Descripción: Accurate characterization of the molecular mechanisms of the action of ligands is an extremely important issue for their appropriate research, pharmacological, and therapeutic uses. In view of this fact, the aim of the present work was to investigate the mechanisms involved in the actions of mepyramine at the guinea pig H1 receptor stably expressed in Chinese hamster ovary cells. We found that mepyramine is able to decrease the basal constitutive activity of the guinea pig H1 receptor, to bind with high affinity to a Gq/11 protein-coupled form of the receptor and to promote a G protein-coupled inactive state of the H1 receptor that interferes with the Gq/11-mediated signaling of the endogenously expressed ATP receptor, as predicted by the Cubic Ternary Complex Model of receptor occupancy. The effect of mepyramine on ATP-induced signaling was specifically neutralized by Gα11 overexpression, indicating that mepyramine is able to reduce G protein availability for other non-related receptors associated with the same signaling pathway. Finally, we found a loss of mepyramine efficacy in decreasing basal levels of intracellular calcium at high Gα11 expression levels, which can be theoretically explained in terms of high H1 receptor constitutive activity. The whole of the present work sheds new light on H1 receptor pharmacology and the mechanisms H1 receptor inverse agonists could use to exert their observed negative efficacy.

Descripción: We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.

Descripción: Uterine decidualization is characterized by stromal cell proliferation and differentiation, which are controlled by ovarian hormones estradiol and progesterone. Here we report that the proliferative response of UIII rat uterine stromal cells to a short treatment with progestins requires active progesterone receptor (PR) and estrogen receptor β (ERβ) as well as a rapid and transient activation of Erk1-2 and Akt signaling. The optimal R5020 concentration for the proliferative response as well as for activation of the signaling cascades was between 10 and 100 pM. UIII cells are negative for ERα and have low levels of ERβ and PR located mainly in the cytoplasm. Upon progestin treatment PR translocated to the cell nucleus where it colocalized with activated Erk1-2. Neither progestins nor estradiol transactivated the corresponding transfected reporter genes, suggesting that endogenous PR and ERβ are transcriptionally incompetent. A fraction of endogenous PR and ERβ form a complex as demonstrated by coimmunoprecipitation. Taken together, our results suggest that the proliferative response of uterine stromal cells to picomolar concentrations of progestins does not require direct transcriptional effects and is mediated by activation of the Erk1-2 and Akt signaling pathways via cross talk between PR and ERβ. Copyright © 2005 by The Endocrine Society.

Descripción: Background: Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses. Results: We showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism. Conclusions: IGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation. © 2013 Giudice et al.; licensee BioMed Central Ltd.

Descripción: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most of the toxic and endocrine-disruptive actions of aromatic compounds in the ovary. Paradoxically, this receptor has been shown to play important roles in normal female reproductive function as well. Although knowledge of AHR expression regulation in the ovary is of crucial significance to understand the receptor biology and its function in reproductive physiology, there are only limited data in this area. The purpose of the present study was to establish the possible regulation that AHR might undergo in ovarian cells. Here we show that the hormones FSH and estradiol are able to reduce AHR protein and transcript levels in granulosa cells in a way that parallels the changes observed in ovarian tissue across the rat estrous cycle. These findings suggest that estradiol and FSH would be cycle-associated endogenous modulators of AHR expression. In addition, we show that in granulosa cells the receptor is rapidly downregulated via proteasomal degradation following treatment with AHR ligands. However, prolonged treatment with an agonist caused an increase in Ahr mRNA levels. These actions would constitute a regulatory mechanism that both attenuates AHR signal rapidly and replenishes the cellular receptor pool in the long term. In conclusion, our results indicate that AHR expression is regulated by classical hormones and by its own ligands in granulosa cells. © 2006 by the Society for the Study of Reproduction, Inc.

Descripción: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that, besides mediating toxic responses, may have a central role in ovarian physiology. Studying the actions of AHR ligands on granulosa cells function, we have found that beta-naphthoflavone amplifies the comitogenic actions of FSH and 17beta-estradiol in a dose-dependent manner. This amplification was even greater in cells that overexpress the AHR and was reversed by cotreatment with the AHR antagonist alpha-naphthoflavone, suggesting that this effect is mediated by the AHR. The estrogen receptor is likewise implicated in this phenomenon, because a pure antiestrogen abolished the described synergism. However, the more traditional inhibitory AHR-estrogen receptor interaction was observed on the estrogen response element-driven transcriptional activity. On the other hand, alpha-naphthoflavone inhibited dose-dependently the mitogenic actions of FSH and 17beta-estradiol. Beta-naphthoflavone induced the expression of Cyp1a1 and Cyp1b1 transcripts, two well-characterized AHR-inducible genes that code for hydroxylases that metabolize estradiol to catecholestrogens. Nevertheless, the positive effect of beta-naphthoflavone on proliferation was not caused by increased metabolism of estradiol to catecholestrogens, because these compounds inhibited the hormonally stimulated DNA synthesis. This latter inhibition exerted by catecholestrogens suggests that these hydroxylases would play a regulatory point in granulosa cell proliferation. Our study indicates that AHR ligands modulate the proliferation of rat granulosa cells, and demonstrates for the first time that an agonist of this receptor is able to amplify the comitogenic action of classical hormones through a mechanism that might implicate a positive cross-talk between the AHR and the estrogen receptor pathways. © 2006 by the Society for the Study of Reproduction, Inc.

Descripción: The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X L/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E2) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E 2 effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E2-induced leptin expression. Moreover, E2 treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between - 1951 and -1847 bp is both necessary and sufficient to achieve E2 effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E2 on leptin expression. Moreover, E2 action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E2 could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 μM PD98059 and 0.1 μM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E 2 induction of leptin promoter. On the other hand, E2 treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E2 induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways. © 2010 by the Society for the Study of Reproduction, Inc.

Descripción: The G protein-coupled receptor oncogene (vGPCR) of the Kaposi's sarcoma (KS) associated herpesvirus (KSHV), an oncovirus implicated in angioproliferative neoplasms, induces angiogenesis by VEGF secretion. Accordingly, we found that expression of vGPCR in human umbilical vein endothelial cells (HUVEC) leads to immortalization with constitutive VEGF receptor-2/ KDR expression and activation. vGPCR immortalization was associated with anti-senescence mediated by alternative lengthening of telomeres and an anti-apoptotic response mediated by vGPCR constitutive signaling and KDR autocrine signaling leading to activation of the PI3K/AKT pathway. In the presence of the KS growth factor VEGF, this mechanism can sustain suppression of signaling by the immortalizing gene. We conclude that vGPCR can cause an oncogenic immortalizing event and recapitulate aspects of the KS angiogenic phenotype in human endothelial cells, pointing to this gene as a pathogenic determinant of KSHV.

Descripción: The α9 and α10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of α9α10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the α10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian α9α10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native α9α10 receptors. Our main finding in the present work is that, in contrast to the high (pCa 2+/pMonovalents ∼10) Ca 2+ permeability reported for rat α9α10 receptors, recombinant and native chicken α9α10 receptors have a much lower permeability (∼2) to this cation, comparable to that of neuronal α4β2 receptors. Moreover, we show that, in contrast to α10, α7 as well as α4 and β2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca 2+ permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after α9α10 gating at the cholinergic-hair cell synapse. In addition, they suggest that high Ca 2+ permeability of the α9α10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.

Descripción: The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10- mediated responses and for potential inner ear therapeutics based on this interaction. © 2007 Association for Research in Otolaryngology.

Descripción: Background: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GRDNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior. © 2010 Presman et al.

Descripción: In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both α9 and α10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that α9 mRNA is expressed in adult IHCs but that α10 mRNA disappears after the onset of hearing. In the present study, antibodies to α10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of α10, but not α9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Descripción: As for any solid tumour, pituitary adenoma expansion is dependent on neovascularization through angiogenesis. In this process, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) may play an outstanding role. The intention of this work was to study the expression/localization and possible function of VEGF receptors in pituitary adenomas. VEGF receptor mRNA and protein expression was studied by in situ hybridization, immunohistochemistry and RT-PCR in 6 normal human pituitaries, 39 human pituitary adenomas and 4 rodent pituitary adenoma cell lines. VEGFR-1 expressing somatotroph MtT-S cells were used as a model to study the role of VEGF on cell proliferation and to elucidate the underlying mechanism of action. In normal pituitaries, VEGFR-1 was detected in endocrine cells, whereas VEGFR-2 and NRP-1 were exclusively expressed in endothelial cells. In pituitary tumours, a heterogeneous VEGFR expression pattern was observed by IHC. VEGFR-1, VEGFR-2 and NRP-1 were detected in 24, 18 and 17 adenomas respectively. In the adenomas, VEGFR-1 was expressed in epithelial tumour cells and VEGFR-2/NRP-1 in vessel endothelial cells. Functional studies in VEGFR-1-positive MtT-S cells showed that the ligands of VIEGFR-1 significantly stimulated cell proliferation. This effect was mediated through the phosphatidylinositol-3-kinase-signalling pathway and involves induction of cyclin D1 and Bcl-2. Based on our results, we speculate that the ligands of VEGF receptors, such as VEGF-A and placenta growth factor, not only play a role in angiogenesis in pituitary adenomas, but also affect the growth of pituitary tumour cells through VEGFR-1. © 2006 Society for Endocrinology.

Descripción: The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-α (RORα) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORα-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR. Copyright © 2006 by The Endocrine Society.

Descripción: Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS. © 2007 Glezer et al.

Descripción: Although homomeric channels assembled from the α9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both α9 and α10 subunits. To gain insight into α10 subunit function in vivo, we examined olivocochlear innervation and function in α10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in α10 +/+ and α10+/- mice, with no detectable responses to ACh in α10+/+ mice. In contrast, a proportion of α10-/- outer hair cells showed small ACh-evoked currents. In α10-/- mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual α9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, α10-/- mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that α9-/- and α10-/- mice have overlapping but nonidentical phenotypes. Moreover, α10 nAChR subunits are required for normal olivocochlear activity because α9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in α10-/- mutant mice. © 2007 by The National Academy of Sciences of the USA.

Descripción: The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054 ± 0.020 μm/s. Individual runs had a mean velocity of ∼0.15 μm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites. © 2007 Federation of European Biochemical Societies.

Descripción: The cellular resistance to tumor necrosis factor (TNF) of most cell types has been attributed to both a protective pathway induced by this cytokine and the preexistence of protective factors in the target cell. NF-κB has been postulated as one of the principal factors involved in antiapoptotic gene expression control on TNF-resistant cells. We have previously shown that glucocorticoids protect the naturally TNF-sensitive L-929 cells from apoptosis. Here we analyze the role of NF-κB and glucocorticoids on TNF-induced apoptosis in L-929 cells. We found that inhibition of NF-κB enhanced the sensitivity to TNF-induced apoptosis. Glucocorticoids inhibited NF-κB transactivation via IκB induction. Moreover, glucocorticoids protected from TNF-induced apoptosis even when NF-κB activity was inhibited by stable or transient expression of the superrepressor IκB. These results demonstrate that although glucocorticoids inhibit NF-κB transactivation in these cells, this is not required for their protection from TNF-induced apoptosis. (C) 2000 Elsevier Science B.V.