Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

CCN2/CTGF increases expression of miR-302 microRNAs, which target the TGF beta type II receptor with implications for nephropathic cell phenotypes

Signalling interplay between transforming growth factor-beta (TGF beta) and CCN2 [also called connective tissue growth factor (CTGF)] plays a crucial role in the progression of diabetic nephropathy and has been implicated in cellular differentiation. To investigate the potential role of microRNAs (miRNAs) in the mediation of this signalling network, we performed miRNA screening in mesangial cells treated with recombinant human CCN2. Analysis revealed a cohort of 22 miRNAs differentially expressed by twofold or more, including members of the miR-302 family. Target analysis of miRNA to 3'-untranslated regions (3'-UTRs) identified TGF beta receptor II (T beta RII) as a potential miR-302 target. In mesangial cells, decreased T beta RII expression was confirmed in response to CCN2 together with increased expression of miR-302d. T beta RII was confirmed as an miR-302 target, and inhibition of miR-302d was sufficient to attenuate the effect of CCN2 on T beta RII. Data from the European Renal cDNA Biopsy Bank revealed decreased T beta RII in diabetic patients, suggesting pathophysiological significance. In a mouse model of fibrosis (UUO), miR-302d was increased, with decreased T beta RII expression and aberrant signalling, suggesting relevance in chronic fibrosis. miR-302d decreased TGF beta-induced epithelial mesenchymal transition (EMT) in renal HKC8 epithelial cells and attenuated TGF beta-induced mesangial production of fibronectin and thrombospondin. In summary, we demonstrate a new mode of regulation of TGF beta by CCN2, and conclude that the miR-302 family has a role in regulating growth factor signalling pathways, with implications for nephropathic cell fate transitions.

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Mast cell tryptase controls paracellular permeability of the intestine: role of protease-activated receptor 2 and beta-arrestins

Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves protease-activated receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.

Estrogen and thyroid hormone receptor interactions: physiological flexibility by molecular specificity

The influence of thyroid hormone on estrogen actions has been demonstrated both in vivo and in vitro. In transient transfection assays, the effects of liganded thyroid hormone receptors (TR) on transcriptional facilitation by estrogens bound to estrogen receptors (ER) display specificity according to the following: 1) ER isoform, 2) TR isoform, 3) the promoter through which transcriptional facilitation occurs, and 4) cell type. Some of these molecular phenomena may be related to thyroid hormone signaling of seasonal limitations upon reproduction. The various combinations of these molecular interactions provide multiple and flexible opportunities for relations between two major hormonal systems important for neuroendocrine feedbacks and reproductive behaviors.

Distinct behavioral phenotypes in male mice lacking the thyroid hormone receptor alpha1 or beta isoforms

Thyroid hormones influence both neuronal development and anxiety via the thyroid hormone receptors (TRs). The TRs are encoded by two different genes, TRalpha and TRbeta. The loss of TRalpha1 is implicated in increased anxiety in males, possibly via a hippocampal increase in GABAergic activity. We compared both social behaviors and two underlying and related non-social behaviors, state anxiety and responses to acoustic and tactile startle in the gonadally intact TRalpha1 knockout (alpha1KO) and TRbeta (betaKO) male mice to their wild-type counterparts. For the first time, we show an opposing effect of the two TR isoforms, TRalpha1 and TRbeta, in the regulation of state anxiety, with alpha1 knockout animals (alpha1KO) showing higher levels of anxiety and betaKO males showing less anxiety compared to respective wild-type mice. At odds with the increased anxiety in non-social environments, alpha1KO males also show lower levels of responsiveness to acoustic and tactile startle stimuli. Consistent with the data that T4 is inhibitory to lordosis in female mice, we show subtly increased sex behavior in alpha1KO male mice. These behaviors support the idea that TRalpha1 could be inhibitory to ERalpha driven transcription that ultimately impacts ERalpha driven behaviors such as lordosis. The behavioral phenotypes point to novel roles for the TRs, particularly in non-social behaviors such as state anxiety and startle.

Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma

In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30 min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis. (C) 2009 Elsevier Ltd. All rights reserved.