Cystatin M/E, Cathepsin V and Asparaginyl Endopeptidase: New actors of the proteolytic balance in lung fibrosis - 08/04/25

Idiopathic pulmonary fibrosis (IPF) is a chronic disease with unknown etiology, characterized by progressive and irreversible fibrous thickening in the interstitial spaces of the lung. Alterations in alveolar epithelia induce the TGF-β1-dependent differentiation and proliferation of fibroblasts into myofibroblasts. Extracellular matrix (ECM) overproduction by myofibroblasts causes a dysregulated remodeling in fibrotic foci, which is closely associated with an altered protease/antiprotease balance [1Taggart C., Mall M.A., Lalmanach G., Cataldo D., Ludwig A., Janciauskiene S., et al. Eur Respir J 2017 ;  49 : 1501200

Cliquez ici pour aller à la section Références]. Among proteases involved, some cysteine cathepsins are potent collagenases and elastases which participate in ECM remodeling. During IPF, the cathepsin/cystatin (i.e. endogenous cathepsin inhibitors) balance is impaired in favor of cystatins. The secretion of human Cystatin C (CysC) is upregulated during myodifferentiation, promoting collagen I deposition [2Kasabova M., Joulin-Giet A., Lecaille F., Saidi A., Marchand-Adam S., Lalmanach G. Human cystatin C: a new biomarker of idiopathic pulmonary fibrosis? Proteomics Clin Appl 2014 ;  8 : 447-453 [cross-ref]

Cliquez ici pour aller à la section Références]. In addition, expression levels of both fibronectin and elastin are altered during fibrogenesis. Accordingly, this led us to investigate the involvement of Cathepsin V (CatV, the most potent human elastase) [3Lecaille F., Chazeirat T., Saidi A., Lalmanach G., Cathepsin V. Molecular characteristics and significance in health and disease Mol Aspects Med 2022 ;  88 : 101086

Cliquez ici pour aller à la section Références], Asparaginyl endopeptidase (AEP) (a.k.a. Legumain (LGMN), participate in extracellular matrix degradation) [4Dall E., Brandstetter H. Structure and function of legumain in health and disease Biochimie 2016 ;  122 : 126-150 [cross-ref]

Cliquez ici pour aller à la section Références] and of Cystatin M/E (CysM) (a dual tight-binding inhibitor of both enzymes) [5Lalmanach G., Kasabova-Arjomand M., Lecaille F., Saidi A. Cystatin M/E (Cystatin 6): A Janus-Faced Cysteine Protease Inhibitor with Both Tumor-Suppressing and Tumor-Promoting Functions Cancers (Basel) 2021 ;  13 : 1877 [cross-ref]

Cliquez ici pour aller à la section Références], in the differentiation of human lung fibroblasts.

Characterization of CysC, CysM, CatV and AEP was conducted by immunochemical analysis of control and IPF bronchoalveolar lavage fluid (BALF) and lung biopsies. CCD-19Lu human lung fibroblasts were used as a model of TGF-β1-dependent myodifferentiation to study the regulation of these proteases and cystatins during pulmonary fibrosis. SiRNAs targeting the proteins of interest and the effects on ECM were evaluated as previously. Also, kinetic assays were performed.

We observed that CysC and CysM are oversecreted in IPF patients BALF. Similar results were observed in supernatants of TGF-β1-differentiated human lung fibroblasts. Furthermore, silencing of CatV and AEP increased the expression levels of ECM proteins, elastin and fibronectin, respectively.

Our data support that CysC and CysM are key molecular players involved in the TGF-β1-dependent myofibrogenesis. Secreted forms could impair both fibronectin degradation and elastinolytic activities of extracellular cysteine proteases. Results obtained from IPF lung biopsies further confirmed this imbalance between protease and cystatins. Since, these proteases are involved in the ECM remodeling, we suggest that this alteration of protease/antiprotease balance may subsequently promote fibrotic phenotype found in IPF.