A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Influence of RNA circularity on Target RNA-Directed MicroRNA Degradation.

A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to 'sponge' or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here, we studied whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ when in a circular versus linear form. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.

Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET.

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Automated quantification of protein periodic nanostructures in fluorescence nanoscopy images: abundance and regularity of neuronal spectrin membrane-associated skeleton.

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS.

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function.

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.

B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and ß-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.

Underlying mechanisms of cAMP- and glucocorticoid-mediated inhibition of FasL expression in activation-induced cell death.

Glucocorticoids (GCs) and cAMP-dependent signaling pathways exert diverse and relevant immune regulatory functions, including a tight control of T cell death and homeostasis. Both of these signaling molecules inhibit TCR-induced cell death and FasL expression, but the underlying mechanisms are still poorly understood. Therefore, to address this question, we performed a comprehensive screening of signaling pathways downstream of the TCR, in order to define which of them are targets of cAMP- and GC-mediated inhibition. We found that cAMP inhibited NF-κB and ERK pathways through a PKA-dependent mechanism, while Dexamethasone blocked TCR-induced NF-κB signaling. Although GCs and cAMP inhibited the induction of endogenous FasL mRNA expression triggered by TCR activation, they potentiated TCR-mediated induction of FasL promoter activity in transient transfection assays. However, when the same FasL promoter was stably transfected, the facilitatory effect of GCs and cAMP became inhibitory, thus resembling the effects on endogenous FasL mRNA expression. Hence, the endogenous chromatinization status known to occur in integrated or genomic vs. episomic DNA might be critical for proper regulation of FasL expression by cAMP and GCs. Our results suggest that the chromatinization status of the FasL promoter may function as a molecular switch, controlling cAMP and GC responsiveness and explaining why these agents inhibit FasL expression in T cells but induce FasL in other cell types.

The corticotropin-releasing hormone network and the hypothalamic-pituitary-adrenal axis: molecular and cellular mechanisms involved.

Corticotropin-releasing hormone (CRH) plays a key role in adjusting the basal and stress-activated hypothalamic-pituitary-adrenal axis (HPA). CRH is also widely distributed in extrahypothalamic circuits, where it acts as a neuroregulator to integrate the complex neuroendocrine, autonomic, and behavioral adaptive response to stress. Hyperactive and/or dysregulated CRH circuits are involved in neuroendocrinological disturbances and stress-related mood disorders such as anxiety and depression. This review describes the main physiological features of the CRH network and summarizes recent relevant information concerning the molecular mechanism of CRH action obtained from signal transduction studies using cells and wild-type and transgenic mice lines. Special focus is placed on the MAPK signaling pathways triggered by CRH through the CRH receptor 1 that plays an essential role in CRH action in pituitary corticotrophs and in specific brain structures. Recent findings underpin the concept of specific CRH-signaling pathways restricted to specific anatomical areas. Understanding CRH action at molecular levels will not only provide insight into the precise CRH mechanism of action, but will also be instrumental in identifying novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders.

Immunology, signal transduction, and behavior in hypothalamic-pituitary-adrenal axis-related genetic mouse models.

A classical view of the neuroendocrine-immune network assumes bidirectional interactions where pro-inflammatory cytokines influence hypothalamic-pituitary-adrenal (HPA) axis-derived hormones that subsequently affect cytokines in a permanently servo-controlled circle. Nevertheless, this picture has been continuously evolving over the last years as a result of the discovery of redundant expression and extended functions of many of the molecules implicated. Thus, cytokines are not only expressed in cells of the immune system but also in the central nervous system, and many hormones present at hypothalamic-pituitary level are also functionally expressed in the brain as well as in other peripheral organs, including immune cells. Because of this intermingled network of molecules redundantly expressed, the elucidation of the unique roles of HPA axis-related molecules at every level of complexity is one of the major challenges in the field. Genetic engineering in the mouse offers the most convincing method for dissecting in vivo the specific roles of distinct molecules acting in complex networks. Thus, various immunological, behavioral, and signal transduction studies performed with different HPA axis-related mutant mouse lines to delineate the roles of beta-endorphin, the type 1 receptor of corticotropin-releasing hormone (CRHR1), and its ligand CRH will be discussed here.

Glucocorticoids inhibit GATA-3 phosphorylation and activity in T cells.

Glucocorticoid (GC) immunosuppression and anti-inflammatory action involve the regulation of several transcription factors (TFs). GCs inhibit the acute production of T-helper (Th) 1 and Th2 cytokines but ultimately favor a shift toward Th2 phenotype. GCs inhibit the transcriptional activity of T-bet Th1 TF by a transrepression mechanism. Here we analyze GC regulation of GATA-3, the master driver of Th2 differentiation. We found that GCs inhibit GATA-3 transcriptional activity. We demonstrate that this mechanism does not involve physical interaction between the glucocorticoid receptor (GR) and GATA-3 or reduction of GATA-3 binding to DNA, as described previously for T-bet. Instead, GCs inhibit GATA-3 activity by inhibition of p38 mitogen-activated protein kinase induced GATA-3 phosphorylation. GCs also inhibit GATA-3 mRNA and protein expression. Finally, GATA-3 inhibition affects the interleukin-5 gene, a central Th2 cytokine. The IC(50) of dexamethasone is 10 nM with a maximum effect at 100 nM. All inhibitory actions were blocked by the GR antagonist RU38486 (1 uM), proving the specificity of GR action. In view of the crucial role of GATA-3 in T-cell differentiation and inflammation, we propose that the mechanism of GATA-3 inhibition compared with that in T-bet may have relevant implications in understanding and modulating the anti-inflammatory and Th-regulatory properties of GCs.

Mecanismos moleculares de acción de algunas drogas inmunosupresoras / Molecular mechanisms of action of some immunosuppresive drugs

Los tratamientos utilizados para desordenes inmunológicos son de origen empírico, utilizando drogas inmunosupresoras identificadas a través de la selección de un gran número de compuestos naturales y sintéticos. Las drogas inmunosupresoras son ampliamente utilizadas en tratamientos clínicos de desordenes autoinmunes, en la prevención de rechazo a transplantes así como también en desordenes de carácter no autoinmune tales como las alergias. El diseño de las terapias inmunosupresoras está basado en controlar una respuesta inmune exacerbada. La base fisiopatológica de este concepto es en modular la acción de células mononucleares, siendo el principal punto de control las células T. Estas drogas inhiben la función normal de protección del sistema inmune llevando a la aparición de complicaciones en las terapias de inmunosupresión. Las drogas inmunosupresoras tienen diferentes blancos en el proceso de inmunidad celular. Según su modo de acción pueden clasificarse en cuatro categorías: drogas antinflamatorias de la familia de los corticosteroides, inmunosupresoras específicas inhibidoras de la calcineurina, citotóxicas o antiproliferativas y anticuerpos específicos. En este trabajo describimos el mecanismo de acción molecular de agentes inmunosupresores tales como, esteroides, ciclosporina, tacrolimo, azatioprina, ciclofosfamida, sirolimus, mofetil mecofenolato, leflunomida y anticuerpos específicos, para contribuir a la comprensión de cómo utilizar y mejorar estos agentes.

A number of natural and synthetic substances are used in the treatment of immunological disorders. The immunosuppressive drugs are widely utilized in clinical treatments of autoimmune disorders, in the prevention of transplant rejection as well as in non-autoimmune diseases such as allergy. The design of immunosuppressive therapies is based on the control of the exacerbated immune response. The pathophysiologic mean of this concept is to modulate the action of mononuclear cells, being T cells the main targets. Immunosuppressive agents have different molecular targets, and an important drawback in their use is that they also inhibit the normal immune system response. Depending on their mode of action, immunosuppressive drugs can be classified in four different groups: antinflammatory drugs of the corticosteroid family, inhibitors of the calcineurin pathway, cytototoxic or antiproliferative drugs and specific antibodies. In this article, we focus on the molecular action of immunosuppressive drugs such as steroids, cyclosporine, tacrolimus, azathioprine, cyclophosphamide, sirolimus, mycophenolate mofetil, leflunomide and specific antibodies, providing data to characterize and improve the use of these agents.

[Molecular mechanisms of action of some immunosuppressive drugs]. / Mecanismos moleculares de acción de algunas drogas inmunosupresoras.

A number of natural and synthetic substances are used in the treatment of immunological disorders. The immunosuppressive drugs are widely utilized in clinical treatments of autoimmune disorders, in the prevention of transplant rejection as well as in non-autoimmune diseases such as allergy. The design of immunosuppressive therapies is based on the control of the exacerbated immune response. The pathophysiologic mean of this concept is to modulate the action of mononuclear cells, being T cells the main targets. Immunosuppressive agents have different molecular targets, and an important drawback in their use is that they also inhibit the normal immune system response. Depending on their mode of action, immunosuppressive drugs can be classified in four different groups: antinflammatory drugs of the corticosteroid family, inhibitors of the calcineurin pathway, cytototoxic or antiproliferative drugs and specific antibodies. In this article, we focus on the molecular action of immunosuppressive drugs such as steroids, cyclosporine, tacrolimus, azathioprine, cyclophosphamide, sirolimus, mycophenolate mofetil, leflunomide and specific antibodies, providing data to characterize and improve the use of these agents.

The activated glucocorticoid receptor inhibits the transcription factor T-bet by direct protein-protein interaction.

Glucocorticoids (GCs) immunosuppression acts via regulation of several transcription factors (TF), including activating protein (AP)-1, NF-kappaB, and NFAT. GCs inhibit Th1 cytokines and promote a shift toward Th2 differentiation. Th1 phenotype depends on TF T-bet. In this study, we examined GC regulation of T-bet. We found that GCs inhibit T-bet transcriptional activity. We show that glucocorticoid receptor (GR) physically interacts with T-bet both in transfected cell lines and in primary splenocyte cultures with endogenous GR and T-bet. This interaction also blocks GR-dependent transcription. We show both in vitro and in vivo at endogenous binding sites that the mechanism underlying T-bet inhibition further involves reduction of T-bet binding to DNA. Using specific mutations of GR, we demonstrate that the first zinc finger region of GR is required for T-bet inhibition. GCs additionally inhibit T-bet both at mRNA and protein expression levels, revealing another layer of GR action on T-bet. Finally, we examined the functional consequences of GR/T-bet interaction on IFN-gamma, showing that GCs inhibit transcriptional activity of T-bet on its promoter. In view of the crucial role of T-bet in T cell differentiation and inflammation, we propose that GR inhibitory interaction with T-bet may be an important mechanism underlying the immunosuppressive properties of GCs.

Molecular understanding of cytokine-steroid hormone dialogue: implications for human diseases.

Highly sophisticated mechanisms confer upon the immune system the capacity to respond with a certain degree of autonomy. However, the final outcome of an adaptative immune response depends on the interaction with other systems of the organism. The immune-neuroendocrine systems have an intimate cross-communication, making possible a satisfactory response to environmental changes. Part of this interaction occurs through cytokines and steroid hormones. The last step of this crosstalk is at the molecular level. In this article we will focus on the physical and functional interrelationship between cytokine signaling pathway-activated transcription factors (TFs) and steroid receptors in different cell models, where the signals triggered by cytokines and steroid hormones have major roles: (1) the ligand-dependent-activated glucocorticoid receptor (GR) influence the genetic program that specifies lineage commitment in T helper (Th) cell differentiation. How posttranslational modifications of several TFs as well as nuclear hormone receptors could be implicated in the molecular crosstalk between the immune-neuroendocrine messengers is discussed. (2) glucocorticoid (GC) antagonism on the TCR-induced T cell apoptosis. (3) estrogen receptor/TGF-beta family proteins molecular interaction implicated on pituitary prolactinomas pathogenesis. The functional crosstalk at the molecular level between immune and steroids signals is essential to determine an integrative response to both mediators (which in the last instance results in a new gene activation/repression profile) and constitutes the ultimate integrative level of interaction between the immune and neuroendocrine systems.

Bone morphogenetic protein-4 control of pituitary pathophysiology.

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-Beta(TGF-Beta) family, is overexpressed in different prolactinoma models and induces the development of these lineage adenomas. SMAD proteins activated by growth factors of the TGF-Beta and BMP family interact with estrogen receptors to stimulate the proliferation of prolactin and growth hormone-secreting cells. Furthermore, BMP-4 presents differential expression in normal and adenomatous corticotropes and inhibitory action on corticotropinoma cell proliferation. Moreover, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights not only the crucial and opposite role of BMP-4 in the progression of pituitary adenomas but also that BMP-4 and retinoic acid interaction might serve as a potential new mechanism target for therapeutic approaches for Cushing disease.

Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action.

The molecular mechanisms governing the pathogenesis of ACTH-secreting pituitary adenomas are still obscure. Furthermore, the pharmacological treatment of these tumors is limited. In this study, we report that bone morphogenetic protein-4 (BMP-4) is expressed in the corticotrophs of human normal adenohypophysis and its expression is reduced in corticotrophinomas obtained from Cushing's patients compared with the normal pituitary. BMP-4 treatment of AtT-20 mouse corticotrophinoma cells has an inhibitory effect on ACTH secretion and cell proliferation. AtT-20 cells stably transfected with a dominant-negative form of the BMP-4 signal cotransducer Smad-4 or the BMP-4 inhibitor noggin have increased tumorigenicity in nude mice, showing that BMP-4 has an inhibitory role on corticotroph tumorigenesis in vivo. Because the activation of the retinoic acid receptor has an inhibitory action on Cushing's disease progression, we analyzed the putative interaction of these two pathways. Indeed, retinoic acid induces both BMP-4 transcription and expression and its antiproliferative action is blocked in Smad-4dn- and noggin-transfected Att-20 cells that do not respond to BMP-4. Therefore, retinoic acid induces BMP-4, which participates in the antiproliferative effects of retinoic acid. This new mechanism is a potential target for therapeutic approaches for Cushing's disease.

Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas.

Corticotropin-releasing hormone (CRH) coordinates hormonal and behavioral responses to stress. The mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2) mediates several functions in different forebrain structures and recently has been implicated in CRH signaling in cultured cells. To study in vivo CRH-mediated activation of central ERK1/2, we investigated the expression pattern of the phosphorylated ERK1/2(p-ERK1/2) in the mouse brain after intracerebroventricular CRH injections. As shown by immunohistochemistry and confocal microscopy analysis, CRH administration increased p-ERK1/2 levels specifically in the CA3 and CA1 hippocampal subfields and basolateral complex of the amygdala, both structures related to external environmental information processing and behavioral aspects of stress. Other regions such as hypothalamic nuclei and the central nucleus of the amygdala, also related to central CRH system but involved in the processing of the ascending visceral information and neuroendocrine-autonomic response to stress, did not show CRH-mediated ERK1/2 activation. To dissect the involvement of CRH receptor 1 (CRHR1) and CRHR2, we used conditional knockout mice in which Crhr1 is inactivated in the anterior forebrain and limbic structures. The conditional genetic ablation of Crhr1 inhibited the p-ERK1/2 increase, underlining the involvement of CRHR1 in the CRH-mediated activation. These findings underscore the fact that CRH activates p-ERK1/2 through CRHR1 only in selected brain regions, pointing to a specific role of this pathway in mediating behavioral adaptation to stress.

Cytokine signaling/transcription factor cross-talk in T cell activation and Th1-Th2 differentiation.

The secretion of interleukin 2 (IL-2) is a key event in T cell activation. IL-2 allows T cells to enter into the S phase of the cell cycle and divide. After the activation phase takes place, T lymphocytes proliferate and differentiate to generate effector T cells. Thereby, T helper (Th) precursor cells, which are functionally immature, may become Th1 or Th2 effector cells. These subsets are responsible for cell-mediated immunity and humoral responses, respectively. Both T cell activation and Th differentiation are processes that depend on changes in the pattern of gene expression. The expression and changes in the genes responsible for these events are regulated by transcription factors. This review will focus on both the transcription factors involved in the control of IL-2 as well as those that are key to T helper differentiation.

Integrating systemic information at the molecular level: cross-talk between steroid receptors and cytokine signaling on different target cells.

An essential event in immune activation is the increase of cytokines in both plasma and immune tissues. Steroid hormones influence several adaptive responses in both health and disease. Cytokines and steroids have an intimate cross-communication in many systems, making possible a satisfactory adaptive response to environmental changes. The ultimate level of integration of the cytokine-steroids cross-talk is the molecular level. We have demonstrated this in four types of cross-talk mechanisms on different cells in which steroids have major roles: (1) The tumor necrosis factor (TNF)-glucocorticoid receptor (GR) transcriptional interaction in cellular targets of TNF-induced cytotoxicity. TNF potentiates the transactivation activity of GR and the priming with TNF increases the protective action of GR on TNF-induced cytotoxicity. (2) The GR-T cell receptor (TCR) antagonism in GR-TCR-induced T cell apoptosis and its modulation by cAMP. cAMP inhibits the TCR-induced apoptosis through a PKA-CREB-dependent mechanism and potentiates glucocorticoid-induced apoptosis by means of a CREB-independent mechanism. (3) The GR influence on Th1-Th2 cytokine expression and differentiation. Glucocorticoids inhibit the induction of GATA-3 and T-bet transcription factors. (4) The influence of ER/Smad-4 signaling cross-communication on prolactinoma pathogenesis. Physical and functional interactions between Smad-4 and estrogen receptors take place in prolactinoma cells, providing a molecular explanation to link the tumorigenic action of these two important players of prolactinoma pathogenesis. The molecular cross-talk between steroids and transcription factors is the mechanism that provides the basis for the outcome of adaptive responses integrating the systemic information provided by hormones and cytokines.