MiR-199a Downregulation as a Driver of the NOX4/HIF-1α/VEGF-A Pathway in Thyroid and Orbital Adipose Tissues from Graves' Patients.

Julie Craps,Virginie Joris,Antonella Boschi,Marie-Christine Many,Catherine Behets,Michel Mourad,Antoine Buemi,Chantal Daumerie,Alessandra Camboni,Chantal Dessy,Benoit Lengelé,Lelio Baldeschi

doi:10.3390/ijms23010153

Abstract

Graves’ disease (GD) is an autoimmune thyroiditis often associated with Graves’ orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.

Highlights

Introduction distributed under the terms andGraves’ disease (GD) is an autoimmune thyroiditis that causes hyperthyroidism due to excess stimulation of thyrocytes by circulating thyrotropin (TSH) receptor-specific stimulatory autoantibodies (TRAb) [1]
The present work illustrates the role of NOX4 as a non-mitochondrial source of ROS in thyroids and orbital fats from GD/Graves’ orbitopathy (GO) patients
Several defense mechanisms co-exist, allowing the cells to cope with the burden of ROS production, with the NOX/DUOX family composed of DUOX1, DUOX2, NOX2 and NOX4 being the major source of non-mitochondrial ROS [15,38,39]

Summary

Introduction

Introduction distributed under the terms andGraves’ disease (GD) is an autoimmune thyroiditis that causes hyperthyroidism due to excess stimulation of thyrocytes by circulating thyrotropin (TSH) receptor-specific stimulatory autoantibodies (TRAb) [1]. Remarkable increase in the eyelid aperture, remarkable exophthalmos, periorbital and conjunctival hyperaemia, and edema are observed. These symptoms mainly result from an increase in intraorbital pressure leading to secondary orbital and periorbital venous congestion [1,2]. In tissues affected by the GD autoimmune process, OS and increased angiogenesis have been observed [3,4,5,6,7] Antioxidant therapies, such as selenium supplementation, have been shown to slow down the progression of mild to moderately severe, non-active (clinical activity score < 2) GO of recent (

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