The aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as hypoxia-inducible factor (HIF)-1β, plays a pivotal role in the adaptive responses to (micro-)environmental stresses such as dioxin exposure and oxygen deprivation (hypoxia). ARNT belongs to the group of basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) transcription factors, which act as heterodimers. ARNT serves as a common binding partner for the aryl hydrocarbon receptor (AhR) as well as HIF-α subunits. HIF-α proteins are regulated in an oxygen-dependent manner, whereas ARNT is generally regarded as constitutively expressed, meaning that neither the arnt mRNA nor the protein level is influenced by hypoxia (despite the name HIF-1β). However, there is emerging evidence that tumor cells derived from different entities are able to upregulate ARNT, especially under low oxygen tension in a cell-specific manner. The objective of this review is therefore to highlight and summarize current knowledge regarding the hypoxia-dependent upregulation of ARNT, which is in sharp contrast to the general point of view described in the literature. Elucidating the mechanism behind this rare cellular attribute will help us to gain new insights into HIF biology and might provide new strategies for anticancer therapeutics. In conclusion, putative treatment effects on ARNT should be taken into account while studying the HIF pathway. This step is of great importance when ARNT is intended to serve as a loading control or as a reference.

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.

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The differentiation of adipose stem/progenitor cells (ASCs) into adipocytes contributes to adipose tissue expansion in obesity. This process is regulated by numerous signalling pathways including MAPK signalling. In the present study, we show that weight loss (WL) interventions induce upregulation of Sprouty1 ( SPRY1 ), a negative regulator of MAPK signalling, in human ASCs and elucidate the role of the Sprouty1/MAPK interaction for adipogenic differentiation. We found that the Sprouty1 protein levels are low in proliferating ASCs, increasing in density arrested ASCs at the onset of adipogenic differentiation and decreasing in the course of adipogenesis. Knock-down (KD) of Sprouty1 by RNA interference led to elevated MAPK activity and reduced expression of the early adipogenic transcription factor CCAAT/enhancer-binding protein β ( C/EBP β ), concomitant with an abrogation of adipogenesis. Intriguingly, co-treatment of Sprouty1 KD ASCs with differentiation medium and the pharmacological MEK inhibitor U0126 blunted ERK phosphorylation; however, failed to rescue adipogenic differentiation. Thus, the effects of the Sprouty1 KD are not reversed by inhibiting MAPK signalling although the inhibition of MAPK signalling by U0126 did not prevent adipogenic differentiation in wild type ASCs. In conclusion, we show that Sprouty1 is induced after WL in ASCs of formerly obese people acting as a negative regulator of MAPK signalling, which is necessary to properly trigger adipogenesis at early stages by a C/EBP β dependent mechanism.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated β-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPβ and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.