Estrogen receptor modulators and estrogen receptor beta immunolabelling in human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) exposed to the female sex hormone estradiol show different kinds of effects including increased elasticity, activation of plasma membrane Na⁺/H⁺ exchange, prostacyclin production, prevention of apoptosis and many others. The aim of this study was the systematic analysis of the immunolabelling of estrogen receptors (ERs), ERα and ERβ, in HUVEC after stimulation with different commercially available ER modulators and ER agonists or antagonists. HUVEC response to these substances was shown to be regulated via ERβ. ERα immunolabelling or up-regulation was abrogated after application of estrogen derivatives, selective estrogen receptor modulators (SERM) and ER agonists or antagonists. Immunolabelling of ERβ was significantly increased by estradiol, estrone, ethinylestradiol and tumour necrosis factor alpha (TNFα). SERM, such as Tamoxifen, and pure antagonists, such as ICI 182.780, stimulated ERβ in HUVEC at low concentrations, whereas higher concentrations inhibited ERβ immunolabelling. The pure estrogen receptor agonist 2,3-bis (4-hydroxyphenyl) proprionitrile (DPN) exhibited its activating potential at low concentrations. In contrast, higher concentrations resulted in a down-regulation of ERβ. Estrogenic effects in HUVEC, independent of stimulation or inhibition, are mediated via the ERβ. SERM such as Tamoxifen and ER antagonists such as ICI 182.780 act as ER activators in low concentrations, whereas higher concentrations lead to inhibitory effects.     

Genomic and non-genomic actions of estrogen on synaptic plasticity in SH-SY5Y cells

Estrogen modulates synaptic plasticity, an important mechanism of memory storage. Previously, we have reported that estrogen rapidly increases the expression of Arc (activity-regulated cytoskeleton associated protein), a key protein for synaptic plasticity, via non-genomic phosphoinositide-3 kinase (PI-3K)-, mitogen-activated protein kinase (MAPK)-, and estrogen receptor (ER)-dependent pathways in SH-SY5Y cells. The present study aimed to investigate the role of each ER subtype, α and β, in synaptic plasticity in SH-SY5Y cells. The specific agonist of ERβ (DPN) markedly induced Arc expression that mimics treatment with estrogen, but not ERα (PTT). Determination of subcellular localization of ERβ using immunocytochemistry shows that ERβ was retained in the cytoplasm of the untreated cells. In estrogen-treated cells, the membrane and cytosolic ERβ gradually decreased, while nuclear ERβ progressively increased in time-dependent manner, suggesting estrogen-dependent nuclear translocation of ERβ. Nuclear accumulation of ERβ at 6–12 h post-estrogen treatment, leads to increased PSD-95 and SYP mRNA expression, indicating the classical genomic estrogenic action on synaptic plasticity. However, the block of PI-3K signaling by Wortmannin partially suppressed estrogen (48 h)-induced PSD-95 and SYP expression, suggesting a crosstalk mechanism between genomic and non-genomic actions of estrogen on synaptic plasticity. Therefore, the estrogen-enhanced synaptic plasticity is ERβ-dependent and involves the crosstalk mechanism of non-genomic and genomic estrogenic actions.     

Epigallocatechin-3-gallate-mediated Tollip induction through the 67-kDa laminin receptor negatively regulating TLR4 signaling in endothelial cells

Background

Green tea polyphenol epigallocatechin-3-gallate (EGCG) has the potential to impact a variety of inflammation-related diseases; however, the anti-inflammatory action of EGCG in endothelial cells has not been understood. Recently, we demonstrated that the 67-kDa laminin receptor (67LR) acts as a cell-surface EGCG receptor.

Aim

This research was carried out to clarify the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in lipopolysaccharide (LPS)-stimulated endothelial cells.

Results

RNAi-mediated silencing of 67LR resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced activation of downstream signaling pathways. Also, we found that EGCG induced a rapid upregulation of Toll-interacting protein (Tollip), a negative regulator of TLR signaling, through 67LR in endothelial cells. RNAi-mediated silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Additionally, silencing of Tollip resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced expressions of cell-associated adhesion molecules, such as ICAM-1 and VCAM-1.

Conclusion

Taken together, these novel findings provide new insights into an understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.     

Regulator of G-protein signaling 2 inhibits acid-induced mucin5AC hypersecretion in human airway epithelial cells

Mucus hypersecretion is a common pathological feature of inflammatory airway diseases. Previous studies have shown that acidic microenvironment of inflamed airways may provoke the pathophysiology of inflammatory airway diseases. However, the acidic-sensing and negative regulatory mechanisms that mediate mucus hypersecretion in inflamed airway remain elusive. Thus, we sought to explore the role of ovarian cancer G-protein-coupled receptor 1 (OGR1) in acid-induced mucin5AC (MUC5AC) hypersecretion in human airway epithelium and the inhibitory effect of regulator of G-protein signaling 2 (RGS2) in this process. We found that airway acidification increased [Ca²⁺]_i, which was required for MUC5AC secretion. Knocking-down OGR1 and G_q with siRNAs and pretreating cells with phospholipase C inhibitor effectively attenuated acid-induced cellular responses. Moreover, the overexpression of wild-type RGS2 attenuated acid-induced cellular responses. In contrast, reducing RGS2 with siRNA enhanced the increases in acid-induced cellular responses. These data suggest that airway acidification can induce MUC5AC hypersecretion through an OGR1-mediated mechanism and RGS2 can inhibit acid-induced MUC5AC hypersecretion at OGR1 receptor level.     

Defective STING expression potentiates IL-13 signaling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps

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