AJAP1 enhances apoptosis in epithelial cells to potentially impact 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 forms the outermost cell layer of the heart. It is composed of a single layer of specialized epithelial cells that make significant cellular contributions to cardiovascular development. During embryonic development, cells delaminate from the epicardium and give rise to multipotent epicardial-derived cells (EPDCs), which undergo epithelial-to-mesenchymal transition and differentiate into non-myocyte cardiac lineages. The epicardium is also a source of paracrine signals essential for fetal cardiac growth, coronary vessel patterning, and cardiac tissue regeneration. Therefore, it is essential to identify the molecular signature of the epicardium for regulating behaviors required for its formation and EPDC differentiation. Our lab identified adherens junction associated protein 1 (AJAP1) as a novel cardiac transcript in the Tbx5-deficient mouse model (Tbx5epi-/-) which is characterized by defective epicardium formation, impaired coronary vessel development, and reduced AJAP1 expression. Previously, we observed AJAP1 expression in the epithelial-like cells of the embryonic mouse heart epicardium. This prompted us to explore the impact of AJAP1 on epithelial cell behaviors that are required for epicardium formation and EPDC differentiation.
Methods: The goal of this study is to determine how AJAP1 regulates epithelial cell behaviors, including cell proliferation and apoptosis, that are necessary for epicardium formation and EPDC differentiation. We conducted experiments on the HMEpiC primary human epithelial cell line with endogenous AJAP1 expression. 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. Knockdown of AJAP1 expression was confirmed by quantitative PCR and immunofluorescence. We investigated the effect of AJAP1 on cell proliferation and apoptosis. To measure the proliferation of our cells, we plated 3.2x102 – 3.2x104 control or AJAP1 KD HMEpiCs in 96-well plates for 48 hours prior to performing the MTS Cell Proliferation Assay. To investigate apoptosis in our cell population, we transfected 1.5x105 HMEpiCs per well on glass coverslips for 96 hours prior to paraformaldehyde fixation and permeabilization of control or AJAP1 KD HMEpiCs. We used the In Situ Cell Death Detection Kit to conduct a TUNEL assay on these cells to detect apoptosis via immunofluorescence. TUNEL-positive cells were viewed and quantified within a series of high-powered fields under a fluorescent microscope Also, cells were counterstained with DAPI to visualize the nuclei in all cells.
Results: Initially, we observed morphological changes in AJAP1 KD HMEpiCs that included a reduction in cell density, nuclear fragmentation, and membrane blebbing that was not observed in control HMEpiCs. Our TUNEL assay revealed a significant 2.1-fold increase in apoptotic cells in AJAP1 KD HMEpiCs versus control HMEpiCs. No differences in cell proliferation were observed between control and AJAP1 KD HMEpiCs.
Discussion: Our data reveal a novel role for AJAP1 in potentially regulating epicardium formation and EPDC differentiation through appropriate maintenance of epithelial cell apoptosis. This evidence further supports an impact of AJAP1 on epithelial cell behaviors that may be crucial during formation of the epicardium and EPDC differentiation during mammalian cardiac development.
Embargo Period
5-23-2024
AJAP1 enhances apoptosis in epithelial cells to potentially impact epicardium and EPDC formation
Philadelphia, PA
Introduction: The epicardium forms the outermost cell layer of the heart. It is composed of a single layer of specialized epithelial cells that make significant cellular contributions to cardiovascular development. During embryonic development, cells delaminate from the epicardium and give rise to multipotent epicardial-derived cells (EPDCs), which undergo epithelial-to-mesenchymal transition and differentiate into non-myocyte cardiac lineages. The epicardium is also a source of paracrine signals essential for fetal cardiac growth, coronary vessel patterning, and cardiac tissue regeneration. Therefore, it is essential to identify the molecular signature of the epicardium for regulating behaviors required for its formation and EPDC differentiation. Our lab identified adherens junction associated protein 1 (AJAP1) as a novel cardiac transcript in the Tbx5-deficient mouse model (Tbx5epi-/-) which is characterized by defective epicardium formation, impaired coronary vessel development, and reduced AJAP1 expression. Previously, we observed AJAP1 expression in the epithelial-like cells of the embryonic mouse heart epicardium. This prompted us to explore the impact of AJAP1 on epithelial cell behaviors that are required for epicardium formation and EPDC differentiation.
Methods: The goal of this study is to determine how AJAP1 regulates epithelial cell behaviors, including cell proliferation and apoptosis, that are necessary for epicardium formation and EPDC differentiation. We conducted experiments on the HMEpiC primary human epithelial cell line with endogenous AJAP1 expression. 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. Knockdown of AJAP1 expression was confirmed by quantitative PCR and immunofluorescence. We investigated the effect of AJAP1 on cell proliferation and apoptosis. To measure the proliferation of our cells, we plated 3.2x102 – 3.2x104 control or AJAP1 KD HMEpiCs in 96-well plates for 48 hours prior to performing the MTS Cell Proliferation Assay. To investigate apoptosis in our cell population, we transfected 1.5x105 HMEpiCs per well on glass coverslips for 96 hours prior to paraformaldehyde fixation and permeabilization of control or AJAP1 KD HMEpiCs. We used the In Situ Cell Death Detection Kit to conduct a TUNEL assay on these cells to detect apoptosis via immunofluorescence. TUNEL-positive cells were viewed and quantified within a series of high-powered fields under a fluorescent microscope Also, cells were counterstained with DAPI to visualize the nuclei in all cells.
Results: Initially, we observed morphological changes in AJAP1 KD HMEpiCs that included a reduction in cell density, nuclear fragmentation, and membrane blebbing that was not observed in control HMEpiCs. Our TUNEL assay revealed a significant 2.1-fold increase in apoptotic cells in AJAP1 KD HMEpiCs versus control HMEpiCs. No differences in cell proliferation were observed between control and AJAP1 KD HMEpiCs.
Discussion: Our data reveal a novel role for AJAP1 in potentially regulating epicardium formation and EPDC differentiation through appropriate maintenance of epithelial cell apoptosis. This evidence further supports an impact of AJAP1 on epithelial cell behaviors that may be crucial during formation of the epicardium and EPDC differentiation during mammalian cardiac development.