GSK-3 Inhibition Modulates Metalloproteases in a Model of Lung Inflammation and Fibrosis.

Francesco Cinetto,Carla Felice,Gianpietro Semenzato,Ilaria Caputo,Jessica Ceccato,Riccardo Scarpa,Daniela Cangiano,Marcello Rattazzi,Maria Piazza,Fiorella Calabrese,Carmela Gurrieri,Francesca Lunardi,Barbara Montini,Fabrizio Vianello,Carlo Agostini

doi:10.3389/fmolb.2021.633054

Abstract

Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine–threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFβ following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy.

Highlights

We aim to investigate the in vivo and in vitro roles of Glycogen synthase kinase 3 (GSK-3) inhibition in the modulation of matrix metalloproteinase (MMP)-9 and MMP-2 and of their inhibitors tissue inhibitors of metalloproteinases (TIMPs)-1 and TIMP-2 in the development of lung fibrosis
We assessed gene expression levels of TIMP-1 and TIMP-2, the physiologic inhibitors of MMP-9 and MMP-2, respectively. Both transcripts were detected at low levels in inflammatory cells collected from bronchoalveolar lavage fluid (BALF), we found that TIMP-1 and TIMP-2 expressions were augmented in BLM-treated mice compared to control mice (p < 0.0001)
We further investigated the role of GSK-3 in the early phase of extracellular matrix (ECM) remodeling, which is known to play a pivotal role in Idiopathic pulmonary fibrosis (IPF), focusing upon the modulation of MMPs and TIMPs that are essential in the physiological turnover of the matrix and in the repair of the disrupted basement membrane (BM)

Summary

Introduction

Idiopathic pulmonary fibrosis (IPF) is characterized by extensive lung parenchyma remodeling due to the abnormal deposition of extracellular matrix (ECM) by fibroblasts and the migration of epithelial cells and myofibroblasts through the disrupted basement membrane (BM) into the alveolar spaces (Selman et al, 2001).Metalloproteases in Lung FibrosisIn this context, matrix metalloproteinases (MMPs), a family of extracellular and zinc-dependent enzymes, are proposed to play a crucial role through their proteolytic activity (Woessner, 1991).MMP activity is regulated at multiple levels including gene transcription and extracellular activation of the zymogen and inactivation by specific inhibitors referred to as tissue inhibitors of metalloproteinases (TIMPs) (Chakraborti et al, 2003). Idiopathic pulmonary fibrosis (IPF) is characterized by extensive lung parenchyma remodeling due to the abnormal deposition of extracellular matrix (ECM) by fibroblasts and the migration of epithelial cells and myofibroblasts through the disrupted basement membrane (BM) into the alveolar spaces (Selman et al, 2001). In this context, matrix metalloproteinases (MMPs), a family of extracellular and zinc-dependent enzymes, are proposed to play a crucial role through their proteolytic activity (Woessner, 1991). Evidence suggests that inducible MMP-9 may have multiple roles in the lung, with studies implicating it in wound repair of human respiratory epithelium as well as in pathological processes including alveolar bronchiolization in bleomycin-induced lung injury (Buisson et al, 1996; Aoudjit et al, 1998; Legrand et al, 1999; Lemjabbar et al, 1999)

Objectives

Methods

Results

Conclusion