Inflammation of the airways involves recruitment and activation of immune cells that communicate with surrounding respiratory epithelium, lung parenchyma and smooth muscle. Airway smooth muscle (ASM) cells may contribute to this response through synthesis and secretion of a variety of chemical mediators. p38 MAP kinases are important contributors to the inflammatory response in immune cells, but their role in the secretory response of ASM is not defined. Our major hypothesis is that p38 MAP kinases are necessary for the secretory response of ASM. p38 MAPKs probably directly regulate transcription via phosphorylation of transcription factors, but may also act indirectly by several other mechanisms. We propose to test a novel indirect mechanism whereby p38 MAPK activates MK2 to phosphorylate HSP27, which influences mediator gene expression. HSP27 phosphorylation may be necessary for proper cytoskeletal structure and function required for mediator signal transduction.
SPECIFIC AIM 1 is to define the secretory response of cultured human bronchial smooth (hBSM) muscle cells stimulated with a defined cytokine mixture (IL-1b, TNFb, IFNy) or bronchioalveolar lavage (BAL) fluid from asthmatic patients. Expression of cytokine, chemokine and signal transduction genes will be assayed by cDNA microarrays, quantitative RT-PCR, western blotting and ELISA.
SPECIFIC AIM 2 is to determine the contribution of the p38 MAPK pathway in the secretory response by manipulating this pathway with adenoviral vectors and chemical kinase inhibitors. The secretory response of hBSM cells will be elicited with a cytokine mixture or BAL fluid and the expression profile of cytokine, chemokine and signal transduction genes will be examined. Adenoviral vectors will be used manipulate the function of: a and b isoforms of p38 MAPKs; p21-activated protein kinase 1 (PAKI), MKK.3 and MKK6b, which are upstream activators of p38 MAPKs; MAP kinase activated protein kinase 2 (MK2), a substrate of p38 MAPK; and HSP27, a substrate of MK2 important in regulating the actin cytoskeleton. Comparing multiple steps in the p38 MAPK signaling pathway will help define the degree of redundancy, extent of crosstalk and the step(s) in the pathway most sensitive to inhibition. The role of p38 MAPK in the inflammatory response of ASM, an important non-immune cell type in the lungs, will be defined by these studies and may eventually suggest new targets for effective and more selective treatments of inflammatory airway diseases.
Showing the most recent 10 out of 16 publications
