AJAP1 Gene Silencing Alters Protein Expression in Epithelial Cells

Abstract

IntroductionThe epicardial layer of the heart is formed during cardiovascular development from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate out of the PE to surround and adhere to the myocardium to form a layer of epithelial cells that compose the epicardium. A subset of the epicardial cells delaminate and migrate into the subepicardial space where they undergo epithelial‐mesenchymal transition (EMT) to become epicardial‐derived cells (EPDCs). These EPDCs invade the myocardium and differentiate into a subset of cells that make up the coronary vessels. During this entire process, the epicardium receives input from several transcription factors including Tbx5. Mice with a conditional deletion of Tbx5 exhibit impaired epicardium formation and coronary vessel development. Tbx5‐deficient mouse hearts have a significant decrease in mRNA expression of adherens junction associated protein (Ajap1). Our studies show that knockdown of endogenous AJAP1 (AJAP1 KD) mRNA expression in epithelial cells leads to increased cell adhesion, reduced cell migration, enhanced expression of epithelial markers of EMT, and altered expression of transcripts associated with adherens junctions, tight junctions and EMT. Specifically, our qPCR array data shows a significant alteration in ZEB2 mRNA expression in AJAP1 KD cells versus negative control cells.Study ObjectiveThe goal of this study was to examine the role of AJAP1 in regulating protein expression of ZEB2 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZEB2 protein is an important functional mediator of AJAP1 in epithelial cells for undergoing EMT.MethodsWe utilized the primary human mammary epithelial cell line (HMEpiC) as an in vitro system to examine contributions of AJAP1 to epithelial cell biology. We silenced AJAP1 mRNA expression in HMEpiCs using small interfering RNAs and examined protein expression of ZEB2. We compared ZEB2 expression in negative control versus AJAP1 KD HMEpiCs.ResultsReduced expression of AJAP1transcripts produced noticeable changes in protein expression of ZEB2 in epithelial cells, and it had significant effects on expression of adherens junction‐related transcripts. Western blot analysis revealed an increase in ZEB2 protein expression in AJAP1 KD versus negative control HMEpiCs.ConclusionOur data indicate that AJAP1 contributes to epithelial cell function, and it does this potentially by mediating ZEB2 protein expression. ZEB2 is known to regulate EMT, which is a biological process that is crucial for the formation of the epicardium and its subsequent formation of the coronary vasculature.