HIF-1α Directly Controls WNT7A Expression During Myogenesis.

Federica Cirillo,Michele Ferrara,Giuseppe Ciconte,Elia Angelino,Paola Rota,Giulia Resmini,Adriana Tarantino,Luigi Anastasia,Michelle M Monasky,Marco Piccoli,Carlo Pappone,Andrea Graziani,Andrea Ghiroldi

doi:10.3389/fcell.2020.593508

Abstract

Herein we unveil that Hypoxia-inducible factor-1α (HIF-1α) directly regulates WNT7A expression during myogenesis. In fact, chromatin immunoprecipitation (ChiP) and site-directed mutagenesis experiments revealed two distinct hypoxia response elements (HREs) that are specific HIF-1α binding sites on the WNT7A promoter. Remarkably, a pharmacological activation of HIF-1α induced WNT7A expression and enhanced muscle differentiation. On the other hand, silencing of WNT7A using CRISPR/Cas9 genome editing blocked the effects of HIF-1α activation on myogenesis. Finally, treatment with prolyl hydroxylases (PHDs) inhibitors improved muscle regeneration in vitro and in vivo in a cardiotoxin (CTX)-induced muscle injury mouse model, paving the way for further studies to test its efficacy on acute and chronic muscular pathologies.

Highlights

Skeletal muscle regeneration is a complex process which occurs throughout the lifespan thanks to an adult stem cell reservoir present in the adult muscle
Quantitative ChIP assays were performed to identify any binding regions of hypoxia-inducible factor-1α (HIF-1α) on the WNT7A promoter, by dividing it into seven putative binding sequences from −1,000 to −197 bp, each containing at least one Hypoxia Responsive Element (HRE; 5 -CGTG-3 ; Figure 1C)
While no significant binding on WNT7A promoter was observed under normoxia, results showed that, under hypoxia, HIF-1α bound to Seq.1 (−1,000 −843 bp) and Seq.4 (−653 −489 bp; Figure 1D and Supplementary Figure S3), which contain one and three HRE sequences, respectively (Figure 1E)

Summary

Introduction

Skeletal muscle regeneration is a complex process which occurs throughout the lifespan thanks to an adult stem cell reservoir present in the adult muscle. People have struggled to therapeutically exploit Wnt7a and, to date, little is known about its upstream regulators to possibly envision other strategies to activate myogenesis (von Maltzahn et al, 2011). In this context, we previously reported that subjecting murine skeletal myoblasts C2C12 to a hypoxic pre-conditioning (1% O2 for 24 h) induced the upregulation of the hypoxia-inducible factor-1α (HIF-1α) as well as an activation of the non-canonical WNT-pathway, including Wnt7a, eventually leading to an activation of myogenesis and the formation of hypertrophic myotubes (Cirillo et al, 2017). We failed to recognize the mechanism of the observed effects

Methods

Results

Conclusion