Biological role of the proapoptotic transcription factor CHOP in Idiopathic Pulmonary Fibrosis (IPF)

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive diffuse parenchymal lung disease (DPLD), with a median survival time of less than three years following diagnosis. The precise mechanism of IPF is still not fully understood but chronic injury of alveolar epithelial type-II cells (AECII) is increasingly accepted as key event. We have recently identified severe Endoplasmic Reticulum (ER) stress leading to apoptosis as a major pathomechanism for such alveolar epithelial cell injury. In line with this, the pro-apoptotic transcription factor CHOP, which functions as a crucial mediator of ER stress-induced apoptosis, was strongly upregulated in AECII from patients with IPF. We thus hypothesize that epithelial induction of the CHOP protein – which is hardly detectable under physiological conditions – plays a key role in the evolution of IPF.

Aim: We therefore aim to fully disclose the transcriptional regulation of CHOP expression in vitro and in vivo and to clarify the biological role of epithelial CHOP induction in pulmonary fibrosis.

Methodology: Promotor analysis of the human CHOP gene in silico (MatInspektor and TFSEARCH software) and in vitro (reporter gene assays for several distal/proximal CHOP promoter fragments, cloning of these constructs upstream from the luciferase gene into the pGL3-basic reporter vector, assessment of luciferase activity in transfected A549 and MLE15 cells in the presence or absence of ER stress-inducing agents); transient overexpression studies of FLAG- and c-myc-tagged CHOP constructs in lung epithelial cell lines, and expression analysis of CHOP and downstream targets of CHOP as well as apoptosis markers.

Results and Outlook: The in silico-analysis of the 2.7-kb 5'-flanking region of the human CHOP gene revealed approximately 15 previously unknown different putative transcription factor binding sites, in addition to the known and well-conserved ER stress-response elements (ERSE) and Amino-Acid-response elements (AARE). Several CHOP promotor constructs differing in size and amount of putative regulatory elements are already cloned, and are going to be assessed. In addition, CHOP could be successfully overexpressed in MLE12, MLE15 and A549 cells, with maximum of protein expression by 48–72 hours. At the moment, the cellular consequences are going to be assessed.

Based on the data generated herein, we expect to be able to develop novel, AECII specific, antiapoptotic treatment strategies for treatment of IPF.