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Cystic Fibrosis Pathogens Activate
Ca2+ -dependent mitogen-activated
Protein Kinase Signaling Pathways in
Airway Epithelial Cells
by Aubrey Osborne
and Freda Young
Background
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Cystic fibrosis (CF) is an autosomal recessive
disease that primarily affects the airway epithelium.
Caused by chronic infection leading to
inflammation.
People with CF have a short life expectancy
(approx. 32 years).
Disease symptoms are caused by over 750 different
mutations in the cystic fibrosis transmembrane
receptor (CFTR).
Most current treatments target symptoms,
prevention of further infection, and increasing
stamina. Gene and protein repair therapies are in
the experimental stages.
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Goals
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The major goal of this study was to identify
the molecular mechanisms that lead to
inflammation in the airway epithelial cells.
Ratner et al. sought to determine how the
two major pathogens, Pseudomonas
aeruginosa and Staphylococcus aureus, are
involved in the symptoms of CF.
Hypothesis
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Respiratory cells are known to induce an IL-8
response when asialylated glycolipid receptors are
stimulated.
Other pathogenic species are known to induce Ca2+
concentration increases which activate gene
transcription via NF-kB in response to pilin binding.
Ratner et al. hypothesize that S. aureus and P.
aeruginosa (the major pathogens in CF) bind to an
asialylated glycolipid receptor and induce a Ca2+
mediated response via some MAP kinase cascade.
They hypothesize that NF-kB is also involved in IL-8
gene expression.
Major Questions
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4.
Is IL-8 production a direct effect of Ca2+
concentration increases?
Is Ca2+ fluctuation alone, independent of
receptor stimulation, enough to induce an
IL-8 response?
Do S. aureus and P. aeruginosa stimulate
inflammatory responses via the same
pathway? If so, what are the major
signaling cascades utilized?
Does stimulation of the receptor activate
NF-kB?
Pathway
Ligand
asialoGM1 receptor
Ca2+ Conc. Increase
MAP kinase cascade
NF-kB
IL-8
Evidence of Ca2+ Increases
Baseline Calcium Levels
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Addition of P.
aeruginosa
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Calcium imaging of
monolayers of
airway epithelial
cells
Increased Calcium Levels
Conclusions:
-asialoGM1 is
the receptor
-pilin is the
portion of the
bacteria that
serves as the
ligand
-calcium
increases upon
activation of
the receptor
Is IL-8 production a direct effect of
Ca2+ concentration increases?
BAPTA
EGTA
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BAPTA (intracellular
Ca2+ chelator)
results in decreased
IL-8 production
NiCl2 and EGTA
(external Ca2+
chelators) did not
alter IL-8
NiCl
Conclusion:
Increases in intracellular
2
Ca2+ concentrations cause increased ILexpression
Control
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8 expression.
Are Ca2+ fluxes alone enough to
evoke a IL-8 response?
Conclusion:
Intracellular Ca2+
concentration
fluxes evoke IL-8
response with and
without receptor
stimulation.
Transiently increase
intracellular
Ca2+concentrations
Do S. aureus and P. aeruginosa stimulate
inflammatory responses via the same pathway? If so,
what are the major signaling cascades utilized?
ERK activation via
receptor stimulation and
Calcium increase
ERK
P.aeurginosa
ERK
Control
Control
S. aureus
ERK
Control
P.aeurginosa
S. aureus
P38 pathway
activated by
both pathogens
ERK pathway
activated by both
pathogens
Do S. aureus and P. aeruginosa stimulate
inflammatory responses via the same
pathway? If so, what are the major
signaling cascades utilized? (cont.)
Control
p38 kinase
inhibitor
inhibitor
MAPK/ERK
Results from enzyme-linked immunosorbent assays (ELISA)
p38 kinase inhibitor
S. aureus
P. aeruginosa
Control
p38 kinase inhibitor
MAPK/ERK
inhibitor
Control
Anti-aGM1
Activation of p38
pathway is inhibited
most in the S. aureusstimulated cell.
MAPK/ERK inhibitor
Activation of
MAP/ERK pathway is
inhibited most in the
P. aeruginosastimulated cell.
Does stimulation of the
receptor activate NF-kB?
Intracellular Ca2+ inhibitor
decreased NF-kB levels
Receptor
activation
yields
increased
NF-kB levels
Conclusion: Yes, based on
results from luciferase
reporter construct.
Conclusions
1.Is IL-8 production a direct effect of Ca2+
concentration increases? Yes.
2.Is Ca2+ fluctuation alone, independent of
receptor stimulation, enough to induce an IL-8
response? Yes.
3.Do S. aureus and P. aeruginosa stimulate
inflammatory responses via the same pathway?
Yes If so, what are the major signaling cascades
utilized? MAP/ERK and p38
4.Does stimulation of the receptor activate NF-kB?
Yes.
Conclusions (cont.)
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Normally, aGM1 receptors are expressed in very low levels.
Expression is vastly increase in damaged and regenerating
cells as well as in the cells of those with the CFTR mutation
that causes CF.
The inflammation caused by the activation of the aGM1
receptor likely involves redundancy of pathways and cross-talk
between pathways.
It has been suggested that proximal activation of this pathway
may use a G-protein dependent mechanism using IP3.
Parts of this study suggest that proteolysis-dependent IkB
pathways may be responsible for activation of NF-kB.
The complexity of the known pathways and the unknown
portions of the pathways show why treatment options for CF
are limited.
References
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Ratner et al. (2001). Cystic Fibrosis Pathogens Activate Ca2+ dependent mitogen-activated Protein Kinase Signaling
Pathways in Airway Epithelial Cells. J. of Biological Chem.
276(22): 19267-19275.
Cystic Fibrosis. (2005). <www.webmd.com>