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Lab Focus

Lab Focus. Characterizing G protein-coupled receptor signaling and function in primary airway cells Relevant modulators include: a) putative pathogenic mediators b) therapies c) disease state d) genetic variation. Systems.

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Lab Focus

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  1. Lab Focus • Characterizing G protein-coupled receptor signaling and function in primary airway cells • Relevant modulators include: a) putative pathogenic mediators b) therapies c) disease state d) genetic variation

  2. Systems • Primary cell cultures (airway smooth muscle, fibroblasts, epithelial cells, T cells) • Murine and human airways • Coordination with in vivo measurements and clinical profile • more artificial cell lines as expression systems

  3. Endpoints • Signal transduction pathway delineation • Receptor modifications, protein trafficking • Protein induction, autocrine/paracrine factor production/secretion • Transcriptional regulation • Bioassays

  4. Endpoints • Mesenchymal cells growth • T cell proliferation and maturation • Chemotaxis/migration • ASM/airway contraction ex vivo • Ex vivo human (e.g., HAE) • In vivo mouse (inflammation, airway RL) • Pharmacologic screens

  5. Effects of inhaled beta-agonist, segmental antigen challenge on acutely dissociated airway epithelia Penn et al. AJP Lung 1996

  6. 110 kD 90 kD 50 kD 35 kD A B Flag M1 Ab Anti-CysLT1R Ab 110 kD 90 kD 50 kD 35 kD COS pcDNA3 HEK 293 pcDNA3 COS FLAG-CysLT1R HEK 293 FLAG-CysLT1R COS pcDNA3 HEK 293 pcDNA3 COS FLAG-CysLT1R HEK 293 FLAG-CysLT1R C

  7. A B C D

  8. PKC exerts negative feedback regulation of CysLT1R function in ASM at the receptor locus

  9. GFP A PKI-GFP RevAB-GFP GFP PKI-GFP RevAB-GFP GFP B Relative cell count GFP GFP GFP

  10. D E (↓28%) (↓33%) (↓36%) (↓41%) (↓76%) (↓84%) (↓23%) (↓23%) (↓27%) (↓31%) (↓34%) (↓56%)

  11. A B C

  12. 100 100 100 101 101 101 102 102 102 103 103 103 104 Vehicle 10mM ISO 100nM ISO VASP-phospho-Ser157 (FITC/AL488) 10mM ISO 100nM ISO Phospho-VASP, % inhibition

  13. 100 100 100 100 100 100 101 101 101 101 101 101 102 102 102 102 102 102 103 103 103 103 103 103 104 104 104 104 104 104 CRTH2- CRTH2+ CD56- CD56+ PGE2 PGE2 PGE2 PGE2 ISO ISO ISO ISO VASP-phospho-Ser157 (FITC/AL488) VASP-phospho-Ser157 (FITC) Vehicle Vehicle PGE2 or ISO PGE2 or ISO Isotype control IL-13 mAb 65% IL-13 (PE) Vehicle ISO db-cAMP VASP-phospho-Ser157 (FITC/AL488)

  14. Current Projects

  15. I. GPCR signaling and function in ASM Goal: Demonstrate PKA is both the major effector AND feedback regulator of ASM beta-2-adrenoceptors (b2ARs)

  16. Figure 11. Aim 3 strategy for assessing regulation of mechanisms affecting ASM contraction by global and compartmentalized cAMP/PKA/PDE.

  17. II. Arrestin function in ASM Goal: Demonstrate what roles arrestin isoforms play in desensitizing beta-2-adrenoceptors in ASM

  18. PLC AC Ca2+ PKA CONTRACTION PKA CONTRACTION Ca2+ ↑↑# active receptors barr-1 or barr-2 inhibition # active receptors

  19. A B WT barr1 (rat) barr2 (rat) βarr2-KO 60 kDa C D

  20. A B MCh KCl

  21. III. OGR1, a novel GPCR that responds to low pH in ASM and capable of mediating contraction or relaxation Goal: Figure out how and find small molecules that function like agonists

  22. Proton-sensing GPCRs may represent another mechanisms mediating effects of more subtle decreases in airway pH

  23. IV. “Optimizing beta-adrenoceptor signaling bias in asthma.” Goal: Determine the good and bad of b2AR signaling in the lung and find the better beta-agonist (antagonist?)

  24. Integrative studies by the Bond suggest a bAR paradox in asthma • Chronic treatment with “beta-blocker” nadolol deters allergen-induced murine asthma • b2AR-/- mice fail to develop asthma phenotype after OVA challenge/sensitization • Taking out systemic epinephrine also deters development of murine asthma

  25. Effect of ß2-AR agonists and antagonists on mucous metaplasia S/C Vehicle Control S/C Nadolol S/C Albuterol Am J Respir Cell Mol Biol. 38: 256-262, 2008.

  26. Summary of Chronic B2AR Drug Effects All parameters were significantly (P<0.05) when comparing nadolol vs salbutamol *Also different (P<0.05) compared to S/C NTX

  27. Effect of Sensitization and Challenge on Mucous Metaplasia In Wild Type and Beta-2 AR Null Mice CONTROL S/C – Vehicle S/C – Nad 28d FVB/N 2-AR (-/-) FVB/N Nguyen et al., Proc. Nat. Acad. Sci. 101: 4948 – 4953, 2009.

  28. Human Trials: Effect of Chronic Nadolol on MCh PC20 Hanania et al., Pulm Pharmacol Ther. 21: 134-141, 2008

  29. Today: “Bias”

  30. Typical Fellow Research Experience in Penn lab • Show up or else • Work hard, play hard • Understand scientific question to be addressed • Master select techniques • Generate data for publication • Present data at ATS, publish

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