PYK2 mediates anti-apoptotic AKT signaling in response to benzo[a]pyrene diol epoxide in mammary epithelial cells.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. The carcinogenicity of BaP has been partially attributed to the formation of the BaP diol epoxide (BPDE), which has been shown to stably bind DNA and act as an initiator. BaP is a complete carcinogen, but the mechanisms for tumor promotion are less well characterized. Previous studies have demonstrated that BPDE enhanced anti-apoptotic signaling through Akt; however, mechanisms for Akt activation by BPDE are not well defined. In the current studies, we found that BPDE increased intracellular Ca2+ concentration in the human mammary epithelial cell line MCF-10A. A peak in Ca2+ concentration at 20 min was followed by increased phosphorylation of Pyk2 at Tyr881 and increased total tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Consistent with activation of the EGFR, Akt and ERK1/2 phosphorylation was detected in MCF-10A cells treated with BPDE. Pharmacological methods to prevent Ca2+ elevation and EGFR activity, and small-interfering RNA against Pyk2, prevented Akt phosphorylation by BPDE, which suggested that Ca2+, Pyk2 and EGFR activation lay upstream of Akt. In addition, we found that BPDE increased p53 activity and apoptosis in MCF-10A; however, transient transfection of constitutively active Akt attenuated both BPDE-dependent apoptosis and p53 activity. In contrast, apoptosis was enhanced by inhibitors of phosphatidyl inositol 3-kinase (PI3-K). This work demonstrates a novel mechanism for Akt activation by BPDE that occurs through increased Ca2+ concentration, and implicates Ca2+, Pyk2, EGFR and Akt as a potential pathway by which BPDE can inhibit apoptosis and act as a promoter of carcinogenesis.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A).

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure prior to chick embryo incubation (GD 0) induces dilated cardiomyopathy, and reduces myocardial hypoxia, vascular endothelial growth factor A (VEGF-A) expression, and coronary vascularization. We investigated whether reduced coronary vascularization 1) occurs in the absence of changes in cardiac morphology and 2) is associated with altered secretion of VEGF-A and/or an antivasculogenic factor. METHODS: Chicken eggs were treated with control (corn oil) or TCDD (0.075-0.3 pmol of TCDD/gm) on GD 5. In vivo cardiac morphology and artery number were determined on GD 10, while in vitro vascular outgrowth and VEGF-A secretion were determined from cardiac explants on GD 6. Effects of recombinant VEGF-A (rcVEGF-A), soluble flt-1 (sFlt-1) receptor plus rcVEGF-A, and control conditioned media were assessed in TCDD explants, while effects of TCDD-conditioned media was assessed in control explants. RESULTS: TCDD reduced coronary artery number in vivo by 53 +/- 8% and induced a dose-related reduction in tube outgrowth in vitro, but had no effect on cardiac morphology. All TCDD doses reduced explant VEGF-A secretion equally (43 +/- 3%), compared to control. sFlt-1 blocked outgrowth in control cultures and blocked rcVEGF-A-mediated rescue of outgrowth in TCDD explants. Control conditioned media partially rescued outgrowth from TCDD explants, while conditioned media from TCDD explants had no effect on controls. CONCLUSIONS: TCDD inhibition of coronary vascularization can occur in the absence of changes in cardiac morphology and is associated with reduced VEGF-A secretion but not an antivasculogenic factor. Since control media only partly rescues TCDD's inhibitory effect, we suggest that TCDD-exposed endothelial cells are less responsive to vasculogenic stimuli.

2,3,7,8-tetrachlorodibenzo-p-dioxin reduces myocardial hypoxia and vascular endothelial growth factor expression during chick embryo development.

BACKGROUND: Previous research has demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cardiomyocyte growth arrest, thinner ventricle walls, and reduced number and size of coronary arteries during chick embryogenesis. Coronary vascular development is believed to be mediated, in part, by myocardial oxygen gradients and a subsequent increase in hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor-A (VEGF-A) expression. We investigated whether TCDD inhibition of coronary development was associated with altered myocardial oxygen status and reduced cardiac HIF-1alpha and VEGF-A. METHODS: Chick embryos were exposed to 15% or 20% O2 for 24 hr from incubation days 9-10 or were injected with control (corn oil) or 0.24 pmol TCDD/gm egg on day 0. On day 9, embryos were injected with control (0.9% NaCl) or EF5, a tissue hypoxia marker, and cardiac binding of EF5 was determined by immunohistochemistry on day 10. In addition, embryo hearts were analyzed for VEGF-A mRNA by in situ hybridization and quantitative RT-PCR, and for HIF-1alpha mRNA by quantitative RT-PCR. RESULTS: Cardiac binding of EF5 was significantly increased in embryos exposed to 15% O2, compared to embryos exposed to 20% O2. In contrast, TCDD-exposed embryos exhibited significantly reduced binding of EF5 in the heart, compared to controls. Similarly, cardiac expression of HIF-1alpha and VEGF-A were increased following hypoxia and tended to be decreased following TCDD exposure. CONCLUSIONS: These results suggest that the myocardium may be a target of TCDD toxicity, resulting in reduced myocardial hypoxia, and HIF-1alpha and VEGF-A expression believed necessary for normal coronary development.

Vascular endothelial growth factor rescues 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibition of coronary vasculogenesis.

BACKGROUND: We previously demonstrated that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces coronary vascular development in chick embryos in vivo. In the current study, we assessed whether TCDD inhibits early events in coronary endothelial tube formation and outgrowth, and whether this inhibition occurs through a vascular endothelial growth factor (VEGF)-dependent mechanism. METHODS: Fertile chicken eggs were treated with control (corn oil) or TCDD (0.3 pmol TCDD/g) on incubation day 0. On embryonic day 6, cardiac ventricle explants were cultured on a three-dimensional collagen gel, when coronary angioblasts are present, but prior to their assembly into endothelial tubes. Endothelial cells migrating out from explants were identified by immunohistochemistry, and endothelial tube number and length were quantitated. In addition, on incubation days 6 and 8, cardiac VEGF mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Endothelial tube length and number were significantly reduced (40% +/- 1.7% and 36% +/- 3%, respectively) in TCDD explants, compared to controls. Recombinant exogenous VEGF, as well as hypoxic stimulation with CoCl2 or 10% O2, significantly increased the length and number of outgrowing tubes in TCDD cultures, and this stimulation was prevented by a VEGF neutralizing antibody. In contrast, VEGF neutralizing antibody reduced the length and number of tubes only in control cultures, and had no inhibitory effect on tube outgrowth from TCDD explants. Finally, hearts from TCDD-treated embryos exhibited a significant reduction in both VEGF mRNA and protein, compared to controls. CONCLUSIONS: These data suggest that TCDD inhibits early coronary vascular outgrowth via a VEGF-dependent mechanism.