Chronic changes in the environment of lung parenchymal cells induce structural and functional adaptations that contribute to airway obstruction and hyperreactivity. Airway smooth muscle adapts to inflammation by changing from contractile cells to secretory or proliferating and migrating cells. This is termed """"""""phenotypic plasticity"""""""" which requires changes in gene and protein expression over a period of many hours, days or weeks. Airway smooth muscle also undergoes a more rapid """"""""mechanical plasticity"""""""" that occurs in a matter of minutes. Nearly constant isometric force can be generated over a very wide range of tissue lengths. In both cases, plasticity is defined as a persistent change in cell structure or function in response to a change in the environment. Environmental stimuli that trigger muscle plasticity include neurotransmitters, prostanoids, cytokines and mechanical strain. These signals are transduced by multiple protein kinase signaling cascades, some of which target the actin cytoskeleton. Hypothesis: The major hypothesis is that dynamic changes in the actin cytoskeleton required for phenotypic and mechanical plasticity of human airway smooth muscle are mediated in part by small heat shock proteins HSP27, HSP22 and HSP20. These proteins associate with actin filaments and actin attachment structures in striated and smooth muscles, but their precise functions are poorly defined. Phosphorylation and formation of homo- and heteropolymers among the heat shock proteins are thought to regulate their binding to actin filaments, which could alter the number and position of actin attachment sites at the cell membrane and at cytoplasmic dense bodies. Approach: To test this hypothesis, mechanical and chemical stimuli will be used to stimulate airway smooth muscle in vitro. Cell and tissue mechanics, cell attachment and spreading, proliferation and survival will be assayed as readouts of mechanical and phenotypic adaptations. Adenoviral, retroviral and plasmid-mediated gene transfer techniques will be used in gain of function and dominant negative overexpression approaches.
Specific Aim 1 is to: (1) Define the signals and protein kinases that regulate polymer size and actin binding of HSP27 in human airway smooth muscle cells; (2) Define the binding partners for HSP27 using immunocytochemistry, immunoprecipitation and yeast 2-hybrid screening (3) Define the functions of HSP27 phosphorylation in tissue and cell mechanics, cell attachment and spreading, cell proliferation and cell survival.
Specific Aim 2 is to: (1) Determine whether HSP20 is inducible by cytokines and mechanical stressors, (2) Determine whether HSP20 copolymerizes with HSP27 and (3) Define the necessity of HSP20 phosphorylation for contraction, tissue stiffness, cell attachment and spreading. The results will test directly the notion that small heat shock proteins are important participants in mechanical and phenotypic adaptations of airway smooth muscle.
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