Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

Fatemeh Sadeghi,Reza Falak,Gholam Ali Kardar,Mohammad Reza Bolouri,Farzad Nasri,Maryam Sadri

doi:10.1186/s40659-020-00293-4

Abstract

BackgroundHypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition.MethodsWe inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT.ResultsHypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and β-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and β-catenin and protein level of β-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of β-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant.ConclusionbHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Highlights

Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia
The efficiency of transfection and the viability of HEK293T cell after transfection The rate of transfection of HEK293T with control pIRES2-EGFP and inhibitory bHLH (ibHLH) pIRES2-EGFP were 78% and 87% respectively, 48 h post-transfection (Fig. 4) and the transfection assay had no significant effect on the viability of HEK293T cells (Fig. 5)
The induction of hypoxia with Cobalt chloride (CoCl2) Treating HEK293T with CoCl2 significantly increased the expression of vascular endothelial growth factor (VEGF) as the main downstream gene of HIF-1a, at both 150 μM (p < 0.01) and 200 μM (p < 0.001) concentrations after 48 h (Fig. 3a). CoCl2 at concentration

Summary

Introduction

Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. In the past two decades, gene-therapy has received great attention in the fields of medical sciences. It could be more favorable for the treatment of highly prevalent diseases such as cancer, the most effective results have been reported from monogenic diseases, as they are caused by a mutation in a single gene [1]. The overlapping of different signaling pathways should be clearly considered [1, 3]

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Discussion

Conclusion