AJAP1 Gene Silencing in Epithelial Cells Alters Protein Expression
Location
Philadelphia, PA
Start Date
11-5-2022 1:00 PM
End Date
11-5-2022 4:00 PM
Description
Introduction: The epicardial layer of the heart is formed from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate away from the PE and they 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 an impairment in 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 tight junction protein 1 (TJP1) mRNA expression in AJAP1 KD versus negative control cells. The TJP1 transcript encodes the protein zonula occludens 1 (ZO1).
Study Objective: The goal of this study was to examine the role of AJAP1 in regulating protein expression of ZO1 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZO1 protein is an important mediator of AJAP1 in epithelial cells for its role in forming tight junctions for cell-cell interactions.
Methods: We 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 the protein expression of ZO1. We compared ZO1 expression in negative control versus AJAP1 KD HMEpiCs.
Results: Reduced expression of AJAP1 transcripts produced noticeable changes in protein expression of ZO1 in epithelial cells. Western blot analysis revealed an increase in ZO1 protein expression in AJAP1 KD versus negative control HMEpiCs.
Conclusion: Our data indicates that AJAP1 contributes to epithelial cell function by potentially mediating ZO1 protein expression. ZO1 contributes to the formation of tight junctions and is known to maintain epithelial cell integrity and polarity. These biological processes are crucial for the maintenance of epithelial cells during epicardium formation.
Embargo Period
5-25-2022
AJAP1 Gene Silencing in Epithelial Cells Alters Protein Expression
Philadelphia, PA
Introduction: The epicardial layer of the heart is formed from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate away from the PE and they 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 an impairment in 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 tight junction protein 1 (TJP1) mRNA expression in AJAP1 KD versus negative control cells. The TJP1 transcript encodes the protein zonula occludens 1 (ZO1).
Study Objective: The goal of this study was to examine the role of AJAP1 in regulating protein expression of ZO1 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZO1 protein is an important mediator of AJAP1 in epithelial cells for its role in forming tight junctions for cell-cell interactions.
Methods: We 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 the protein expression of ZO1. We compared ZO1 expression in negative control versus AJAP1 KD HMEpiCs.
Results: Reduced expression of AJAP1 transcripts produced noticeable changes in protein expression of ZO1 in epithelial cells. Western blot analysis revealed an increase in ZO1 protein expression in AJAP1 KD versus negative control HMEpiCs.
Conclusion: Our data indicates that AJAP1 contributes to epithelial cell function by potentially mediating ZO1 protein expression. ZO1 contributes to the formation of tight junctions and is known to maintain epithelial cell integrity and polarity. These biological processes are crucial for the maintenance of epithelial cells during epicardium formation.