Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells

Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.     

Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells

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Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation

LNCaP prostate cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and an Ah-responsive reporter gene. Similar results were obtained with the selective AhR modulator 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF); however, TCDD but not 6-MCDF induced degradation of the AhR protein. TCDD and 6-MCDF inhibited growth of LNCaP cells, and inhibitory AhR-androgen receptor (AR) crosstalk was investigated in cells transfected with constructs containing the androgen-responsive probasin promoter (-288 to +28) (pPB) or three copies of the -244 to -96 region of this promoter (pARR(3)). Ten nanomolar dihydrotestosterone (DHT) and 17 beta-estradiol (E2) induced transactivation in LNCaP cells transfected with pPB or pARR(3); however, inhibitory AhR-AR crosstalk was observed only with the latter construct. 6-MCDF and TCDD did not inhibit DHT- or E2-induced transactivation in ZR-75 human breast cancer cells, indicating that these interactions were promoter and cell context-dependent. Both E2 and DHT stabilized AR protein in LNCaP cells; however, cotreatment with TCDD or 6-MCDF decreased AR protein levels. These results indicate that inhibitory AhR-AR crosstalk in prostate cancer cells is complex and for some responses, AR protein stability may play a role.     

Aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha in MCF-7 breast cancer cells

The aryl hydrocarbon receptor (AhR) binds with high affinity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatics, but also binds with lower affinity to structurally diverse exogenous and endogenous chemicals. One study reported that 3-methylcholanthrene (3MC) activated the estrogen receptor (ER) through the AhR, which acts as co-regulatory protein, whereas a recent report showed that 3MC directly bound and activated ERalpha. This study also shows that the AhR agonists benzo[a]pyrene, 3,3',4,4'-tetrachlorobiphenyl, chrysin, 6-methyl-1,3,8-trichlorodibenzofuran, and 3,3'-diindolylmethane also induce ERalpha-dependent transactivation. Moreover, in chromatin immunoprecipitation assays, these compounds induce binding of AhR and ERalpha to the CYP1A1 and pS2 gene promoters, which is consistent with their activities as both selective AhR modulators (SAhRMs) and selective ER modulators (SERMs).     

Aryl hydrocarbon receptor pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of matrix metalloproteinase-9

Background  

Abberant aryl hydrocarbon receptor (AhR) expression and AhR pathway activation are involved in gastric carcinogenesis. However, the relationship between AhR pathway activation and gastric cancer progression is still unclear. In present study, we used 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), a classic and most potent ligand of AhR, to activate AhR pathway and investigated the effect of AhR pathway activation on human gastric cancer AGS cell invasion and explored the corresponding mechanism.     

Aryl hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine epidermis

An immunoregulatory role of aryl hydrocarbon receptor (AhR) has been shown in conventional αβ and γδ T cells, but its function in skin γδ T cells (dendritic epidermal T cells [DETC]) is unknown. In this study, we demonstrate that DETC express AhR in wild-type mice, and are specifically absent in the epidermis of AhR-deficient mice (AhR-KO). We show that DETC precursors are generated in the thymus and home to the skin. Proliferation of DETC in the skin was impaired in AhR-KO mice, resulting in a >90% loss compared with wild type. Surprisingly, DETC were not replaced by αβ T cells or conventional γδ T cells, suggesting a limited time frame for seeding this niche. We found that DETC from AhR-KO mice failed to express the receptor tyrosine kinase c-Kit, a known growth factor for γδ T cells in the gut. Moreover, we found that c-kit is a direct target of AhR, and propose that AhR-dependent c-Kit expression is potentially involved in DETC homeostasis. DETC are a major source of GM-CSF in the skin. Recently, we had shown that impaired Langerhans cell maturation in AhR-KO is related to low GM-CSF levels. Our findings suggest that the DETCs are necessary for LC maturation, and provide insights into a novel role for AhR in the maintenance of skin-specific γδ T cells, and its consequences for the skin immune network.     

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.     

Protein kinase Cdelta signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.     

Aryl hydrocarbon receptor activation impairs the priming but not the recall of influenza virus-specific CD8+ T cells in the lung

The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR(+/+) recipients, and the generation of CD45.2AhR(-/-)-->CD45.1AhR(+/+) chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.     

Aryl hydrocarbon receptor (AhR)-linked inhibition of luteal cell progesterone secretion in 2,3,7,8-tetrachlorodibenzo-p-dioxin treated cells.

In this study, we tested firstly, the hypothesis that decrease of progesterone secretion by luteal cells under the influence of 2,3,7,8-tetrachlorodibezo-p-dioxin (TCDD) is due to influence on specific enzymatic steps in the biosynthetic pathway of steroidogenesis and secondly, involvement of aryl hydrocarbon receptor (AhR) or estradiol receptor (ER) in this process. Luteal cells isolated from mature porcine corpora lutea were cultured with 25-hydroxycholesterole (25-OH) or pregnenolone (P5) as a substrate. Additionally aminoglutethimide, the inhibitor of P540scc or trilostane the inhibitor of 3 beta-HSD was added to basal and stimulated cells. The synergistic action of TCDD with aminoglutethimide in decreasing of progesterone secretion was observed. In pregnenolone treated cells 1.6 fold decrease of progesterone secretion was observed that in both TCDD alone and together with trilostane treated cells. In the second part of experiments to show the involvement of AhR and ER in TCDD action on progesterone secretion alpha- naphtophlavone, the AhR blockers and 4-hydroxytamoxifen (4-OH-TMX), the inhibitor of ER were used. alpha-naphtophlavone, the inhibitory effect of TCDD while 4-OH-TMX had no effect on TCDD-treated cells. These experiments suggest TCDD decreased progesterone secretion by luteal cells by reduction of the activity of mitochondrial enzymes, which converts cholesterol into pregnenolone. Moreover points to AhR dependent but not ER-dependent mechanisms in TCDD action in luteal cells.     

Aryl hydrocarbon receptor activation during influenza virus infection unveils a novel pathway of IFN-gamma production by phagocytic cells

The contribution of environmental factors is important as we consider reasons that underlie differential susceptibility to influenza virus. Aryl hydrocarbon receptor (AhR) activation by the pollutant dioxin during influenza virus infection decreases survival, which correlates with a 4-fold increase in pulmonary IFN-gamma levels. We report here that the majority of IFN-gamma-producing cells in the lung are neutrophils and macrophages not lymphocytes, and elevated IFN-gamma is associated with increased pulmonary inducible NO synthase (iNOS) levels. Moreover, we show that even in the absence of dioxin, infection with influenza virus elicits IFN-gamma production by B cells, gammadelta T cells, CD11c(+) cells, macrophages and neutrophils, as well as CD3(+) and NK1.1(+) cells in the lung. Bone marrow chimeric mice reveal that AhR-mediated events external to hemopoietic cells direct dioxin-enhanced IFN-gamma production. We also show that AhR-mediated increases in IFN-gamma are dependent upon iNOS, but elevated iNOS in lung epithelial cells is not driven by AhR-dependent signals from bone marrow-derived cells. Thus, the lung contains important targets of AhR regulation, which likely influence a novel iNOS-mediated mechanism that controls IFN-gamma production by phagocytic cells. This suggests that AhR activation changes the response of lung parenchymal cells, such that regulatory pathways in the lung are cued to respond inappropriately during infection. These findings also imply that environmental factors may contribute to differential susceptibility to influenza virus and other respiratory pathogens.