Epidermal Growth Factor-activated Aryl Hydrocarbon Receptor Nuclear Translocator/HIF-1β Signal Pathway Up-regulates Cyclooxygenase-2 Gene Expression Associated with Squamous Cell Carcinoma

Kwang-Yu Chang,Meng-Ru Shen,Mei-Yi Lee,Wen-Lin Wang,Wu-Chou Su,Wen-Chang Chang,Ben-Kuen Chen

doi:10.1074/jbc.m806210200

Abstract

Hypoxia-inducible factor (HIF) accumulates when tumors grow under hypoxic conditions. The genesis of tumors, however, usually involves normoxic conditions. In this study, we were interested in examining the potential role of aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta in tumor growth under normoxic conditions, specifically when cells are treated with epidermal growth factor (EGF), which is known to affect the gene expression of tumor growth-related protein COX-2 (cyclooxygenase-2). The results showed that EGF receptor inhibitor, AG1478, abolished EGF-induced nuclear accumulation of ARNT as well as the expression of COX-2. ARNT small interfering RNA inhibited the promoter activity, mRNA level, and protein expression of COX-2 in cells treated with EGF. In contrast, CoCl(2)-induced HIF-1alpha exhibited no effect on COX-2 expression. EGF also stimulated the formation of the ARNT.c-Jun complex as well as the complex binding to the COX-2 promoter. ARNT small interfering RNAs blocked EGF-activated cell migration. Moreover, COX-2 and ARNT were cohorts present distinctively in clinical specimens of human cervical squamous cell carcinoma and were almost nondetectable in adjacent normal or noncancerous cervical tissues. Our results revealed that ARNT plays an important role in EGF-regulated COX-2 gene expression and may thus be related to either a cause or a consequence of tumorigenesis in cervical cancer.

Highlights

1␤)) is a member of the basic helix-loop-helix Per/aryl hydrocarbon receptor (AhR)/ARNT/Sim family of transcription factors and a general partner factor for bHLHPAS proteins such as the AhR, HIF-1␣, and single-minded (SIM) proteins [1]
These results demonstrate that the development of Von Hippel-Lindau-associated vascular tumors in the liver depends on functional ARNT but not in an HIF-1␣/hypoxia-dependent manner
Coexpression of COX-2 and ARNT was found clinically in human cervical cancer tissue. These results provide the new regulatory mechanism of HIF-1 factor involved in growth factor-induced gene expression under normoxia

Summary

Introduction

1␤)) is a member of the basic helix-loop-helix Per/aryl hydrocarbon receptor (AhR)/ARNT/Sim (bHLH-PAS) family of transcription factors and a general partner factor for bHLHPAS proteins such as the AhR, HIF-1␣ (hypoxia-inducible factor-1␣), and single-minded (SIM) proteins [1]. Our results suggest a model in which c-Jun expression, induced by EGF, could recruit either c-Fos or other transcription factors to the promoter and regulate gene expression of COX-2. In order to confirm the functional role of endogenous ARNT in regulating the EGF-induced COX-2 expression, ARNT short interfering RNA (siRNA) oligonucleotide was transfected into A431 cells.

Results

Conclusion