Synthesis of Poly d(G-C) Oligonucleotides

Model sequences for evaluation of the GC dimer sequence repetition on synthesis success were prepared and analyzed by HPLC. Contiguous d(G-C) or d(C-G) sequences have a deleterious effect on DNA oligonucleotide synthesis. The critical number seems to be about 6 GCs in a row. If the GCs are separated by other nucleotides, the effect is not as severe.     

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Vasoactive intestinal peptide suppresses ovarian granulosa cell apoptosis in vitro by up-regulating Bcl-2 gene expression

Vasoactive intestinal peptide (VIP) is an endogenous peptide showing a rich profile of biological activities. Within ovaries, VIP directly regulates the ovarian functions, including granulosa cells (GCs) development. In the present study, the effects of VIP on proliferation and apoptosis in goose granulosa cells were demonstrated and its underlying mechanism investigated. A strategy of RNAi-mediated "gene silencing" of Bcl-2 (RV-Bcl-2), over-expression of Bcl-2 (JLV-Bcl-2) synthesis, and exogenous VIP was used to treat goose GCs. The results showed the amounts of Bcl-2 protein were negatively correlated with apoptosis of goose GCs in all experimental groups. Compared with other control groups, apoptosis was decreased in goose GCs following treatment of 100 nM VIP, and the amount of Bcl-2 protein was increased (P < 0.05) increased. However, VIP failed to exert an effect on cell proliferation (P > 0.05). In conclusion, the exogenous VIP plays an important role in inhibiting apoptosis of goose GCs via inducing Bcl-2 gene expression.     

Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells

Brain-derived neurotrophic factor (BDNF) is involved in regulating the growth of ovarian follicles, maturation of the oocyte, and development of the early embryo through its receptor, tyrosine kinase receptor B (TrkB). However, it is still unclear as to how BDNF influences proliferation and steroidogenesis of bovine granulosa cells (GCs). In this paper, we confirmed that BDNF and TrkB were expressed in bovine GCs, and that proliferation and steroidogenesis by bovine GCs were reduced by knockdown of BDNF or inhibition of TrkB. With respect to GC proliferation, BDNF enhanced cellular viability and the percentage of cells in the S phase. BDNF also activated both protein kinase B (PKB, also known as AKT) and the extracellular signal-regulated protein kinase 1/2 (ERK1/2)-signaling pathway. Through the AKT-signaling pathway, BDNF increased the expression of proliferation-related genes, including cyclin A1 (CCNA1), cyclin E2 (CCNE2), cyclin D1 (CCND1), and cyclin-dependent kinase 1 (CDK1). However, through the ERK1/2 signaling pathway, BDNF only increased the expression of CCNA1 and CCNE2. Regarding steroidogenesis by bovine GCs, BDNF promoted progesterone (P ₄) synthesis, but had no effect on estradiol; it also activated the AKT-signaling pathway and increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 (HSD3B1). In summary, our data are the first to show that BDNF promotes the proliferation of bovine GCs through TrkB–AKT and ERK1/2 signaling pathways and increases P₄ synthesis by bovine GCs through the TrkB–AKT signaling pathway.     

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.     

Annexin 1: a paracrine/juxtacrine mediator of glucorticoid action in the neuroendocrine system

Glucocorticoids (GCs) play an essential role in the maintenance of homeostasis. In normal circumstances their secretion is tightly regulated by a complex servo mechanism through which the steroids suppress the synthesis and release of ACTH and its hypothalamic releasing factors (CRH and AVP) and thereby reduce the positive drive to the adrenal cortex. The feedback actions of GCs on hormone release develop rapidly (within minutes), well before any changes in hormone synthesis are apparent. By using immunoneutralization, gene targeting and pharmacological strategies in in vivo and in vitro models, we have identified annexin 1, a Ca(2+)- and phospholipid-binding protein, as a key mediator of the early inhibitory actions of GCs on peptide release. This brief review outlines this work and describes molecular and cellular studies which have provided insight into the mechanism of annexin 1-dependent GC signalling in the neuroendocrine system.     

Glucocorticoids inhibit lipopolysaccharide-mediated inflammatory response by downregulating microRNA-155: a novel anti-inflammation mechanism

Glucocorticoids (GCs) are among the most widely used and effective therapies for many chronic inflammatory diseases. Although attempts have been made to identify important protein-coding genes and pathways involved in the anti-inflammatory effect of GCs, knowledge of genomic aberrations associated with noncoding genes, such as micro-RNAs (miRNAs), and their contributions is relatively limited. In this study, a systematic screening of the miRNA expression profile by microarray showed that GCs inhibited the expression of miR-155 in lipopolysaccharide (LPS)-induced macrophage inflammatory responses. Overexpression of miR-155 markedly reversed the suppressive action of GCs, whereas inhibition of miR-155 exhibited an effect similar to that of GCs on LPS-treated RAW264.7 cells, indicating miR-155 to be a functional regulator in the anti-inflammatory effect of GCs. Furthermore, GCs inhibited miR-155 expression in a GC receptor- and NF-κB-dependent manner. Bioinformatics analysis and luciferase assay revealed that the NF-κB binding site located in the promoter region of the B-cell integration cluster was important in mediating the GC-driven suppression of miR-155 in response to LPS stimulation. In addition, the combination of treatment with GCs and inhibition of miR-155 enhanced the anti-inflammatory effect of GCs on LPS-stimulated RAW264.7 cells. Therefore, we identify miR-155 to be a novel target through which GCs exert their anti-inflammatory effect on the LPS-induced macrophage inflammatory response. These findings may provide a basic rationale for new approaches in the effort to develop anti-inflammatory therapeutics.     

Glucocorticoid-induced contractility and F-actin content of human lung fibroblasts in three-dimensional culture

Fibroblast contractility plays a useful role in the wound healing process but contributes to architectural distortion in the lungs. Glucocorticoids (GCs) have been reported to reduce dermal fibroblast contractility, which may result in delaying wound healing, but the effects on lung fibroblasts are unknown. In this study, we examined how human lung fibroblast contractility is altered in the presence of GCs. Lung fibroblast cell lines (n = 5) were established from normal parts of surgically resected lung tissue. The effects of GCs on contractility were investigated with a type I collagen gel contraction assay. Filamentous actin (F-actin) content was detected by confocal microscopy and measured with a fluorescent phalloidin binding assay. GCs augmented fibroblast contraction in a concentration-dependent manner, with an approximate EC(50) of 1.8 x 10(-8) M, whereas other steroid derivatives had no effects. GC contractility needed de novo protein synthesis. The GC-induced increase in contractility was found to be consistent with an increase in F-actin content. In conclusion, lung fibroblast contractility was enhanced with GCs through an upregulation of lung fibroblast F-actin.     

Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles

The development of an ovarian follicle requires a complex set of reciprocal interactions between the oocyte and granulosa cells in order for both types of cells to develop properly. These interactions are largely orchestrated by the oocyte via paracrine factors such as growth differentiation factor 9 (GDF9). To examine these interactions further, a study was conducted of the effects of oocytes at different stages of development on proteins synthesized by mouse granulosa cells during the transition of granulosa cells (GCs) from preantral, secondary (2 degrees ) follicles (2 degrees GCs) to mural granulosa cells (3 degrees GCs) of antral tertiary (3 degrees ) follicles. The ability of recombinant GDF9 to mimic the effects of oocytes was also determined. Effects were evaluated by high- resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis. Coculture of the 2 degrees GCs with growing oocytes (GOs) from 2 degrees follicles brought about many of the changes in granulosa cell phenotype associated with the 2 degrees to 3 degrees follicle transition. GDF9 likewise brought about many of these changes, but only a subset of GDF9-affected protein spots were also affected by coculture with GOs. Coculture of 2 degrees GCs with the nearly fully grown oocytes (FGOs) from 3 degrees follicles had a reduced effect on 2 degrees GC phenotype, in comparison with coculture with GOs. For some proteins, oocyte coculture or GDF9 treatment appeared to have opposite effects on 2 degrees GCs and 3 degrees GCs. Additional effects of GDF9 and oocytes were seen in cultures of 2 degrees GCs for proteins other than those that differed between untreated control 2 degrees and 3 degrees GCs. These results indicate that GOs and GDF9 can each induce 2 degrees GCs to shift their phenotype toward that of 3 degrees GCs. The ability of the oocyte to produce this effect is diminished with oocyte development. The transition in the GC phenotype promoted by oocytes appears stable because differences in 2 degrees GCs promoted by oocytes and GDF9 were observed in untreated 3 degrees GCs. We conclude that the influence of the oocyte on GCs changes with the progression of their development, and so too does the response of the GCs to the oocyte. Moreover, by acting on the 2 degrees GCs, GOs are able to influence stably the phenotype of 3 degrees GCs. Thus, at or near the 2 degrees to 3 degrees follicle transition, signals from the growing oocyte contribute to the development of the mural GC phenotype.     

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.

Guard cells synthesize starch using carbon precursors originating in the plastid and/or imported from the cytosol depending on the time of the day.     

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.     

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders.     

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 signaling pathway

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)–GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 pathway.     

Rapid inhibition of leptin signaling by glucocorticoids in vitro and in vivo

Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of (125)I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade.     

Liposomal encapsulation of glucocorticoids alters their mode of action in the treatment of experimental autoimmune encephalomyelitis

Glucocorticoids (GCs) are widely used to treat acute relapses of multiple sclerosis (MS). In this study, we demonstrate that liposomal encapsulation augments the therapeutic potency of GCs as they ameliorate experimental autoimmune encephalomyelitis (EAE) to the same extent as free GC, but at strongly reduced dosage and application frequency. Importantly, this is accompanied by an altered mode of action. Unlike free GCs, which mainly target T lymphocytes during EAE therapy, liposomal GCs only marginally affect T cell apoptosis and function. In contrast, liposomal GCs efficiently repress proinflammatory macrophage functions and upregulate anti-inflammatory genes associated with the alternatively activated M2 phenotype. The GC receptor (GR) per se is indispensable for the therapeutic efficacy of liposomal GC. In contrast to free GCs, however, the individual deletion of the GR either in T cells or myeloid cells has little effect on the efficacy of liposomal GCs in the treatment of EAE. Only the combined deletion of the GR in both cellular compartments markedly compromises the therapeutic effect of liposomal GCs on disease progression. In conclusion, encapsulation of GC does not only enhance their efficacy in the treatment of EAE but also alters their target cell specificity and their mode of action compared with free GCs.     

Germinal center function in the spleen during simian HIV infection in rhesus monkeys

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C-->T and G-->A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.