(Applicant?s Abstract) In asthma and airway remodeling, cytokines can directly modulate myocyte fimction by amplifying and perpetuating inflammatory responses, and by inducing airway smooth muscle (ASM) growth. Although many studies have identified mechanisms that regulate inflammatory cell trafficking and activation in asthma, few have asserted a role for myocytes in modulating airway inflammation and none examined the critical signaling pathways that regulate ASM synthetic functions. The goal of this proposal is to define the cellular and molecular signaling processes that regulate cytokine-induced synthetic functions [defined as cell adhesion molecule (CAM) expression, and cytokine, chemokine, and autocrine growth factor secretion] in human ASM. Recent studies by the P.I?s laboratory support the role of IL-1beta and TNF-alpha in modulating synthetic functions in ASM. Preliminary data from this proposal now demonstrate that IL-1beta and TNF-alpha activate NF-kB, sphingomyelinase (SMase), and mitogen-activated protein kinase (MAPK) pathways in ASM, and that selective inhibition of these pathways has differential effects on the modulation of synthetic functions by cytokines. In addition, ASM express CAMs and RANTES after segmental allergen challenge (SAC) in asthmatics. Collectively, these findings support a central hypothesis that pro-inflammatory cytokines regulate CAM expression and cytokine, chemokine, and growth factor secretion through coordinated activation of NF-kB, SMase, and MAPK pathways. Recently established techniques for the transfection and microinjection of human ASM cells will enable delineation of the precise molecular mechanisms by which cytokines activate these pathways and regulate synthetic functions.
In Aim 1, the role of NF-kB will be explored by inhibiting or activating NF-kB through the use of dominant negative and constitutively active cDNA constructs and assessing attendant alterations in CAM expression, cytokine and autocrine growth factor secretion in cells stimulated with pro- inflammatory cytokines.
In Aim 2, the role of cytokine-induced sphingolipid generation in modulating synthetic functions will be investigated by characterizing the products of SMase in response to cytokine stimulation. In addition, direct stimulation or inhibition of SMase will be achieved via transfection or microinjection techniques to determine whether SMase activity is required or sufficient to mediate changes in ASM synthetic functions.
In Aim 3, the role of MAPK in modulating cytokine regulation of synthetic functions will be examined. Activation of the p42/p44, JNK, and p38 pathways will be assessed using both inununoblot analysis and in vitro kinase assays. Selective inhibition of the MAPK pathways using molecular and pharmacological means will help establish the requirement/sufficiency of a given MAPK pathway in promoting cytokine effects on ASM synthetic functions.
In Aim 4, synthetic function of ASM will be examined after SAC in asthmatics. Immunocytochemical staining of ASM from allergic asthmatics and normal subjects undergoing SAC will establish whether synthetic responses in ASM occur in vivo. Achievement of these aims will significantly advance our understanding of the pathogenesis of asthma and airway remodeling. By understanding the mechanisms by which ASM synthetic responses are regulated, new issues in the pathogenesis of asthma will be addressed and therapeutic measures to prevent these alterations can be developed.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center (P50)
Project #
5P50HL067663-05
Application #
7125074
Study Section
Special Emphasis Panel (ZHL1)
Project Start
Project End
Budget Start
2005-09-01
Budget End
2010-06-30
Support Year
5
Fiscal Year
2005
Total Cost
$346,007
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Jude, Joseph A; Tirumurugaan, Krishnaswamy G; Kang, Bit Na et al. (2012) Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases. Am J Respir Cell Mol Biol 47:427-35
Goncharova, Elena A; Lim, Poay N; Chisolm, Amelia et al. (2010) Interferons modulate mitogen-induced protein synthesis in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 299:L25-35
Acharya, Pinak S; Zukas, Alicia; Chandan, Vishal et al. (2006) Fibroblast activation protein: a serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis. Hum Pathol 37:352-60
Amrani, Yassine; Tliba, Omar; Deshpande, Deepak A et al. (2004) Bronchial hyperresponsiveness: insights into new signaling molecules. Curr Opin Pharmacol 4:230-4
Howarth, Peter H; Knox, Alan J; Amrani, Yassine et al. (2004) Synthetic responses in airway smooth muscle. J Allergy Clin Immunol 114:S32-50
Chen, Hang; Tliba, Omar; Van Besien, Christopher R et al. (2003) TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl. J Appl Physiol 95:864-72; discussion 863
Amrani, Yassine; Panettieri, Reynold A (2003) Airway smooth muscle: contraction and beyond. Int J Biochem Cell Biol 35:272-6
Tliba, Omar; Tliba, Samira; Da Huang, Chien et al. (2003) Tumor necrosis factor alpha modulates airway smooth muscle function via the autocrine action of interferon beta. J Biol Chem 278:50615-23
Huang, Chien-Da; Tliba, Omar; Panettieri Jr, Reynold A et al. (2003) Bradykinin induces interleukin-6 production in human airway smooth muscle cells: modulation by Th2 cytokines and dexamethasone. Am J Respir Cell Mol Biol 28:330-8
Amrani, Yassine; Tliba, Omar; Choubey, Divaker et al. (2003) IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway. Am J Physiol Lung Cell Mol Physiol 284:L1063-71

Showing the most recent 10 out of 13 publications