Nongenomic Interaction Research Articles

Protein Kinase C Alpha (PKCα) overexpression leads to a better response to retinoid acid therapy through Retinoic Acid Receptor Beta (RARβ) activation in mammary cancer cells.

Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment. PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR. Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARβ expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression. Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARβ receptor is required to ensure the effect on this process.

Uterine fibroid management: Today and tomorrow.

Current treatments for fibroids are mainly surgical and expensive, so alternatives need to be found. It is, therefore, vital to develop and evaluate alternatives to surgical procedures, especially when fertility preservation is the goal. Selective progesterone receptor modulators (SPRMs) are synthetic compounds that have either an agonistic or antagonistic impact on target tissues determined by their binding to progesterone receptors. Their mixed activity depends on recruitment of cofactors that regulate transcription along so-called genomic pathways, as well as nongenomic interactions with other signaling pathways. There is no doubt that surgery remains indicated in some instances, but we must now establish whether use of SPRMs (notably ulipristal acetate) allows less invasive surgery or even complete avoidance of surgery. Long-term intermittent administration of ulipristal acetate will undoubtedly change our approach to the management of uterine fibroids according to the International Federation of Gynecology and Obstetrics classification, which provides a comprehensive basis for different treatment options. When considering less invasive techniques (uterus-sparing options like myomectomy), the choice is guided by the size, number and location of fibroids, as well as the personal experience of the gynecologist and available equipment. There is now a growing body of evidence pointing to the crucial role of progesterone pathways in the pathophysiology of uterine fibroids. SPRMs should, therefore, be considered an alternative to surgical therapy, or at least an adjunct to surgery, as illustrated in the algorithms. © 2019 Japan Society of Obstetrics and Gynecology.

TGF-β Stimulation of EMT Programs Elicits Non-genomic ER-α Activity and Anti-estrogen Resistance in Breast Cancer Cells.

Estrogen receptor-α (ER-α) activation drives the progression of luminal breast cancers. Signaling by transforming growth factor-β (TGF-β) typically opposes the actions of ER-α; it also induces epithelial-mesenchymal transition (EMT) programs that promote breast cancer dissemination, stemness, and chemoresistance. The impact of EMT programs on nongenomic ER-α signaling remains unknown and was studied herein. MCF-7 and BT474 cells were stimulated with TGF-β to induce EMT programs, at which point ER-α expression, localization, and nongenomic interactions with receptor tyrosine kinases and MAP kinases (MAPKs) were determined. Cell sensitivity to anti-estrogens both before and after traversing the EMT program was also investigated. TGF-β stimulated MCF-7 and BT474 cells to acquire EMT phenotypes, which enhanced cytoplasmic accumulation of ER-α without altering its expression. Post-EMT cells exhibited (i) elevated expression of EGFR and IGF1R, which together with Src formed cytoplasmic complexes with ER-α; (ii) enhanced coupling of EGF, IGF-1 and estrogen to the activation of MAPKs; and (iii) reduced sensitivity to tamoxifen, an event reversed by administration of small molecule inhibitors against the receptors for TGF-β, EGF, and IGF-1, as well as those against MAPKs. EMT stimulated by TGF-β promotes anti-estrogen resistance by activating EGFR-, IGF1R-, and MAPK-dependent nongenomic ER-α signaling.

Glucocorticoid-mediated activation of GSK3β promotes tau phosphorylation and impairs memory in type 2 diabetes

Type 2 diabetes is increasingly recognized as a risk factor for Alzheimer's disease, but the underlying mechanisms remain poorly understood. Hyperphosphorylation of the microtubule-associated protein tau has been reported in rodent models of diabetes, including db/db mice, which exhibit insulin resistance and chronically elevated glucocorticoids due to leptin receptor insufficiency. In this report, we investigated endocrine mechanisms for hippocampal tau phosphorylation in db/db and wild-type mice. By separately manipulating peripheral and intrahippocampal corticosterone levels, we determined that hippocampal corticosteroid exposure promotes tau phosphorylation and activates glycogen synthase kinase 3β (GSK3β). Subsequent experiments in hippocampal slice preparations revealed evidence for a nongenomic interaction between glucocorticoids and GSK3β. To examine whether GSK3β activation mediates tau phosphorylation and impairs memory in diabetes, db/db and wild-type mice received intrahippocampal infusions of TDZD-8, a non-ATP competitive thiadiazolidinone inhibitor of GSK3β. Intrahippocampal TDZD-8 blocked tau hyperphosphorylation and normalized hippocampus-dependent memory in db/db mice, suggesting that pathological synergy between diabetes and Alzheimer's disease may involve glucocorticoid-mediated activation of GSK3β.

Progesterone regulates the proliferation of breast cancer cells - in vitro evidence.

Reports state that surgery performed at different phases of the menstrual cycle may significantly affect breast cancer treatment outcome. From previous studies, we identified differentially expressed genes in each menstrual cycle phase by microarray, then subjected them to functional in vitro analyses. Microarray studies disclosed genes that are upregulated in the luteal phase and follicular phase. TOB-1 is a tumor suppressor gene and was expressed exclusively in the luteal phase in our microarray study. Therefore, we further functionally characterized the protein product of TOB-1 in vitro. To our knowledge, no studies have yet been conducted on reactive oxygen species-regulated tumor suppressor interactions in accordance with the biphasic nature of progesterone. This work demonstrates that progesterone can produce reactive oxygen species in MCF-7 cells and that TOB-1 exerts a series of non-genomic interactions that regulate antiproliferative activity by modulating the antioxidant enzyme superoxide dismutase. Furthermore, this study implicates PTEN as an interacting partner for TOB-1, which may regulate the downstream expression of cell cycle control protein p27 via multiple downstream signaling pathways of progesterone through a progesterone receptor, purely in a time- and concentration-dependent manner. These results support the hypothesis that surgery conducted during the luteal phase of the menstrual cycle may facilitate improved patient survival.

Does low serum 25 OH vitamin D interact with very strenuous physical activity, facilitating development of rhabdomyolysis?

BackgroundVitamin D plays an important and increasingly understood role in muscle health and performance. Vitamin D exhibits a nuclear receptor for transcription interaction and a transmembrane receptor, giving it genomic and non-genomic interactions. Vitamin D receptors have been described that affect muscle function. Vitamin D has also been correlated with muscle performance. Beyond its role in normal muscle function, vitamin D deficiency can interact with statins to produce myalgia–myositis, which can be reversed by normalizing serum vitamin D [7,13]. Exertional rhabdomyolysis may occur in diverse settings including marathons, ice skating, bicycling and swimming. Our hypothesis is that subjects with pre-existing low serum 25OHD are selected out for exertional rhabdomyolysis during strenuous activities. MethodsWe review exercise induced severe rhabdomyolysis in association with heat stroke with subsequent disseminated intravascular coagulation in a thin, athletic, dark skinned, conditioned young man, occurring in the setting of a 5K race, subsequently found to have severe 25 OH vitamin D deficiency (6ng/ml, laboratory lower normal limit >30ng/ml). ConclusionWe suggest that when very low vitamin D is documented, it be normalized before major prolonged exertion. We hypothesize that normalization of vitamin D before heavy exertion could perhaps prevent the severe muscle damage events and sequelae as was the case for this patient.

Mechanistic Basis and Functional Roles of Long-Term Plasticity in Auditory Neurons Induced by a Brain-Generated Estrogen

The classic estrogen 17β-estradiol (E2) was recently identified as a novel modulator of hearing function. It is produced rapidly, in an experience-dependent fashion, by auditory cortical neurons of both males and females. This brain-generated E2 enhances the efficiency of auditory coding and improves the neural and behavioral discrimination of auditory cues. Remarkably, the effects of E2 are long-lasting and persist for hours after local rises in hormone levels have subsided. The mechanisms and functional consequences of this E2-induced plasticity of auditory responses are unknown. Here, we addressed these issues in the zebra finch model by combining intracerebral pharmacology, biochemical assays, in vivo neurophysiology in awake animals, and computational and information theoretical approaches. We show that auditory experience activates the MAPK pathway in an E2-dependent manner. This effect is mediated by estrogen receptor β (ERβ), which directly associates with MEKK1 to sequentially modulate MEK and ERK activation, where the latter is required for the engagement of downstream molecular targets. We further show that E2-mediated activation of the MAPK cascade is required for the long-lasting enhancement of auditory-evoked responses in the awake brain. Moreover, a functional consequence of this E2/MAPK activation is to sustain enhanced information handling and neural discrimination by auditory neurons for several hours following hormonal challenge. Our results demonstrate that brain-generated E2 engages, via a nongenomic interaction between an estrogen receptor and a kinase, a persistent form of experience-dependent plasticity that enhances the neural coding and discrimination of behaviorally relevant sensory signals in the adult vertebrate brain.

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells

Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17β-estradiol (E2) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT2 Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E2 including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E2 rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ERα at ser118 in cells with a functional IGF-IR versus those without. E2 also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies.

Relaxant effect of trans-resveratrol on isolated porcine coronary arteries.

Recent studies provided evidence that trans-resveratrol (3,4',5-trihydroxystilbene, found in high concentrations in some red wines, may possibly decrease the risk of coronary heart disease mortality. The aim of this study, performed with large epicardial porcine coronary arteries (PCA) strips, was to investigate the relaxant effect of trans-resveratrol on these main conductance vessels, which have been described to be pathologically prone for vasospastic contractions. The data show that the tonic component of the biphasic contractions induced by histamine, as well as the contractions induced by F- ions (10 mmol/l), which activate G proteins downstream of the receptors, could dose-dependently be inhibited by trans-resveratrol (0.1-100 mumol/l). The EC50 values of the dose-response curves established for the inhibition of the sustained component of histamine-induced contractions were very similar to those obtained for the relaxations of fluoride-induced contractions: 0.45 +/- 0.08 and 0.29 +/- 0.05 mumol/l, resp. (n = 6). Ouabain (10 mumol/l)-induced contractions and rhythmic contractions elicited by tetraethylammonium (12 mmol/l) were also strongly inhibited by trans-resveratrol (20 mumol/l). It may be inferred from the results obtained in this study, that the relaxation of the coronary conductance vessels induced by trans-resveratrol is possibly based on a nongenomic interaction with steroid-like receptors located on the cell membrane.

Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40–50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα–BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

Differential effects of estrogen and progesterone on potassium channels expressed in Xenopus oocytes

Hypothesis Potassium (K +) channel activation contributes in part to estrogen-mediated vasorelaxation. However, the underlying mechanism is still unclear. We hypothesize that estrogen increases K + currents via membrane-associated, non-genomic interaction and that steroid hormones have differential effects on different types of K + channels. Experimental Human large-conductance Ca 2+-activated K + channels (BK Ca) and human voltage-gated K + channels (K V1.5) were expressed in Xenopus oocytes, and K + currents elicited by voltage clamp were measured. Results Both 17β-estradiol and BSA-conjugated 17β-estradiol increased the BK Ca current in a concentration-dependent manner and this effect was abolished by tetraethylammonium ions and iberiotoxin (putative BK Ca channel blockers). 17β-estradiol-stimulated increase in the BK Ca current was unaffected by treatment with ICI 182,780 (classic estrogen receptor antagonist), tamoxifen (estrogen receptor agonist/antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). In contrast, progesterone reduced the BK Ca current in the absence or presence of NS 1619 (BK Ca channel activator). Progesterone also inhibited 17β-estradiol-stimulated increase in the BK Ca current. Finally, progesterone but not 17β-estradiol reduced the K V1.5 current. Conclusions The present results show that 17β-estradiol stimulates BK Ca channels without affecting K V1.5 channels. This effect is ICI 182,780-insensitive and is likely mediated via a membrane-bound binding site. Progesterone inhibits both BK Ca- and K V1.5-encoded currents. The present results suggest that inhibition of K + channels may contribute in part to its reported antagonism against 17β-estradiol-mediated vascular relaxation via BK Ca channels.

Anti‐apoptotic effects of allopregnanolone on P19 neurons

Progesterone and its metabolites are potent allosteric modulators of GABA(A) receptor function, through a direct, non-genomic interaction with specific receptor subtypes. In addition, fluctuations in the concentration of progesterone, and allopregnanolone in particular, have been shown to modulate GABA(A) receptor gene expression and activity. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons which express specific neuronal markers, including GABA(A) and N-methyl-d-aspartate (NMDA) receptors. Apoptotic cell death, induced in the presence of NMDA, was efficiently prevented by allopregnanolone and dehydroepiandrosterone (DHEA) but not DHEA sulfate. Apoptosis was accompanied by cytochrome c release to the cytoplasm and Bax translocation to the mitochondria, while the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL remained unchanged. In the presence of the most potent neurosteroid, allopregnanolone, DNA fragmentation as well as cytochrome c and Bax translocation were prevented. On the other hand, short-term exposure (1-20 microm, 24 h) of P19-derived neurons to allopregnanolone and DHEA significantly increased the levels of alpha1 and beta2 mRNAs of GABA(A) receptor, while the levels of NR1 mRNA of NMDA receptor were not altered. These results suggest that neurosteroids, interfering with the mitochondrial apoptotic pathway, are able to act as survival factors in neuronal cells, while they contribute to GABA(A) receptor plasticity modulating the expression of its subunits.

Synthesis, metabolism and levels of the neuroactive steroid, 3 α-hydroxy-4-pregnen-20-one (3 αHP), in rat pituitaries

The neuroactive steroid, 3 α-hydroxy-4-pregnen-20-one (3 αHP), is a metabolite of progesterone and a precursor of 3 α-hydroxy-5 α-pregnan-20-one (5 αP3 α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA A receptor complex, 3 αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca 2+-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3 αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3 αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with 3H- or 14C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3 αHP along with 5 αP3 α, 5 α-pregnane-3,20-dione, 20 α-hydroxy-5 α-pregnan-3-one, 3 β-hydroxy-5 α-pregnan-20-one, 5 α-pregnane-3 α( β), 20 α-diols, 20 α-hydroxy-4-pregnen-3-one, and 4-pregnene-3 α( β), 20 α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3 α-hydroxysteroid oxidoreductase (3 α-HSO), 20 α-HSO, 3 β-HSO, and 5 α-reductase. The activities of 5 α-reductase and 3 α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3 αHP had been formed. The accumulation of 3 αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7- 3H]3 αHP showed that pituitary cells convert about 29% and 8% of the 3 αHP to progesterone and 5 αP3 α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3 αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3 αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3 αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3 αHP, from progesterone, and in turn to metabolize 3 αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3 αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3 αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.