AJAP1 regulates epithelial cell adhesion required for epicardium and EPDC formation
Location
Philadelphia, PA
Start Date
1-5-2024 1:00 PM
End Date
1-5-2024 4:00 PM
Description
Introduction: The epicardium participates in cardiovascular development through its contribution of multipotent epicardial-derived cells (EPDCs). During cardiovascular development, cells delaminate from the epicardium to form EPDCs that undergo epithelial-to-mesenchymal transition and have the capacity to differentiate into multiple cardiac lineages. This makes epicardial cells an ideal progenitor source for cardiac tissue regeneration. It is vital to identify molecules in epicardial cells that regulate behaviors required for its formation and differentiation into multipotent EPDCs. Previously, our lab identified AJAP1 as a novel cardiac transcript in the Tbx5-deficient mouse model of defective epicardium formation (Tbx5epi-/-). AJAP1 encodes the adherens junction associated protein 1 cell adhesion molecule. Tbx5epi-/- embryonic mouse hearts exhibit a delayed adhesion of epicardial cells to the myocardium, impaired development of coronary vessels, and reduced AJAP1 mRNA expression. We have shown that AJAP1 is expressed in epithelial cells of the epicardium in the developing mouse heart. In vitro knockdown of AJAP1 in primary human epithelial cells inhibits cell migration. Despite these findings, a role for AJAP1 in cardiovascular development has not been identified.
Methods: Given that AJAP1 is expressed in epithelial cells of the mammalian heart, we sought to determine the functions of AJAP1 in the HMEpiC primary human mammary epithelial cell line. We silenced endogenous AJAP1 expression with small interfering RNAs (siRNA) designed to target human AJAP1. HMEpiCs were transfected with AJAP1 siRNAs or a non-targeting control siRNA. Silencing of AJAP1 expression was confirmed by quantitative PCR and immunofluorescence. We investigated the impact of AJAP1 on cell-to-extracellular matrix (ECM) adhesion by conducting cell adhesion assays with these siRNA-transfected cells. 6x104 control or AJAP1 knockdown (KD) HMEpiCs were plated on collagen I, collagen IV, laminin, or fibronectin matrices. These ECMS were chosen for the cell-matrix adhesion assays because they represent matrices that are abundant during mammalian cardiovascular development. Cells were allowed to adhere to the ECM for 1, 4 or 24 hours. At each respective time point, cell adhesion was stopped by methanol fixation of cells. Adherent cells were stained with crystal violet solution, and several high-powered fields were imaged on a microscope to quantify cell-matrix adhesion.
Results: We analyzed cell-ECM matrix adhesion in control versus AJAP1 KD HMEpiCs and observed significant differences in cell-matrix adhesion. In comparison to control HMEpiCs, we observed a significant 1.8-fold increase in adhesion of AJAP1 KD HMEpiCs to collagen I matrix at the 24-hour time point. On the laminin matrix, we observed a 1.2-fold increase in adhesion of AJAP1 KD HMEpiCs versus control HMEpiCs at the 4-hour time point. We detected a 1.6-fold increase in adhesion of AJAP1 KD HMEpiCs to the fibronectin matrix at the 24-hour time point versus control HMEpiCs. Cell-matrix adhesion did not differ between control and AJAP1 KD HMEpiCs on the collagen IV matrix.
Discussion: Our data reveal a new role for AJAP1 in regulating epithelial cell migration through appropriate maintenance of cell-matrix adhesion. The impact of AJAP1 on this epithelial cell behavior may be crucial during formation of the epicardium and EPDC differentiation during mammalian cardiac development.
Embargo Period
5-23-2024
AJAP1 regulates epithelial cell adhesion required for epicardium and EPDC formation
Philadelphia, PA
Introduction: The epicardium participates in cardiovascular development through its contribution of multipotent epicardial-derived cells (EPDCs). During cardiovascular development, cells delaminate from the epicardium to form EPDCs that undergo epithelial-to-mesenchymal transition and have the capacity to differentiate into multiple cardiac lineages. This makes epicardial cells an ideal progenitor source for cardiac tissue regeneration. It is vital to identify molecules in epicardial cells that regulate behaviors required for its formation and differentiation into multipotent EPDCs. Previously, our lab identified AJAP1 as a novel cardiac transcript in the Tbx5-deficient mouse model of defective epicardium formation (Tbx5epi-/-). AJAP1 encodes the adherens junction associated protein 1 cell adhesion molecule. Tbx5epi-/- embryonic mouse hearts exhibit a delayed adhesion of epicardial cells to the myocardium, impaired development of coronary vessels, and reduced AJAP1 mRNA expression. We have shown that AJAP1 is expressed in epithelial cells of the epicardium in the developing mouse heart. In vitro knockdown of AJAP1 in primary human epithelial cells inhibits cell migration. Despite these findings, a role for AJAP1 in cardiovascular development has not been identified.
Methods: Given that AJAP1 is expressed in epithelial cells of the mammalian heart, we sought to determine the functions of AJAP1 in the HMEpiC primary human mammary epithelial cell line. We silenced endogenous AJAP1 expression with small interfering RNAs (siRNA) designed to target human AJAP1. HMEpiCs were transfected with AJAP1 siRNAs or a non-targeting control siRNA. Silencing of AJAP1 expression was confirmed by quantitative PCR and immunofluorescence. We investigated the impact of AJAP1 on cell-to-extracellular matrix (ECM) adhesion by conducting cell adhesion assays with these siRNA-transfected cells. 6x104 control or AJAP1 knockdown (KD) HMEpiCs were plated on collagen I, collagen IV, laminin, or fibronectin matrices. These ECMS were chosen for the cell-matrix adhesion assays because they represent matrices that are abundant during mammalian cardiovascular development. Cells were allowed to adhere to the ECM for 1, 4 or 24 hours. At each respective time point, cell adhesion was stopped by methanol fixation of cells. Adherent cells were stained with crystal violet solution, and several high-powered fields were imaged on a microscope to quantify cell-matrix adhesion.
Results: We analyzed cell-ECM matrix adhesion in control versus AJAP1 KD HMEpiCs and observed significant differences in cell-matrix adhesion. In comparison to control HMEpiCs, we observed a significant 1.8-fold increase in adhesion of AJAP1 KD HMEpiCs to collagen I matrix at the 24-hour time point. On the laminin matrix, we observed a 1.2-fold increase in adhesion of AJAP1 KD HMEpiCs versus control HMEpiCs at the 4-hour time point. We detected a 1.6-fold increase in adhesion of AJAP1 KD HMEpiCs to the fibronectin matrix at the 24-hour time point versus control HMEpiCs. Cell-matrix adhesion did not differ between control and AJAP1 KD HMEpiCs on the collagen IV matrix.
Discussion: Our data reveal a new role for AJAP1 in regulating epithelial cell migration through appropriate maintenance of cell-matrix adhesion. The impact of AJAP1 on this epithelial cell behavior may be crucial during formation of the epicardium and EPDC differentiation during mammalian cardiac development.