AJAP1 Silencing in Epithelial Cells Alters Markers of Biologic Function

Abstract

Introduction The epicardium and coronary vessels of the embryonic mammalian heart are derived from epithelial-like progenitor cells of the proepicardium (PE). These epithelial cells migrate from the PE during development to envelop and adhere to the myocardium to form the epicardium. Epithelial cells maintain their cellular integrity through adherens and tight junctions before undergoing epithelial-mesenchymal transition (EMT) to become epicardial-derived cells that invade the myocardium and transform into the vascular smooth muscle cells and vascular endothelial cells of the coronary vessels. Adherens junction-associated protein 1 (AJAP1) is a cell adhesion molecule that is expressed in the epicardium and coronary vessels, and it regulates epithelial cell behaviors. Study Objective Our goal was to identify and assess the molecular target(s) of AJAP1-modifiable epithelial cell behaviors that are important for formation of the epicardium and coronary vessels during cardiovascular development. We speculate that AJAP1 regulates epithelial cell behaviors by altering the expression of transcripts of adherens junctions, tight junctions and EMT markers. Methods Primary human mammary epithelial cells (HMEpiCs) retain characteristics of epithelial cells in vivo. We examined the in vitro contributions of AJAP1 to epithelial cell behaviors necessary for epicardium formation using HMEpiCs. We silenced AJAP1 mRNA expression in HMEpiCS with small interfering RNAs (siRNAs) and this produced changes in HMEpiC behavior. We obtained qPCR array plates to assay AJAP1-gene-silenced versus control HMEpiCs for differential expression of transcripts classified as markers of adherens junctions, tight junctions and EMT. This array expression data was subjected to gene ontology (GO) analysis to query the targeted genes and classify them by distinct biologic functions. Results A reduction in AJAP1 mRNA expression produced discernible changes in epithelial cell morphology and functions. A comparison of adherens junction, tight junction and EMT qPCR arrays revealed transcripts with altered expression in AJAP1-silenced versus control HMEpiCs. GO analysis showed these transcripts are involved in several biological processes including blood vessel development, cell migration, cell-cell adhesion, cytoskeleton organization and cell junction organization. This analysis correlates with our previous cell behavioral data. Conclusion Our results indicate that AJAP1 plays an important role in epithelial cells to enable expression of genes involved in an array of biological processes such as cell migration, cell-cell adhesion, cytoskeleton organization and cell junction organization. These biological processes are important for formation of the epicardium and coronary vessels during cardiovascular development.