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ER-Stress Transcription Factor C/EBP Homologous Protein (CHOP) Causes Alveolar Epithelial Apoptosis and Provokes Fibrotic Responses

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A4365 - ER-Stress Transcription Factor C/EBP Homologous Protein (CHOP) Causes Alveolar Epithelial Apoptosis and Provokes Fibrotic Responses
Author Block: M. Korfei1, O. Klymenko1, M. Huehn1, J. Wilhelm1, R. Wasnick1, C. Ruppert1, I. Henneke1, S. Hezel1, K. Guenther1, I. Shalashova1, P. Mahavadi1, C. Samakovlis2, W. Seeger3, A. Guenther1; 1Department of Internal Medicine and Biomedical Research Center Seltersberg (BFS), German Center for Lung Research (DZL), Justus-Liebig-University Giessen, Giessen, Germany, 2Excellence Cluster Cardiopulmonary System (ECCPS), German Center for Lung Research (DZL), Justus-Liebig-University Giessen, Giessen, Germany, 3Department of Internal Medicine and Excellence Cluster Cardiopulmonary System (ECCPS), German Center for Lung Research (DZL), Justus-Liebig-University Giessen, Giessen, Germany.
Rationale: Severe endoplasmic reticulum (ER)-stress in alveolar epithelium is thought to underlie idiopathic pulmonary fibrosis (IPF), for which no cure exists to date. C/EBP homologous protein (CHOP), a transcription factor mediating ER-stress-induced apoptosis, is upregulated in type-II alveolar epithelial cells (AECII) of IPF-patients. It is still unclear, how CHOP induction in epithelial cells is regulated, and to which extent alveolar epithelial Chop-overexpression contributes to the fibroproliferative responses in IPF. Methods: Whole genome transcriptomics of AECII-like MLE12-cells overexpressing the main ER-stress-transducers Atf4, Atf6 and spliced (s)Xbp1 versus thapsigargin-treatment was performed. We fully disclosed the transcriptional regulation of CHOP-expression in (lung) epithelial cells by using Luciferase reporter gene-assays for distinct CHOP promoter constructs. Luciferase activity was quantified in response to ER-stress-inducing agents, overexpression of transcription factors (TFs), serial deletion and mutation of TF binding-sites. Overexpression of Chop was achieved by adenoviral gene transfer in primary murine AECII (transient), or in stably-transfected MLE12-cells (conditional). We also developed transgenic mice expressing Chop exclusively in AECII by using the Tetracycline-On-system (SP-C rtTA/tetO7-Chop). Results: Single or combined overexpression of the active ER-stress factors Atf4, p50Atf6 or sXbp1 in MLE12-cells did not result in a substantial Chop induction, as compared to the ER-stress-inducer thapsigargin. Employing reporter gene-assays, we could show that, next to the conventional amino-acid-response (AARE)- and ER-stress-response-elements (ERSE) within the CHOP promoter, additional AP-1 and c-Ets-1 binding-sites are necessary for CHOP induction during ER-stress. Deletion analysis and point mutations of the CHOP promoter revealed that both AP-1 and c-Ets-1 binding-sites act in concert to induce CHOP-expression. Further, we found AP-1 and c-Ets-1 to be upregulated in AECII under ER-stress conditions and in human IPF. Moreover, singular Chop-overexpression in vitro resulted in caspase-3-activation and apoptosis of AECII. In addition, incubation of lung fibroblasts with supernatant media of Chop-overexpressing AECII led to increased fibroblast-proliferation and collagen-I-production. Importantly, in vivo-data supported the results of in vitro-studies. In response to doxycycline-feeding (4/8 weeks), Chop was induced in AECII and resulted in apoptosis of these cells in SP-C rtTA/tetO7-Chop mice. Employing qPCR, induction of Chop-target genes and enhanced profibrotic gene expression were detected in lungs of double-transgenic mice. However, full-blown fibrosis, in a sense of light-microscopically visible scars, was not observed in Chop transgenic mice, although interstitial collagen-I accumulation and septal thickening were evident. Conclusions: We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII injury and consecutive fibrosis, offering new therapeutic prospects in IPF.
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