by Aubrey Osborne and Freda Young

1. Cystic Fibrosis Pathogens Activate Ca2+ -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. • 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. 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. • 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. • 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.

5. Major Questions • 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?

6. Pathway Ligand asialoGM1 receptor Ca2+ Conc. Increase MAP kinase cascade NF-kB IL-8

7. Evidence of Ca2+ Increases Baseline Calcium Levels • Addition of P. aeruginosa • Calcium imaging of monolayers of airway epithelial cells 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 Ca2+ concentration increases? • BAPTA (intracellular Ca2+ chelator) results in decreased IL-8 production • NiCl2 and EGTA (external Ca2+ chelators) did not alter IL-8 expression NiCl2 Conclusion: Increases in intracellular Ca2+ concentrations cause increased IL-8 expression. BAPTA EGTA Control

10. AreCa2+ 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

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 ERK P.aeurginosa Control Control ERK S. aureus Control P38 pathway activated by both pathogens P.aeurginosa ERK pathway activated by both pathogens S. aureus

12. 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. S. aureus Anti-aGM1 P. aeruginosa Control MAPK/ERK inhibitor MAPK/ERK inhibitor p38 kinaseinhibitor p38 kinaseinhibitor Control Control MAPK/ERK inhibitor p38 kinase inhibitor Results from enzyme-linked immunosorbent assays (ELISA)

13. 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.

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. • 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.

16. References • 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>