Membrane progesterone receptors (mPRs) have been detected in breast cancer cells and tissues, but their roles in cancer progression remain unclear. Here, we demonstrate the localization, signaling, and antiapoptotic actions of mPRs in two nuclear progesterone receptor (PR)-negative breast cancer cell lines, SKBR3 and MDA-MB-468 (MB468), and mPR expression in human breast tumor biopsies. mPRα, mPRβ, and mPRγ subtypes were detected in both cell lines as well as in breast tumor tissues from 13 individuals irrespective of nuclear steroid receptor expression. Competitive receptor binding studies with a selective PR ligand, R5020, and an mPR agonist, Org OD 02-0 confirmed the presence of functional mPRs on both cancer cell lines. Progesterone treatment of either cell line caused rapid activation of an inhibitory G protein, as well as activation of p42/44 MAP kinase. Treatment with progesterone or Org OD 02-0 significantly decreased cell death and apoptosis in response to serum starvation, whereas testosterone, 17β-estradiol, dexamethasone, and R5020 and RU486 were ineffective. Progesterone treatment of MB468 cells also increased mitochondrial membrane potential and Akt activity, but no decrease in caspase 3 activity was observed. Knockdown of mPRα expression in MB468 cells by siRNA transfection blocked the inhibitory effects of progesterone on cell death. The results indicate that progesterone can act through mPRs to inhibit apoptosis in breast cancer cells. The involvement of mPRs in the development or progression of breast tumor growth through inhibition of cell death is an intriguing possibility and requires further investigation.
The effects of 17β‐estradiol (E2) on dopamine (DA) transport could explain gender and life‐stage differences in the incidence of some neurological disorders. We tested the effects of E2 at physiological concentrations on DA efflux in nerve growth factor‐differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G‐protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter‐specific loss of 3H‐DA from pre‐loaded cells; a 9–15 min 10−9 M E2 treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non‐genomic response, and an E2‐dendrimer conjugate (limited to non‐nuclear actions) caused DA efflux within 5 min. Efflux dose–responses for E2 were non‐monotonic, also characteristic of non‐genomic estrogenic actions. ERα siRNA knockdown abolished E2‐mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E2‐mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER‐selective agonists/antagonists demonstrated that ERα is the predominant mediator of E2‐mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E2 also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non‐genomic estrogenic effects on DAT activity.
Studies of the nuclear transcriptional regulatory activities of nonphysiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E 2 ) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-α on pituitary cells, and can provoke Ca ++ influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E 2 . It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.
Nongenomic estrogenic mechanisms offer an opportunity to explain the conundrum of environmental estrogen and plant estrogen effects on cells and animals at very the low concentrations which are prevalent in our environments and diets. Heretofore the actions of these compounds have not been adequately accounted for by laboratory tests utilizing assays for actions only via the genomic pathway of steroid action and the nuclear forms of estrogen receptor α and β. Membrane versions of these receptors, and the newly described GPR30 (7TMER) receptor protein provide explanations for the more potent actions of xenoestrogens. The effects of estrogens on many tissues demand a comprehensive assessment of the receptors, receptor levels, and mechanisms that might be involved, to determine which of these estrogen mimetic compounds are harmful and which might even be used therapeutically, depending upon the life stage at which we are exposed to them.
Recent studies by several research groups have shown that G-protein estrogen receptor-1, GPER, formerly known as GPR30, mediates 17β-estradiol (E2) activation of signal transduction pathways in a variety of human cancer cells and displays E2 binding typical of a membrane estrogen receptor. However, the importance of GPER as an estrogen receptor has been questioned by Otto and coworkers. Some of the pitfalls in investigating the functions of recombinant steroid membrane receptors that may explain the negative results of these investigators are discussed. The characteristics of GPER have also been investigated in a teleost fish, Atlantic croaker, where it has been shown to mediate E2 inhibition of oocyte maturation. Investigations on newly discovered homologous proteins from distantly-related vertebrate groups are valuable for determining their fundamental, evolutionarily-conserved functions. Therefore, the functions of croaker and human GPERs were compared. The comparisons show that croaker and human GPER have very similar estrogen binding characteristics, typical of estrogen membrane receptors, and activate the same estrogen signaling pathways via stimulatory G proteins (Gs) resulting in increased cAMP production. These results suggest that the estrogen binding and estrogen signaling functions of GPER arose early in vertebrate evolution, prior to the divergence of the teleosts from the tetrapods, more than 200 million years ago. The finding that estrogen membrane signaling through GPER has been conserved for such a long period in two distantly-related vertebrate groups, mammals and fish, suggests that this is a fundamental function of GPER in vertebrates, and likely its major physiological role.
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