MECHANISMS REGULATING FUNCTIONS OF AIRWAY SMOOTH MUSCLE

Project Details

Description

In asthma and airway remodeling, cytokines can directly modulate myocyte function 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-beta and TNF-alpha activate NF-kappaB, 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. Collectively, these findings support a central hypothesis that pro-inflammatory cytokines regulate CAM expression and cytokine, chemokine, and growth factor secretion through coordinate activation of NF-kappaB, 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-kappaB will be explored by inhibiting or activating NF-kappaB 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 immunoblot 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. 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.
StatusFinished
Effective start/end date9/30/997/31/10

Funding

  • National Institutes of Health: $133,430.00
  • National Institutes of Health
  • National Institutes of Health: $133,430.00
  • National Institutes of Health: $335,697.00
  • National Institutes of Health: $338,145.00

ASJC

  • Medicine(all)

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