Cystic Fibrosis Pathogens Cystic Fibrosis Pathogens Activate CaActivate Ca2+ 2+ -dependent mitogen--dependent mitogen-

activated Protein Kinase activated Protein Kinase Signaling Pathways in Airway Signaling Pathways in Airway

Epithelial CellsEpithelial Cells

by Aubrey Osborne by Aubrey Osborne

and Freda Youngand Freda Young

BackgroundBackground

Cystic fibrosis (CF) is an autosomal recessive Cystic fibrosis (CF) is an autosomal recessive disease that primarily affects the airway disease that primarily affects the airway epithelium. epithelium.

Caused by chronic infection leading to Caused by chronic infection leading to inflammation.inflammation.

People with CF have a short life expectancy People with CF have a short life expectancy (approx. 32 years).(approx. 32 years).

Disease symptoms are caused by over 750 Disease symptoms are caused by over 750 different mutations in the cystic fibrosis different mutations in the cystic fibrosis transmembrane receptor (CFTR).transmembrane receptor (CFTR).

Most current treatments target symptoms, Most current treatments target symptoms, prevention of further infection, and increasing prevention of further infection, and increasing stamina. Gene and protein repair therapies stamina. Gene and protein repair therapies are in the experimental stages.are in the experimental stages.

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GoalsGoals

The major goal of this study was to The major goal of this study was to identify the molecular mechanisms identify the molecular mechanisms that lead to inflammation in the airway that lead to inflammation in the airway epithelial cells.epithelial cells.

Ratner et al. sought to determine how Ratner et al. sought to determine how the two major pathogens, the two major pathogens, Pseudomonas aeruginosa Pseudomonas aeruginosa and and Staphylococcus aureusStaphylococcus aureus, are involved in , are involved in the symptoms of CF.the symptoms of CF.

HypothesisHypothesis

Respiratory cells are known to induce an IL-8 Respiratory cells are known to induce an IL-8 response when asialylated glycolipid receptors response when asialylated glycolipid receptors are stimulated.are stimulated.

Other pathogenic species are known to induce Other pathogenic species are known to induce CaCa2+2+ concentration increases which activate concentration increases which activate gene transcription via NF-kB in response to gene transcription via NF-kB in response to pilin binding.pilin binding.

Ratner et al. hypothesize that Ratner et al. hypothesize that S. aureusS. aureus and and P. P. aeruginosa (aeruginosa (the major pathogens in CF) bind the major pathogens in CF) bind to an asialylated glycolipid receptor and to an asialylated glycolipid receptor and induce a Cainduce a Ca2+2+ mediated response via some mediated response via some MAP kinase cascade. They hypothesize that MAP kinase cascade. They hypothesize that NF-kB is also involved in IL-8 gene expression.NF-kB is also involved in IL-8 gene expression.

Major QuestionsMajor Questions

1.1. Is IL-8 production a direct effect of Is IL-8 production a direct effect of CaCa2+2+ concentration increases?concentration increases?

2.2. Is CaIs Ca2+2+ fluctuation alone, independent fluctuation alone, independent of receptor stimulation, enough to of receptor stimulation, enough to induce an IL-8 response?induce an IL-8 response?

3.3. Do Do S. aureusS. aureus and and P. aeruginosa P. aeruginosa stimulate inflammatory responses via stimulate inflammatory responses via the same pathway? If so, what are the same pathway? If so, what are the major signaling cascades utilized?the major signaling cascades utilized?

4.4. Does stimulation of the receptor Does stimulation of the receptor activate NF-kB?activate NF-kB?

Ca2+ Conc. Increase

MAP kinase cascade

NF-kB

IL-8

Ligand

asialoGM1 receptor

Pathway

Evidence of CaEvidence of Ca2+2+ IncreasesIncreases

Addition of Addition of P. P. aeruginosaaeruginosa

Calcium imaging Calcium imaging of monolayers of of monolayers of airway epithelial airway epithelial cellscells

Baseline Calcium Levels

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 Is IL-8 production a direct effect of Caeffect of Ca2+2+ concentration concentration increases?increases?

BAPTA BAPTA (intracellular Ca(intracellular Ca2+ 2+

chelator) results in chelator) results in decreased IL-8 decreased IL-8 productionproduction

NiClNiCl22 and EGTA and EGTA (external Ca(external Ca2+ 2+

chelators) did not chelators) did not alter IL-8 alter IL-8 expressionexpression

Conclusion: Increases in intracellular Ca2+ concentrations cause increased IL-8 expression.

Con

trol

EG

TA

BA

PTA

NiCl2

AreAre CaCa2+ 2+ fluxes alone enough to fluxes alone enough to evoke a IL-8 response? evoke a IL-8 response?

Transiently increase

intracellular Ca2+concentration

s

Conclusion: Intracellular Ca2+ concentration fluxes evoke IL-8 response with and without receptor stimulation.

Do Do S. aureusS. aureus and and P. aeruginosa P. aeruginosa stimulate stimulate inflammatory responses via the same pathway? inflammatory responses via the same pathway? If so, what are the major signaling cascades If so, what are the major signaling cascades utilized?utilized?

ERK activation via receptor stimulation and Calcium increase

ERK

Control

P38 pathway activated by both pathogens

P.aeurginosa

S. aureus

P.aeurginosa

S. aureus

ERK pathway activated by both pathogens

ERK

Control

ERK

Control

MA

PK

/ER

K

inh

ibit

or

p3

8 k

inase

inh

ibit

or

MA

PK

/ERK

in

hib

itor

MA

PK

/ER

K in

hib

itor

p3

8 k

inase

inh

ibit

or

p3

8 k

inase

in

hib

itor

Con

trol

Con

trol

Con

trol

Anti-aGM1 P. aeruginosa

S. aureus

Do Do S. aureusS. aureus and and P. aeruginosa P. aeruginosa stimulate inflammatory responses via stimulate inflammatory responses via the same pathway? If so, what are the same pathway? If so, what are the major signaling cascades utilized? the major signaling cascades utilized? (cont.)(cont.)

Activation of MAP/ERK pathway is inhibited most in the P. aeruginosa-stimulated cell.

Activation of p38 pathway is inhibited most in the S. aureus- stimulated cell.

Results from enzyme-linked immunosorbent assays (ELISA)

Does stimulation of the Does stimulation of the receptor activate NF-kB?receptor activate NF-kB?

Conclusion: Yes, based on results from luciferase reporter construct.

Receptor activation

yields increased

NF-kB levels

Intracellular Ca2+ inhibitor decreased NF-kB levels

ConclusionsConclusions1.Is IL-8 production a direct effect of Ca2+ 1.Is IL-8 production a direct effect of Ca2+

concentration increases? concentration increases? Yes.Yes.

2.Is Ca2+ fluctuation alone, independent of 2.Is Ca2+ fluctuation alone, independent of receptor stimulation, enough to induce an receptor stimulation, enough to induce an IL-8 response? IL-8 response? Yes.Yes.

3.Do 3.Do S. aureusS. aureus and and P. aeruginosa P. aeruginosa stimulate stimulate inflammatory responses via the same inflammatory responses via the same pathway? pathway? YesYes If so, what are the major If so, what are the major signaling cascades utilized? signaling cascades utilized? MAP/ERK and MAP/ERK and p38p38

4.Does stimulation of the receptor activate 4.Does stimulation of the receptor activate NF-kB? NF-kB? Yes.Yes.

Conclusions (cont.)Conclusions (cont.)

Normally, aGM1 receptors are expressed in very low Normally, aGM1 receptors are expressed in very low levels. levels.

Expression is vastly increase in damaged and Expression is vastly increase in damaged and regenerating cells as well as in the cells of those with regenerating cells as well as in the cells of those with the CFTR mutation that causes CF.the CFTR mutation that causes CF.

The inflammation caused by the activation of the The inflammation caused by the activation of the aGM1 receptor likely involves redundancy of pathways aGM1 receptor likely involves redundancy of pathways and cross-talk between pathways. and cross-talk between pathways.

It has been suggested that proximal activation of this It has been suggested that proximal activation of this pathway may use a G-protein dependent mechanism pathway may use a G-protein dependent mechanism using IP3.using IP3.

Parts of this study suggest that proteolysis-dependent Parts of this study suggest that proteolysis-dependent IkB pathways may be responsible for activation of NF-IkB pathways may be responsible for activation of NF-kB. kB.

The complexity of the known pathways and the The complexity of the known pathways and the unknown portions of the pathways show why unknown portions of the pathways show why treatment options for CF are limited.treatment options for CF are limited.

ReferencesReferences

Ratner et al. (2001). Cystic Fibrosis Pathogens Ratner et al. (2001). Cystic Fibrosis Pathogens Activate CaActivate Ca2+ 2+ -dependent mitogen-activated Protein -dependent mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells. J. Kinase Signaling Pathways in Airway Epithelial Cells. J. of Biological Chem. 276(22): 19267-19275.of Biological Chem. 276(22): 19267-19275.

Cystic Fibrosis. (2005). <www.webmd.com>Cystic Fibrosis. (2005). <www.webmd.com>