Desmosomal Cadherin Cleavage in the Regulation of Apoptosis in the Context of Inflammation Público
Yulis, Mark (Spring 2018)
Abstract
The regulation of apoptosis sensitivity is critical to the maintenance of physiological homeostasis and dysregulation of apoptosis is known to be involved in the pathobiology or progression of many disease states. The single pass, transmembrane proteins of the cadherin family have been appreciated as important proteins that regulate intercellular adhesion. In addition to this critical function, cadherins contribute to important signaling events that control cellular homeostasis. Much of the work on cadherin mediated signaling focuses on classical cadherins or on specific disease states such as pemphigus vulgaris. Cadherin mediated signaling has been shown to play critical roles during development, in proliferation, apoptosis, disease pathobiology and beyond. We have previously reported that select pro-inflammatory cytokines induce ectodomain cleavage of the desmosomal cadherin desmoglein-2 (Dsg2) and shedding of the resulting extracellular fragment from intestinal epithelial cells (IECs). The Dsg2 extracellular cleaved fragment (Dsg2 ECF) functions to induce paracrine pro-proliferative signaling in epithelial cells. The goal of this study was to further understand the role of desmoglein cleavage in the regulation of enterocyte homeostasis. To address this, we focused on characterizing Dsg2 intracellular cleavage and the functional effects of the resultant intracellular cleaved fragment. In this study, we showed that exposure of IECs to pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) resulted in Dsg2 intracellular cleavage and generation of a ~55kDa fragment (Dsg2 ICF). Dsg2 intracellular cleavage is mediated by caspase-8 and occurs prior to Dsg2 extracellular cleavage and the execution of apoptosis. Expression of exogenous Dsg2 ICF in model IECs increased sensitivity to apoptotic stimuli and apoptosis execution. Additionally, expression of the Dsg2 ICF repressed the anti-apoptotic Bcl-2 family member proteins Bcl-XL and Mcl1. Taken together, our findings identify a novel mechanism by which pro-inflammatory mediators induce cleavage of Dsg2 to activate apoptosis and eliminate damaged cells, while also promoting release of Dsg2 ECF that enhances proliferation of neighboring cells and epithelial barrier recovery. These findings expand our understanding of the signaling capabilities of desmosomal cadherins and provide a new pathway through which apoptosis can be altered in the context of intestinal inflammation.
Table of Contents
Chapter 1: Introduction 1
Mechanisms and pathways of apoptosis 2
Regulation of apoptosis sensitivity by the Bcl-2 family of proteins 6
Desmosomal junctions 7
Overview of Cadherins as Signaling Mediators 7
Cadherin mediated regulation of proliferation 8
Cadherin mediated modulation of apoptosis 11
Cadherin mediated control of cellular differentiation 12
Other signaling mediated by cadherins 14
Cadherin mediated signaling in the context of disease 16
Cadherin intracellular fragment mediated signaling 20
Cadherin signaling in cells lacking intercellular adhesion 21
The importance of epithelial apoptosis in Inflammatory
Bowel Disease (IBD) pathogenesis 22
Scope and significance of dissertation 24
Figures and table 26
Chapter 2: Desmosomal Cadherin Cleavage and Modulation of
Enterocyte Apoptosis in the Context of Inflammation 38
Introduction 39
Results 41
TNF-α and IFN-γ promote Dsg2 Intracellular Cleavage 41
Calpain Inhibition Leads to Accumulation of Active Caspase-8
and Promotes Dsg2 ICF Generation 43
Caspase-8 is Responsible for Generating the Dsg2 ICF 44
TRAIL promotes Dsg2 cleavage analogous to TNF-α and IFN-γ 45
Expression of the Dsg2 ICF sensitizes epithelial cells to
Apoptosis 45
Discussion 47
Figures 53
Experimental procedures 65
Chapter 3: Discussion 71
A model for Dsg2 cleavage as a beneficial signaling mechanism
during inflammation 73
Dependency of Dsg2 ECF generation on Dsg2 ICF generation 75
Mechanistic basis for Dsg2 ICF signaling 75
The implications of cadherin intracellular fragments as independent
signaling mediators 80
Additional functions of Dsg2 ICF signaling 81
The role of inflammation in Dsg2 cleavage 83
Dsg2 cleavage during intrinsic apoptosis 84
Placing the Dsg2 ICF and its signaling functions into the broader
context of cadherin mediated signaling 85
Final conclusions 87
References 88
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