acting as potent estrogens via nongenomic signaling pathways or by interfering with those actions of multiple physiological estrogens. Collectively, our and other studies have compared a wide range of estrogenic compounds, including some closely structurally related subgroups. The estrogens that have been studied include environmental contaminants of different subclasses, dietary estrogens, and several prominent physiological metabolites. By comparing the nongenomic signaling and functional responses to these compounds, we have begun to address the structural requirements for their actions through membrane estrogen receptors in the pituitary, in comparison to other tissues, and to gain insights into their typical non-monotonic dose-response behavior. Their multiple inputs into cellular signaling begin processes that eventually integrate at the level of mitogen-activated protein kinase activities to coordinately regulate broad cellular destinies, such as proliferation, apoptosis, or differentiation.

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases.

BACKGROUND: Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high levels of the membrane estrogen receptor-alpha. METHODS: We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. RESULTS: Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH3/B6/F10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. CONCLUSION: Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary.

Characterization of the cyclic adenosine monophosphate target site in the oxytocin receptor gene in rabbit amnion.

Oxytocin (OXT) is a potent stimulator of prostaglandin E(2) (PGE(2)) synthesis by rabbit amnion cells obtained near the end of pregnancy. Coincident with a marked increase in sensitivity of PGE(2) synthesis to OXT, the concentration of OXT receptors (OXTRs) is abruptly upregulated about 200-fold at term. This increase can be mimicked in preterm amnion cells in primary culture by the synergistic action of agents that increase cAMP synthesis and by glucocorticoids. To elucidate the mechanism of cAMP action, we cloned the rabbit OXTR gene and isolated a 200-base pair (bp) forskolin-responsive region about 4.7 kilobase upstream from the transcriptional start site using transient transfection assays. This region corresponds to a DNase I-hypersensitive site that appears in amnion tissue only near the end of pregnancy, when OXTRs are upregulated. The effects of forskolin were mediated in part by cAMP response element binding protein (CREB), as coexpression of reporter constructs with dominant negative CREB inhibited reporter expression. In addition, CREB was cross-linked to sites in the 200-bp region only in chromatin isolated from cells near the end of pregnancy, as demonstrated by chromatin immunoprecipitation (ChIP). Because the transient transfection results are consistent with work using tissue extracts (DNase I hypersensitivity and ChIP), we conclude that cAMP, acting through a specific upstream CREB binding site, is critical for the physiological upregulation of OXTRs in the amnion at the end of gestation.

Alkylphenol xenoestrogens with varying carbon chain lengths differentially and potently activate signaling and functional responses in GH3/B6/F10 somatomammotropes.

BACKGROUND: Alkylphenols varying in their side-chain lengths [ethyl-, propyl-, octyl-, and nonylphenol (EP, PP, OP, and NP, respectively)] and bisphenol A (BPA) represent a large group of structurally related xenoestrogens that have endocrine-disruptive effects. Their rapid nongenomic effects that depend on structure for cell signaling and resulting functions are unknown. OBJECTIVES: We compared nongenomic estrogenic activities of alkylphenols with BPA and 17beta-estradiol (E(2)) in membrane estrogen receptor-alpha-enriched GH3/B6/F10 pituitary tumor cells. These actions included calcium (Ca) signaling, prolactin (PRL) release, extracellular-regulated kinase (ERK) phosphorylation, and cell proliferation. METHODS: We imaged Ca using fura-2, measured PRL release via radioimmunoassay, detected ERK phosphorylation by fixed cell immunoassay, and estimated cell number using the crystal violet assay. RESULTS: All compounds caused increases in Ca oscillation frequency and intracellular Ca volume at 100 fM to 1 nM concentrations, although long-chain alkylphenols were most effective. All estrogens caused rapid PRL release at concentrations as low as 1 fM to 10 pM; the potency of EP, PP, and NP exceeded that of E(2). All compounds at 1 nM produced similar increases in ERK phosphorylation, causing rapid peaks at 2.5-5 min, followed by inactivation and additional 60-min peaks (except for BPA). Dose-response patterns of ERK activation at 5 min were similar for E2, BPA, and PP, whereas EP caused larger effects. Only E2 and NP increased cell number. Some rapid estrogenic responses showed correlations with the hydrophobicity of estrogenic molecules; the more hydrophobic OP and NP were superior at Ca and cell proliferation responses, whereas the less hydrophobic EP and PP were better at ERK activations. CONCLUSIONS: Alkylphenols are potent estrogens in evoking these nongenomic responses contributing to complex functions; their hydrophobicity can largely predict these behaviors.

Membrane estrogen receptor-alpha-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells.

BACKGROUND: Estradiol (E2) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E2 can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERalpha (mERalpha)-enriched GH3/B6/F10 rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E2 and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects. METHODS: To investigate the activation of different nongenomic pathways, and determine the involvement of mERalpha, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca2+] by Fura-2 fluorescence imaging in cells treated with E2 or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol). RESULTS: Coumesterol and daidzein increased PRL release similar to E2 in GH3/B6/F10 cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1-10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca2+ uptake, though in unique dose-dependent Ca2+ response patterns for several aspects of this response. A subclone of GH3 cells expressing low levels of mERalpha (GH3/B6/D9) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERalpha. CONCLUSION: Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca2+]) via mERalpha than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E2 and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.

Nongenomic actions of estradiol compared with estrone and estriol in pituitary tumor cell signaling and proliferation.

Physiological estrogens, including estrone (E(1)), estradiol (E(2)), and estriol (E(3)), fluctuate with life stage, suggesting specific roles for them in biological and disease processes. We compared their nongenomic signaling and functional actions in GH3/B6/F10 rat pituitary tumor cells. All hormones caused prolactin release at 1 min; the lowest effective concentrations were 10(-11) M E(2), 10(-10) M E(1), and 10(-7) M E(3). All estrogens increased the oscillation frequency of calcium (Ca) spikes, with the same time delay (approximately 200 s) at all levels (10(-15) to 10(-9) M). At some concentrations, E(1) and E(3) provoked more Ca-responding cells than E(2). The amplitude and volume of Ca peaks were elevated by all hormones at > or = 10(-15) M. All hormones caused cell proliferation, with the lowest effective concentrations of E(2) (10(-15) M) > E(1) (10(-12) M) > E(3) (10(-10) M); E(2) caused higher maximal cell numbers at most concentrations. All estrogens caused oscillating extracellular-regulated kinase (ERK) activations, with relative potencies of E(1) and E(2) > E(3). All estrogens were ineffective in activation of ERKs or causing proliferation in a subline expressing low levels of membrane estrogen receptor-alpha. Dose-response patterns were frequently nonmonotonic. Therefore, the hormones E(1) and E(3), which have been designated "weak" estrogens in genomic actions, are strong estrogens in the nongenomic signaling pathways and functional responses in the pituitary.

Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences.

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed ( approximately 30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Interleukin-1-induced NF-kappaB recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells.

The myometrial oxytocin receptor (OTR) is highly regulated during pregnancy, reaching maximal concentrations near term. These levels are then abruptly reduced in advanced labour and the post-partum period. Our goal was to examine the molecular basis for this reduction, using chromatin immunoprecipitation (ChIP). Interleukin-1alpha (IL1A) treatment of cultured human myometrial cells has previously been shown to reduce steady-state levels of OTR mRNA. We show further that IL1A reduced RNA polymerase II cross-linking to the otr promoter, as reflective of transcriptional inhibition. IL1A also increased the recruitment of nuclear factor kappaB (NF-kappaB) to a site 955 bp upstream from the transcriptional start site. Inhibition of NF-kappaB activation negated the effects of IL1A on polymerase II dissociation, indicating a causal relationship, at least in part, between recruitment of NF-kappaB and detachment of polymerase from the otherwise constitutively active otr promoter. IL1A treatment also resulted in increased histone H4 acetylation in the otr promoter region. Whereas NF-kappaB recruitment and histone acetylation are generally associated with activation of gene expression, our findings show that both processes can be involved in dissociation of RNA polymerase II from an active promoter. The results of these studies suggest that the elevation of IL1 in the myometrium occurring at the end of pregnancy initiates the process of down-regulation of OTRs in advanced labour, resulting in the desensitization of the myometrium to elevated levels of OT in the blood during lactation.

Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector.

An examination of cellular processes involved in myometrial function has been greatly assisted by the use of human myometrial cells in primary culture. However, these cells can be used only for several passages before they senesce, and responses to various agents change with time in culture. The use of transformed cells is limited, as they can be polynucleated and can lose or gain chromosomes. We have developed three telomerase-immortalized cell lines from term-pregnant human myometrium to eliminate variability between passage numbers and allow genetic manipulations of myometrial cells to fully characterize signal pathways. These cells have a normal karyotype and were verified to be uterine smooth muscle by immunocytochemical staining for smooth muscle cell-specific alpha-actin and high affinity oxytocin antagonist binding sites. The three cell lines and the cells in primary culture from which they were derived were examined by cDNA microarray analysis. Of >10 000 expressed genes, there were consistent changes in the expression of approximately 1% in the three immortalized cell lines. We were unable to detect any significant differences between primary and immortalized cells in signal pathways such as epidermal growth factor-stimulated epidermal growth factor receptor phosphorylation, insulin-stimulated Akt phosphorylation, oxytocin and lysophosphatidic acid-stimulated extracellular signal-regulated kinase 1 and 2 phosphorylation, myosin light chain phosphorylation, and interleukin-1 induction of IkappaBalpha degradation. The immortalized cells should be useful for a range of studies, including high throughput analyses of the effects of environmental agents on the human myometrium.