A HIF-1α-driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT.

M Mandl,M-K Lieberum,R Depping

doi:10.1038/cddis.2016.187

Abstract

Oxygen-deprived (hypoxic) areas are commonly found within neoplasms caused by excessive cell proliferation. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) is part of the hypoxia-inducible factor (HIF) pathway, which mediates adaptive responses to ensure cellular survival under hypoxic conditions. HIF signalling leads to metabolic alterations, invasion/metastasis and the induction of angiogenesis in addition to radio-chemoresistance of tumour cells. Activation of the HIF pathway is based on the abundance of HIF-α subunits, which are regulated in an oxygen-dependent manner and form transcriptional active complexes with ARNT or ARNT2 (also referred as HIF-1β and HIF-2β, respectively). ARNT is considered to be unaffected by hypoxia but certain cell lines, including Hep3B cells, are capable to elevate this transcription factor in response to oxygen deprivation, which implies an advantage. Therefore, the aim of this study was to elucidate the mechanism of hypoxia-dependent ARNT upregulation and to determine implications on HIF signalling. Gene silencing and overexpression techniques were used to alter the expression pattern of HIF transcription factors under normoxic and hypoxic conditions. qRT-PCR and western blotting were performed to measure gene and protein expression, respectively. HIF activity was determined by reporter gene assays. The results revealed a HIF-1α-dependent mechanism leading to ARNT upregulation in hypoxia. Forced expression of ARNT increased reporter activity under normoxic and hypoxic conditions. In conclusion, these findings indicate a novel feed-forward loop and suggest that ARNT might be a limiting factor. Augmented HIF signalling in terms of elevated target gene expression might be advantageous for tumour cells.

Highlights

A heterogeneous oxygenation is a characteristic attribute of solid tumours
A previous study published in 1995 by Wang et al.[20] demonstrated the capability of Hep3B cells to upregulate Aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxia. This cellular trait was confirmed by Wolff et al.[18] who showed that the induction of ARNT was more pronounced in an hypoxic environment of 3% O2 as compared with 1% O2
To investigate whether the protein level of the ARNT homologue ARNT2 might be affected by low-oxygen tension as well, Hep3B cells were exposed to 3% O2 or cultured in normoxia for 8h followed by western blot analysis

Summary

Introduction

A heterogeneous oxygenation is a characteristic attribute of solid tumours. Oxygen-deprived, that is hypoxic, areas are commonly found within neoplasms owing to uncontrolled cell proliferation.[1,2,3] In general, tumour hypoxia is considered as a negative prognostic marker associated with resistance to radio-chemotherapy and poor patient outcome.[1,2,4]. To ensure the survival of tumour cells, these are forced to initiate adaptive responses to an insufficient oxygen supply.[2]. This is mediated by the hypoxia-inducible factor (HIF) pathway, which triggers a number of cellular alterations affecting proliferation, metabolism and invasion/metastasis. The induction of angiogenesis is a HIF-dependent mediated process in order to increase the available oxygen concentration.[2] Owing to its tumour-promoting capacity, inhibition of the HIF pathway by several strategies is regarded as a treatment option in cancer therapy.[2,4,5,6,7,8,9,10]. Different HIF-3α splice variants exist, which are able to activate or repress HIF signalling depending on the cellular context.[4,12,13]

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