Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptor Open Access
Wentworth, Christy Colleen (2011)
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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