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In mammals, numerous reports describe an immunomodulating effect of thyroid-active compounds. In contrast, only few reports have been published on this subject in fish. We previously demonstrated that immune cells of rainbow trout (Oncorhynchus mykiss) possess thyroid hormone receptors (THRs) and that exposure of trout to the thyroid hormone 3,3',5-triiodo-. l-thyronine (T3) or the antithyroid drug propylthiouracil (PTU) alters immune cell transcript levels of THR and several immune genes. The present study aims to further characterize the immunomodulating action of thyroid-active compounds in trout immune cells. We report here the use of a custom-designed 60-mer oligo immune-targeted microarray for rainbow trout to analyze the gene expression profiles induced in the head kidney by T3 and PTU. Morphometric analyses of the thyroid showed that PTU exposure increased the size of the epithelial cells, whereas T3 induced no significant effects. Both T3 and PTU had diverse and partly contrasting effects on immune transcript profiles. The strongest differential effects of T3 and PTU on gene expressions were those targeting the Mitogen Associated Protein Kinase (MAPK), NFkB, Natural Killer (NK) and Toll-Like Receptor (TLR) pathways, a number of multipath genes (MPG) such as those encoding pleiotropic transcription factors (atf1, junb, myc), as well as important pro-inflammatory genes (tnfa, tnf6, il1b) and interferon-related genes (ifng, irf10). With these results we show for the first time in a fish species that the in vivo thyroidal status modulates a diversity of immune genes and pathways. This knowledge provides the basis to investigate both mechanisms and consequences of thyroid hormone- and thyroid disruptor-mediated immunomodulation for the immunocompetence of fish. © 2016 Elsevier B.V.

We used small-interference RNA (siRNA) to explore the mechanisms of some vasoactive intestinal peptide (VIP) actions on human breast cancer cells. Transfection of estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast cancer cells with VPAC(1)-receptor siRNA completely abolished VIP stimulatory effect on secretion of the main angiogenic factor, vascular endothelial growth factor (VEGF), and transactivation of epidermal growth factor receptor (EGFR or HER1) and HER2, two members of HER family of tyrosine-kinase receptors. The silencing procedure suggested the involvement of EGFR and HER2 transactivation in VIP-stimulated VEGF secretion. It was further supported by blocking tyrosine kinase activity by the selective HER inhibitors AG-1478 (EGFR) and AG-825 (HER2). Results give value to the specific signaling of VIP through VPAC(1) receptor in human breast cancer cells and support the potential use of VPAC(1)-receptor antagonists in combined targeted therapies for breast cancer. Molecular therapies involving RNA interference of VPAC(1)-receptor expression could also be considered.

Vasoactive intestinal peptide (VIP) and its receptors (VPACs) are involved in proliferation, survival, and differentiation in human breast cancer cells. Its mechanism of action is traditionally thought to be through specific plasma membrane receptors. There is compelling evidence for a novel intracrine mode of genomic regulation by G-protein-coupled receptors (GPCRs) that implies both endocytosis and nuclear translocation of peripheral GPCR and/or the activation of nuclear-located GPCRs by endogenously-produced, non-secreted ligands. Regarding to VPAC receptors, which are GPCRs, there is only a report suggesting them as a dynamic system for signaling from plasma membrane and nuclear membrane complex. In this study, we show that VPAC(1) receptor is localized in cell nuclear fraction whereas VPAC(2) receptor presents an extranuclear localization and its protein expression is lower than that of VPAC(1) receptor in human breast tissue samples. Both receptors as well as VIP are overexpressed in breast cancer as compared to non-tumor tissue. Moreover, we report the markedly nuclear localization of VPAC(1) receptors in estrogen-dependent (T47D) and independent (MDA-MB-468) human breast cancer cell lines. VPAC(1) receptors are functional in plasma membrane and nucleus as shown by VIP stimulation of cAMP production in both cell lines. In addition, VIP increases its own intracellular and extracellular levels, and could be involved in the regulation of VPAC(1)-receptor traffic from the plasma membrane to the nucleus. These results support new concepts on function and regulation of nuclear GPCRs which could have an impact on development of new therapeutic drugs.

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases.

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout ( Oncorhynchus mykiss) express both thyroid receptor α (THRA) and β (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. Invivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells ( cd4, cd8a, trb), B lymphocytes ( mIgM) and macrophages ( csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones. © 2014 Elsevier Ltd.

The widely used herbicide atrazine (ATZ) has been reported to exhibit reproductive toxicity in rats, fish and amphibians, with an avian LD 50 of 5000 mg/kg. In the present work, ATZ was administered as a single oral dose of 25 or 100 mg/kg to female European quail (Coturnix coturnix coturnix) at days 0, 5 and 10 of the experiment, being the animals sampled at days 15, 30 and 45. ATZ significantly increased the expression of hepatic estrogen receptor α (ERα) at both doses at day 30. An important increase was also observed in plasma 17β-estradiol (E2) concentrations. ATZ at 100 mg/kg increased the circulating concentration of vitellogenin (Vtg), but this effect was not related with an increase in hepatic Vtg mRNA levels. ATZ had no effect on the hepatic expression of both cytochrome P450 1A4 (CYP1A4) or the related biotransformation activity ethoxyresorufin-O-deethylase (EROD). These results led to the conclusion that ATZ provokes an estrogenic effect in sexually mature females of European quail. Further studies are necessary to establish the effect on sexual development or reproduction of female and male birds in the wild. © 2012 Elsevier Inc.

9 páginas, 6 figuras, 1 tabla.-- et al., Normal functioning of the endocrine system is essential for the proper development and reproduction of animals. Substances interfering with its homeostasis are called endocrine disruptors (EDs) and may represent a risk for the health of the organism. One of the mechanisms of endocrine disruption that has attracted great attention in recent years concerns alterations in the normal functioning of the estrogen receptor (ER), but far less attention has been paid to those substances interfering with the thyroid axis, which, in fish, plays several critical roles in a variety of biological functions. In aquaculture, feedstuffs can be a source of hormones or persistent pollutants which act as potential EDs. In this study, the main purpose was to assess the possible estrogenic and thyrogenic activities of 32 commercial fish feeds. For the assessment of estrogenicity, a new estrogen receptor specific reporter gene assay using sea bass ERα (sbER α) was developed and validated. Potential thyroidal disruption was screened with a cell line permanently transfected with luciferase as reporter gene under the control of avian thyroid receptor α (THRα). The results obtained showed that 11 and 18 out of 32 assayed feeds were able to activate the sbERα or the avTHRα1, respectively. The present study is pioneer in demonstrating thyrogenic activity in fish diets commercially available and widely used in aquaculture. Given that maintaining the homeostasis in the endocrine system is critical for the proper development and reproduction of fish, any estrogenic or thyrogenic activity caused by the feedstuffs should be taken into account with regards to its potential impact on farmed fish. © 2012 Elsevier B.V., This study was funded by projects RTA 2009-00074-00-00 (MICINN) managed by the INIA, AGL 2010-22247-C03-01 and CSD 2007-00002 managed by IATS-CSIC from the Spanish Ministry of Science and Innovation.

We used an in vivo model of human experimental prostate cancer in order to shed a new light on the effects of vasoactive intestinal peptide (VIP) on tumor growth as well as its pro-metastatic potential in this disease. We used nude mice subcutaneously injected with prostate cancer androgen-independent PC3 cells for 30 days. The regulatory role of VIP on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression as well as on matrix metalloproteinase-2 and 9 (MMP-2 and 9) activities was examined. A selective COX-2 inhibitor, NS-398, and curcumin were used to block VIP effects. Xenografts of VIP-treated PC3 prostate cancer cells in nude mice gave tumors that grew significantly faster than those in the untreated group. It is conceivably a result of both the trophic effect of VIP on prostate cancer cells and the proangiogenic action of the neuropeptide in the growing tumor. We show the overexpression at mRNA and/or protein levels of VIP, its main receptor VPAC 1 , the major angiogenic factor VEGF, and the pro-inflammatory enzyme COX-2 as well as the increased activity of MMP-2 and 9 in tumors derived from VIP-treated PC3 cells as compared with control group. The overexpression of the above biomarkers was suppressed in tumors derived from VIP-treated PC3 cells that had been previously incubated with curcumin or NS-398. Thus, the potential therapeutic role of curcumin and selective COX-2 inhibitors in combination with available VIP antagonists should be considered in prostate cancer therapy as supported by their inhibitory activities on tumor cell growth.

We analyzed the cross-talk between receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) in oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-468) human breast cancer cells. VIP treatment slowly increased the expression levels of EGFR but it rapidly augmented phosphorylation of EGFR and HER2 in both cell lines. This pattern of HERs transactivation was blocked by the specific VIP antagonist JV-1-53, supporting the direct involvement of VIP receptors in formation of P-EGFR and P-HER2. VIP-induced transactivation was also abolished by H89 (protein kinase A inhibitor), PP2 (Src inhibitor) or TAPI-1 (inhibitor of matrix metalloproteases), following a differential pattern. These results shed a new light on the specific signalling pathways involved in EGFR/HER2 transactivation by VPAC receptors and suggest the potential usefulness of VIP receptor antagonists together with current antibodies against EGFR/HER2 and/or tyrosine kinase inhibitors for breast cancer therapy.