Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptor Open Access

Wentworth, Christy Colleen (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/6w924c315?locale=en
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Abstract


Abstract

Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptors
By Christy Colleen Wentworth
Virtually all metazoan life exists in intimate contact with the prokaryotic kingdom, often
in a symbiotic manner. In mammals, commensal bacteria are known to facilitate many
homeostatic processes in the intestine, yet the molecular signaling mechanisms that
mediate these events are largely unknown. The studies described in this thesis directly
address this gap in our knowledge. We show that the bacterial specific peptide N-formyl-
Met-Leu-Phe (fMLF), a component of gut luminal bacteria, is recognized by formyl
peptides receptors (FPRs) located on the apical side of the intestinal epithelial cells.
Recognition of fMLF serves to rapidly and potently activate the extracellular related
kinase (ERK) MAPK signaling pathway in both cultured epithelial cells and the murine
colon, inducing cellular proliferation in these cells. Importantly, no fMLF-induced
activation of the pro-inflammatory NF-κB pathway, or the pro-apoptotic JNK pathway
was detected, thus indicating specificity for FPR mediated signaling events towards ERK
pathway mediated cellular responses. Furthermore, binding of fMLF by FPR induces
rapid generation of reactive oxygen species (ROS) in epithelial cells. Physiological levels
of ROS are known to function as signaling molecules that regulate protein activity by
oxidizing hyper-reactive sensor cysteine residues within a subset of proteins. We show
cellular ROS is rapidly induced by fMLF and is sufficient to oxidize a critical cysteine
residue within the ERK specific MAP kinase phosphatase, DUSP3, rendering the enzyme
catalytically inactive and thus unable to dephosphorylate and inhibit ERK pathway
activity. Collectively, these data show that bacterial fMLF induces homeostatic signaling
and responses in the intestinal epithelium by FPR dependant redox signaling.

Table of Contents



Table of Contents

Chapter 1. Introduction……………………………………………………….....1

Introduction Figures.........……………………………………….......21

Introduction References……………………………………………28



Chapter 2. Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptors..….39

Chapter 2 Figures......………………………………………………..63

Chapter 2 Supplemental Figures………………............……………71

Chapter 2 Figures...................................…………………………….72



Chapter 3. Enteric Commensal Bacteria Induce ERK via FPR Dependent Redox Modulation of DUSP3...78

Chapter 3 Figures………………......………………………………..99

Chapter 3 Supplemental Figures…….………………………………108

Chapter 3 References.............................………....109



Chapter 4. Conclusion…………………………………………………………...115

Conclusion Figures......………………………………………………122

Conclusion References........................................................................125



Appendix I. Salmonella AvrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade...127


Appendix I Figures..............................................................................157

Appendix I References........................................................................167


List of Figures

Chapter 1.
Introduction

Figure 1 Intestinal cells: apical and basolateral aspects....................................33
Figure 2 Diagram of bacterial load in the gut...................................................34
Figure 3 Structure of colonic crypts................................................................35
Figure 4 Structure of N-formyl-Met-Leu-Phe...................................................36
Figure 5 FPR signaling..................................................................................37
Figure 6 MAPK signaling................................................................................38


Chapter 2.
Commensal-epithelial signaling mediated via FPRs

Figure 1 Apically applied enteric bacteria induce ERK phosphorylation in polarized T84 epithelial cells....58
Figure 2 Apically applied L. rhamnosus GG or fMLF specifically activate ERK/Akt pathway signaling.......59
Figure 3 Inhibition of G-protein coupled receptors or formyl peptide receptors attenuates ERK phosphorylation in response to apical L. rhamnosus GG or fMLF stimulation...................................................60
Figure 4 Apical L. rhamnosus GG and fMLF induce phosphorylation of FPR...........61

Figure 5 L. rhamnosus GG or fMLF treatment of murine colon stimulates ERK activation...........62
Figure S1 Basolaterally applied fMLF weakly activates ERK pathway signaling.........................63


Chapter 3.
Commensal Induced ERK Redox Modulation of DUSP3

Figure 1 fMLF induces the generation of ROS in cultured epithelial cells in an FPR dependent manner......84
Figure 2 Dampening of cellular ROS levels attenuates L. rhamnosus GG or fMLF induced ERK pathway activation and cellular proliferation...85

Figure 3 L. rhamnosus GG or fMLF upregulate DUSP3 mRNA and protein....86

Figure 4 L. rhamnosus GG or fMLF- induced generation of ROS oxidizes DUSP3................87

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