Effects of P4 Antagonist RU486 on VEGF and Its Receptors’ Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

Stress hormones promote DNA damage in human oral keratinocytes

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

Repeated stimulation of the HPA axis alters white blood cell count without increasing oxidative stress or inflammatory cytokines in fasting elephant seal pups

ABSTRACT The hypothalamic–pituitary–adrenal (HPA) axis controls the release of glucocorticoids, which regulate immune and inflammatory function by modulating cytokines, white blood cells and oxidative stress via glucocorticoid receptor (GR) signaling. Although the response to HPA activation is well characterized in many species, little is known about the impacts of HPA activation during extreme physiological conditions. Hence, we challenged 18 simultaneously fasting and developing elephant seal pups with daily intramuscular injections of adrenocorticotropin (ACTH), a GR antagonist (RU486), or a combination of the two (ACTH+RU486) for 4 days. We collected blood at baseline, 2 h and 4 days after the beginning of treatment. ACTH and ACTH+RU486 elevated serum aldosterone and cortisol at 2 h, with effects diminishing at 4 days. RU486 alone induced a compensatory increase in aldosterone, but not cortisol, at 4 days. ACTH decreased neutrophils at 2 h, while decreasing lymphocytes and increasing the neutrophil:lymphocyte ratio at 4 days. These effects were abolished by RU486. Despite alterations in white blood cells, there was no effect of ACTH or RU486 on transforming growth factor-β or interleukin-6 levels; however, both cytokines decreased with the 4 day fasting progression. Similarly, ACTH did not impact protein oxidation, lipid peroxidation or antioxidant enzymes, but plasma isoprostanes and catalase activity decreased while glutathione peroxidase increased with fasting progression. These data demonstrate differential acute (2 h) and chronic (4 days) modulatory effects of HPA activation on white blood cells and that the chronic effect is mediated, at least in part, by GR. These results also underscore elephant seals' extraordinary resistance to oxidative stress derived from repeated HPA activation.

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus

Cluster of differentiation 73 (CD73, also known as ecto-5′-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

Progesterone Stimulates Histone Citrullination to Increase IGFBP1 Expression in Uterine Cells

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and an ovine uterine luminal epithelial (OLE) derived cell line. Progesterone (P4) not only maintains the uterine epithelia, but also regulates expression of histotroph genes critical during early pregnancy. Given this, we tested whether P4 stimulates PAD catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD catalyzed histone citrullination and an increase in IGFBP1 expression.

Glucocorticoid guides mobilization of bone marrow stem/progenitor cells via FPR and CXCR4 coupling

Background Our previous studies have proved the efficient exogenous repairing responses via bone marrow stem and progenitor cells (BMSPCs). However, the trafficking of endogenous bone marrow stem and progenitor cells to and from the bone marrow (BM) is a highly regulated process that remains to be elucidated. We aimed to study the relative importance of the hypothalamic-pituitary-adrenal (HPA) axis in the glucocorticoid-induced BMSPC mobilization. Methods The circulating mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) were examined in Crh (+/+, −/−) mice after running stress or glucocorticoid mini-infusion. The MSCs and EPCs were investigated ex vivo after treatment with glucocorticoid and glucocorticoid receptor (GR) antagonist, RU486. The expression of chemotaxis receptors, N-formyl peptide receptor (FPR), and Cys-X-Cys receptor 4 (CXCR4) of MSCs and EPCs as well as their colocalization were investigated after treatment with glucocorticoid, glucocorticoid receptor (GR) antagonist (RU486), and FPR antagonist (Cyclosporin H). Results Forced running stress increased circulating MSCs and EPCs in mice, which was blunted when Crh was knocked out, and positively related to the levels of serum glucocorticoid. Prolonged glucocorticoid mini-infusion imitated the stress-induced increase in circulating MSCs and EPCs in Crh+/+ mice and rescued the impaired mobilization in circulating MSCs and EPCs in Crh−/− mice. Meanwhile, glucocorticoid promoted the chemotaxis of MSCs and EPCs ex vivo via GR, inhibited by RU486 (10 μM). Concurrently, glucocorticoid increased the expression of FPR of MSCs and EPCs, but inhibited their expression of CXCR4, followed by their changing colocalization in the cytoplasm. The GC-induced colocalization of FPR and CXCR4 was blunted by Cyclosporin H (1 μM). Conclusion Glucocorticoid-induced CXCR4-FPR responsiveness selectively guides the mobilization of BMSPCs, which is essential to functional tissue repair. Graphical abstract Schematic view of the role of glucocorticoid on the mobilization of bone marrow-derived stem/progenitor cells subsets in the present study. The HPA axis activation promotes the release of glucocorticoid, which regulates the directional migration of MSCs and EPCs mainly via GR. The possible mechanisms refer to the signal coupling of FPR and CXCR4. Their two-sided changes regulated by glucocorticoid are involved in the egress of MSCs and EPCs from BM, which is helpful for wound healing. MSCs, mesenchymal stem cells; EPCs, endothelial progenitor cells.

Corticosterone Inhibits LPS-Induced NLRP3 Inflammasome Priming in Macrophages by Suppressing Xanthine Oxidase

Following traumatic insult and associated pathogen infection, innate immunity is activated during the perioperative period, especially the NLRP3 inflammasome in macrophages. The neuroendocrine response is also rapidly activated to regulate excessive inflammation; however, the molecular mechanisms are still not completely clear. This study is aimed at investigating the modulation of NLRP3 inflammasome priming by endogenous glucocorticoids (corticosterone, CORT) and its relationship with xanthine oxidase (XO). RAW264.7 murine macrophages were stimulated with LPS (1 μg/ml). LPS-induced NLRP3 expression was pretreated by CORT at different concentrations (0-900 ng/ml). Then, the effect of higher concentrations of CORT (700 ng/ml) on LPS-induced NLRP3 expression and the effect of allopurinol (250 μg/ml) were observed. Finally, the effects of a CORT antagonist (RU486) on XO expression and activity and NLRP3 expression in macrophages were further analyzed. Supernatant levels IL-1β and IL-18 were measured. The results showed that LPS-induced NLRP3 expression was upregulated further by pretreatment with CORT (300 ng/ml) (P<0.05); however, higher concentrations of CORT (greater than 700 ng/ml) downregulated NLRP3 expression (P<0.01) and the expression and activity of XO (P<0.05 and P<0.01, respectively). Allopurinol significantly inhibited NLRP3 expression. However, XO expression and activity, NLRP3 expression, and supernatant IL-1β and IL-18 levels were significantly increased in the RU486 group compared with the CORT group. In conclusion, our results suggested that CORT inhibits LPS-induced NLRP3 inflammasome priming in macrophages. The underlying mechanism is related to the modulation of XO expression and activity, which may be involved in priming and activating the NLRP3 inflammasome.

MON-011 NALCN Expression Is Regulated by Progesterone and Estrogen in Human Myometrial Smooth Muscle Cells

During pregnancy, the uterus transitions from a quiescent state to a highly contractile, excitable state. Both ion channels and hormones are essential for this transition. We recently identified that the Na+ leak channel, non-selective (NALCN) contributes to a leak current in human MSMCs and mice lacking NALCN have prolonged and dysfunctional labor. Additionally, NALCN levels change throughout mouse pregnancy suggesting regulation by hormones of pregnancy, specifically estrogen and progesterone. Here, we tested the hypothesis that P4, a pro-quiescent hormone, and E2, a pro-contractile hormone, regulate NALCN expression and current in the myometrium. In a human immortalized myometrial cells (HM6ERMS2), using qPCR we measured a 2.3 fold decrease and a 5.6 fold increase in NALCN mRNA expression in the presence of E2 and P4, respectively. These findings were also confirmed when NALCN protein expression were measured by immunoblot. Conversely, treatment with the ER antagonist, ICI 182,780, significantly increased NALCN mRNA expression, while treatment with the PR antagonist RU486 significantly decreased NALCN mRNA expression suggesting E2 and P4 work through their respective receptors to regulate NALCN. P4 differentially regulates myometrial activity depending on which progesterone receptor is activated: PRA, promotes contractility, whereas PRB promotes quiescence. Thus to study the effect of each PR, we used a human myometrial cell line stably expressing PRA or PRB, and measured similar increases in NALCN mRNA expression in both cell lines treated with P4. To determine the functional consequences of E2 and P4, we measured NALCN-dependent leak current in MSMCs using whole cell patch clamping. We observed that E2 significantly inhibited while P4 significantly enhanced NALCN current. Finally, we identified estrogen response and progesterone response elements (ERE and PRE) in the NALCN promoter and showed that the PREs contributed to P4 regulation while the ERE did not contribute to the regulation of NALCN expression using luciferase based promoter assays. Overall, our findings show that NALCN is upregulated by P4, the pro-quiescent hormone, and downregulated by E2, the pro-contractile hormone. This data reveals a new mechanism by which NALCN is regulated in the myometrium and may suggest a novel role for NALCN during pregnancy. Further investigation into these novel roles can provide an insight into potential targets to modulate uterine quiescence and contractility.