Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

Molecular characterization and developmental expression of the aryl hydrocarbon receptor from the chick embryo

The aryl hydrocarbon receptor (AhR) was cloned from the chick embryo and its function and developmental expression characterized. Chicken AhR cDNA coded for 858 amino acid protein and 396 bp of 3' UTR. The basic helix loop helix domain exhibited 87-100% amino acid identity to avian, mammalian, and amphibian AhR, and 69-74% to piscine AhR. The PAS (Per-ARNT-Sim) region was slightly less well conserved with (a) 97% identity to other avian sequences, (b) 81-86% to amphibian and mammalian AhR, and (c) 64-69% with piscine AhR. The carboxy terminus diverged the most among species with less than 53% amino acid identity between chicken and any available mammalian and piscine AhR sequences. The chicken AhR RNA and protein were 6.1 kb and 103 kDa, respectively. Chicken AhR dimerized with human AhR nuclear translocator and bound the mammalian dioxin-response element in a ligand-dependent manner. AhR protein was detected in neural ganglia; smooth, cardiac, and skeletal muscle; and epithelium involved in epithelial-to-mesenchymal transformations, such as pituitary, gastrointestinal tract, limb apical-ectodermal ridge, and kidney collecting ducts. AhR mRNA was detected in all tissues expressing protein, except myocardium. Cytochrome P4501A4 mRNA was highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a subset of tissues expressing AhR, including small intestine, liver, kidney, blood vessels, and outflow tract myocardium. In conclusion, the AhR sequence and function is highly conserved between birds and mammals, and although many tissues express AhR during chick embryo development, only a subset are responsive to TCDD induction of CYP1A4.     

Fewer CTL, not enhanced NK cells, are sufficient for viral clearance from the lungs of immunocompromised mice

Activation of the aryl hydrocarbon receptor (AhR) causes numerous defects in anti-viral immunity, including suppressed CTL generation and impaired host resistance. However, despite a reduced CTL response, mice that survive infection clear the virus. Therefore, we examined the contribution of NK cells and pro-inflammatory cytokines to viral clearance in influenza virus-infected mice exposed to TCDD, the most potent AhR agonist. Infection caused transient increases in pulmonary TNFalpha, IL-1, and IFNalpha/beta levels, but neither the kinetics nor magnitude of this response was affected by AhR activation. No IL-18 was detected at any time point examined. Exposure to TCDD enhanced NK cell numbers in the lung but did not affect their IFNgamma production. Furthermore, depletion of NK cells did not alter anti-viral cytolytic activity. In contrast, removal of CD8+ T cells ablated virus-specific cytolytic activity. These results demonstrate that the pulmonary CTL response to influenza virus is robust and few CTL are necessary for viral clearance.     

Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor-dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cells and consequent apoptosis in T cells

We investigated the role of aryl hydrocarbon receptor (AhR) in the regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis in thymic T cells. AhR knockout (KO) mice were resistant to TCDD-induced thymic atrophy and apoptosis when compared with the AhR wild-type mice. TCDD triggered the expression of several apoptotic genes, including FasL in AhR wild-type but not AhRKO mice. TCDD-induced increase in FasL was seen only in thymic stromal but not thymic T cells. When TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas-FasL interactions. Thus, apoptosis in T cells was not detected when TCDD-treated stromal cells from FasL-defective or AhRKO mice were mixed with wild-type T cells or when TCDD-exposed wild-type stromal cells were mixed with Fas-deficient T cells. TCDD treatment, in vivo and in vitro, led to colocalization and translocation of NF-kappaB subunits (p50, p65) to the nucleus in stromal but not T cells from AhR wild-type mice. NF-kappaB activation was not observed in stromal cells isolated from TCDD-treated AhRKO mice. Mutations in NF-kappaB-binding sites on the FasL promoter showed that TCDD regulates FasL promoter activity through NF-kappaB. TCDD treatment in vivo caused activation of the death receptor and mitochondrial pathways of apoptosis. Cross-talk between the two pathways was not necessary for apoptosis inasmuch as TCDD-treated Bid KO mice showed thymic atrophy and increased apoptosis, similar to the wild-type mice. These findings demonstrate that AhR regulates FasL and NF-kappaB in stromal cells, which in turn plays a critical role in initiating apoptosis in thymic T cells.     

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.