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

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.

Hypoxic Upregulation of ARNT (HIF-1β): A Cell-Specific Attribute with Clinical Implications

“…Therefore, it was concluded that augmented HIF signalling in terms of elevated target gene expression might be beneficial for tumour cells. These findings support the concept of ARNT being a limiting factor in at least certain cell models [3].…”

“…[7]) is not sufficient to induce ARNT protein expression in this model. However, other studies clearly confirmed the hypoxia-dependent upregulation of ARNT in Hep3B cells [3,4].…”

“…Moreover, the regulation of ARNT and ARNT2 varied in human hepatocellular carcinoma Hep3B cells. ARNT was elevated in hypoxia, whereas ARNT2 was not affected in this model [3].…”

“…There is mounting evidence indicating a pivotal role of HIF-1α [2][3][4]. It was demonstrated that ARNT was increased in 518A2 human melanoma cells in a HIF-1α-dependent manner under hypoxic conditions [2].…”

“…Therefore, ARNT is considered to be a constitutively expressed gene [1]. Although this notion might be true for the majority of cells/tissues investigated, numerous studies reported the capability of tumour cells to elevate ARNT expression in response to hypoxia [1][2][3][4][5][6][7]. This cellular attribute was found in cells of different tumour types of both human and murine origin.…”