The Aryl Hydrocarbon Receptor (ahr) ligands increase expression of vascular endothelial growth factor in airway epithelial cells

[[abstract]]Rationale: Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis, and may contribute to airway remodeling in various diseases, including asthma and chronic obstructive pulmonary disease (COPD). In recent years, many environmental toxicants, such as 2,3,7,8-tetrachlorodibenzo- -dioxin (TCDD), have been recognized as important environmental factors in thep pathogenesis of asthma and COPD. The aryl hydrocarbon receptor (AhR) is the principle receptor for many environmental toxicants. Our previous study has demonstrated that AhR increased expression of matrix metalloproteinase-1 and interleukin-1 in airway epithelial cells. However, the role of AhR in modulating the expression of VEGF is still elusive. Methods: Airway epithelial cell lines, Beas-2B and HBE-135, were treated with one of the AhR agonists (TCDD and 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester [ITE; an endogenous AhR ligand identified from lung]), and the expressions of VEGF in mRNA and protein levels were assessed with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. To determine the role of canonical AhR pathway, the cells were transfected with short hairpin RNA (shRNA) specific to aryl hydrocarbon receptor nuclear translocator (ARNT) prior to the treatment with an AhR agonist. The level of 15-hydroxyeicosatetraenoic acid (15-HETE) and STAT3 wereassessed with ELISA and immunoblot assay, respectively. The expression of ALOX15 , the gene encoding15-lipoxygenase (15-LOX), was assessed with qRT-PCR. Results Treatment of airway epithelial cells with AhR agonist significantly up-regulated the expression of VEGF and the increase VEGF activity in the condition media was confirmed by its effect on the human umbilical vein endothelial cells (HUVECs), assessed by the wound healing assay (for their migratory activity) and HUVEC tube formation assay. The increased expression of VEGF was not seen in cells transfected with ARNT-specific shRNA. The cells treated with AhR agonists also revealed increased expression of ALOX15 and 15-HETE. Pretreatment of the cells with 15-HETE inhibitors diminished the extent of AhR agonist-induced increased expression of VEGF. The cells treated with AhR agonists also showed activation of STAT3 in a 15-HETE-dependant manner. Conclusions: We have demonstrated that AhR agonists significantly up-regulated the expression of VEGF through an ARNT-dependent (canonical) AhR pathway, followed by the 15-LOX/15-HETE/STAT3 pathway. Further studies, including exploring the detailed mechanisms of this pathway, may enrich our knowledge about the effects of environmental toxicants on the airway epithelial cells and the pathogenesis of asthma and COPD