Regulation and role of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) in type II cells apoptosis and Idiopathic Pulmonary Fibrosis (IPF)

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KlymenkoOleksiy-2016-06-23.pdf (54.02 MB)

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2016

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DOI:
http://dx.doi.org/10.22029/jlupub-14312

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic pulmonary disease of unknown origin with an unavoidable fatal outcome. In principle, IPF is characterized by alveolar epithelial cell damage, increased deposition of extracellular matrix (ECM) in the lung interstitium, and enhanced fibroblast/myofibroblast proliferation and activation. These processes ultimately lead to distortion of normal lung architecture and loss of respiratory function. Recent data show that Endoplasmic reticulum (ER) stress and apoptosis of alveolar epithelial cells type II (AECII) play a key role in both, sporadic and familial forms of IPF. It has been shown that an overload and accumulation of unfolded and misfolded proteins lead to ER stress. C/EBP homologous protein (CHOP) is thought to represent a key regulator of pro-apoptotic responses under ER stress. In IPF, induction and nuclear translocation of CHOP in AECII is regularly found, to a weaker extent also in yet not fibrotic areas of these lungs. Following this line, we hypothesized that ER stress-induced CHOP may significantly contribute to AECII injury and apoptosis and hence fibrosis development in IPF. The present study therefore aimed to (i) characterize the transcriptional regulation of epithelial CHOP expression in vitro, and (ii) to elucidate the biological role of Chop in the induction of epithelial apoptosis and lung fibrosis in vitro and in vivo.For such purpose, the promotor of the human CHOP gene was extensively analyzed employing a luciferase reporter gene assay. We identified a new mechanism for the regulation of CHOP expression during ER stress. According to our data, Ap-1 and c-Ets-1 transcription factors are up-regulated in the lung epithelium under ER-stress conditions, interact with each other and jointly bind to the Chop promotor, thereby inducing the Chop gene expression.Moreover, the role of Chop in the induction of epithelial apoptosis was studied using various in vitro and in vivo models. Exclusive Chop overexpression was achieved in vitro using an inducible "Tetracyclin-On" ("Tet-on")-system in stably transfected epithelial MLE 12 cells and in isolated, primary alveolar type II cells. Both in vitro models revealed induction of cleaved caspase 3 and thus apoptosis in response to Chop overexpression. In addition, incubation of murine lung fibroblasts with supernatants of these stably transfected cells resulted in increased fibroblast proliferation once Chop was induced by doxycyclin application. Moreover, in vivo data support the results of in vitro studies. Conditional Chop overexpression in AECII in vivo was achieved by crossing SP-C rtTA to tetO7 Chop mice. In response to oral doxycyclin feeding, Chop was induced in AECII and led to Caspase 3 cleavage and thus apoptosis of these cells. Furthermore, induction and up-regulation of Chop target genes such as Casp11, Il1b, Il6, Ero1a, Dr5, and Gadd34 was detected on mRNA level by qPCR. In short term periods covering 8 weeks, young transgenic mice with inducible, AECII-specific Chop overexpression did yet not show a distinct fibrotic phenotype, although we could detect significant up-regulation of pro-fibrotic markers. Thus, involvement of Chop in the development of lung fibrosis requires further investigation. Our findings demonstrate for the first time that AP-1 and c-Ets-1 play an important role in the transcriptional regulation of CHOP expression during ER stress. Additionally, we provide evidence that Chop expression alone is sufficient to induce apoptosis in AECII and consecutive fibroproliferation, hence suggesting a pivotal role of CHOP in the pathogenesis of pulmonary fibrosis.

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