ZNF365 is overexpressed in Pulmonary Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology characterized by the expansion of fibroblast population and excessive extracellular matrix (ECM) deposition. Transforming growth factor‐beta 1 (TGF‐β1) is one of the most potent mediators of the fibrotic response and induces fibroblast proliferation, and their differentiation to myofibroblasts, cells characterized by increased contractility, apoptosis resistance and the production of multiple ECM components (mainly collagens).Global gene expression of human lung fibroblasts stimulated with TGFβ performed in our laboratory demonstrated the overexpression of ZNF365, a zinc finger protein that belongs to the C2H2 domain family that has been poorly described in the literature. The purpose of this study is to evaluate ZNF365 expression in IPF lung tissue, the expression of its ortholog Zfp365 in mice lung tissue and its functional effects on human lung fibroblasts and epithelial cells in vitro.Expression of ZNF365 and Zfp365 were evaluated by qPCR and WB in human and mice samples, as well as in fibroblasts and epithelial cells stimulated with TGFβ. Localization of the protein was performed by immunohistochemistry in IPF lungs and in bleomycin‐induced lung fibrosis in mice. For silencing experiments, specific siRNAs and scrambled controls designed for ZNF365 were used. For overexpression, cells were stably transfected with ZNF365. Growth rate and proliferation were analyzed using WST‐1 and CyQUANT reagent, and apoptosis was measured by flow cytometry.We found that ZNF365 is overexpressed in IPF lung tissues and localized in alveolar and bronchial epithelial cells and in fibroblasts/myofibroblasts. The orthologous Zfp365 is overexpressed in lung tissue from bleomycin instilled mice, and the immunoreactive protein was observed in alveolar and airway epithelium. In vitro studies confirmed that ZNF365 is overexpressed in control and IPF fibroblasts stimulated with TGFβ. Likewise, TGFβ induced the upregulation of ZNF365 in a human alveolar epithelial cell line. Functional assays revealed that ZNF365 silencing in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and proliferation compared with the scrambled and WT controls. Preliminary data show that silencing of this protein increases senescence‐asociated β‐galactosidase activity and increases expression of the histone variant γH2A.X, another senescence marker. These results demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and may be involved in the regulation of proliferation and senescence of human lung cells.Support or Funding InformationSupported by PAPIIT 201520