1. Cystic Fibrosis Pathogens Activate Ca 2+ -dependent mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells by Aubrey Osborne and Freda Young

2. Background Cystic fibrosis (CF) is an autosomal recessive disease that primarily affects the airway epithelium. Cystic fibrosis (CF) is an autosomal recessive disease that primarily affects the airway epithelium. Caused by chronic infection leading to inflammation. Caused by chronic infection leading to inflammation. People with CF have a short life expectancy (approx. 32 years). 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). 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. Most current treatments target symptoms, prevention of further infection, and increasing stamina. Gene and protein repair therapies are in the experimental stages. www.webmd.com

3. Goals The major goal of this study was to identify the molecular mechanisms that lead to inflammation in the airway epithelial cells. 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. Ratner et al. sought to determine how the two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, are involved in the symptoms of CF.

4. Hypothesis Respiratory cells are known to induce an IL-8 response when asialylated glycolipid receptors are stimulated. Respiratory cells are known to induce an IL-8 response when asialylated glycolipid receptors are stimulated. Other pathogenic species are known to induce Ca 2+ concentration increases which activate gene transcription via NF-kB in response to pilin binding. Other pathogenic species are known to induce Ca 2+ 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 Ca 2+ mediated response via some MAP kinase cascade. They hypothesize that NF-kB is also involved in IL-8 gene expression. Ratner et al. hypothesize that S. aureus and P. aeruginosa (the major pathogens in CF) bind to an asialylated glycolipid receptor and induce a Ca 2+ mediated response via some MAP kinase cascade. They hypothesize that NF-kB is also involved in IL-8 gene expression.

5. Major Questions 1. Is IL-8 production a direct effect of Ca 2+ concentration increases? 2. Is Ca 2+ fluctuation alone, independent of receptor stimulation, enough to induce an IL-8 response? 3. Do S. aureus and P. aeruginosa stimulate inflammatory responses via the same pathway? If so, what are the major signaling cascades utilized? 4. Does stimulation of the receptor activate NF-kB?

6. Ca 2+ Conc. Increase MAP kinase cascade NF-kB IL-8 Ligand asialoGM1 receptor Pathway

7. Evidence of Ca 2+ Increases Addition of P. aeruginosa Addition of P. aeruginosa Calcium imaging of monolayers of airway epithelial cells Calcium imaging of monolayers of airway epithelial cells Baseline Calcium Levels Increased Calcium Levels

8. Conclusions: -asialoGM1 is the receptor -pilin is the portion of the bacteria that serves as the ligand -calcium increases upon activation of the receptor

9. Is IL-8 production a direct effect of Ca 2+ concentration increases? BAPTA (intracellular Ca 2+ chelator) results in decreased IL-8 production BAPTA (intracellular Ca 2+ chelator) results in decreased IL-8 production NiCl 2 and EGTA (external Ca 2+ chelators) did not alter IL-8 expression NiCl 2 and EGTA (external Ca 2+ chelators) did not alter IL-8 expression Conclusion: Increases in intracellular Ca 2+ concentrations cause increased IL- 8 expression. Control EGTA BAPTA NiCl 2

10. Are Ca 2+ fluxes alone enough to evoke a IL-8 response? Transiently increase intracellular Ca 2+ concentrations Conclusion: Intracellular Ca 2+ concentration fluxes evoke IL-8 response with and without receptor stimulation.

11. 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 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

12. MAPK/ERK inhibitor p38 kinase inhibitor MAPK/ER K inhibitor p38 kinase inhibitor Control Anti-aGM1P. aeruginosa S. aureus Do S. aureus and P. aeruginosa stimulate inflammatory responses via the same pathway? If so, what are the major signaling cascades utilized? (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)

13. Does stimulation of the receptor activate NF-kB? Conclusion: Yes, based on results from luciferase reporter construct. Receptor activation yields increased NF-kB levels Intracellular Ca 2+ inhibitor decreased NF-kB levels

14. 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.

15. Conclusions (cont.) Normally, aGM1 receptors are expressed in very low levels. 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. 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. 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. 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. 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. The complexity of the known pathways and the unknown portions of the pathways show why treatment options for CF are limited.

16. References Ratner et al. (2001). Cystic Fibrosis Pathogens Activate Ca 2+ - dependent mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells. J. of Biological Chem. 276(22): 19267-19275. Ratner et al. (2001). Cystic Fibrosis Pathogens Activate Ca 2+ - dependent mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells. J. of Biological Chem. 276(22): 19267-19275. Cystic Fibrosis. (2005). Cystic Fibrosis. (2005).