Up to 300,000 adults and infants per year in the U.S suffer morbidity and mortality due to acute inflammatory lung diseases such as pneumonia, acute respiratory distress syndrome (ARDS) and respiratory distress of prematurity; while many times that number suffer from chronic, fibrotic lung disease. The proinflammatory cytokine, tumor necrosis factor-a (TNF-a), is a major pathogenic factor in both acute and chronic lung inflammation. In the past 4 years, major advances have been made in understanding signaling mechanisms by which cells respond to TNF-a and that account for the highly pleiotropic and cell type specific effects of the cytokine. The laboratory is applying these advances in molecular biology to understanding how TNF-a and oxidant stress interact to alter pulmonary cell survival, gene expression and function, with an ultimate goal of improving therapy for inflammatory lung disease.

In our laboratory, cell culture and whole animal models are being utilized to determine signaling mechanisms by which members of the NGF/TNF receptor superfamily, of which TNF-a receptor I and II (TNFRI and TNFRII) are representative, regulate gene expression, inflammation and apoptosis in the lung. We have recently demonstrated marked induction of TNF receptor associated factors (TRAF) and Inhibitor of Apoptosis Proteins (IAPs) by TNF-a and by the phorbol ester (TPA) in pulmonary epithelial cells in vitro, in mouse lung following intratracheal TNF-a treatment and in inflammed human and baboon lung tissue. We are pursing studies to establish cellular and developmental localization, regulation and function of the TRAF-1 and cIAP proteins in lung. Recent developments implicate TRAF-1 and cIAPs as anti-apoptotic proteins. Our studies focus on the signal transduction systems that mediate the effects of TNF-a on programmed cell death (apoptosis), proliferation and regulation of transcription factors AP-1 and NFkB in lung cells. Previous work demonstrated inhibiton of surfactant associated protein, SP-B, mRNA levels by TNF-a mediated by shortening of the SP-B mRNA half-life. Mutational analysis, pulmonary epithelial cell lines and RNA gel retardation assays are being utilized to test the hypothesis that cis-active nucleotide sequence(s) interacting with specific trans-acting factor(s) determine levels of SP-B expression by regulating the stability of SP-B mRNA. Effects of TNF-a on pulmonary cell function are also being studied in murine whole animal models. Structural and biochemical abnormalities following intratracheal administration of TNF-a, silica or exposure to hyperoxia, in wildtype and transgenic mice (ie. TNF receptor knockout mice), are being utilized to examine TNF-a mediated regulation of epithelial cell function. An inducible promoter system to allow overexpression of TNF-a in a time and tissue specific manner in cell culture and in mice is being developed in order to investigate the effect of TNF-a on lung at different stages of development.

Pryhuber GS, Church SL, Kroft T, Panchal A and Whitsett JA. 3'-Untranslated region of surfactant protein B mRNA mediates inhibitory effects of TPA and TNF-a on SP-B expression. Am J Physiol 267(Lung Cell Mol Physiol 11): L16-L24, 1994.

Pryhuber GS, Bachurski CJ, Hirsch R, Bacon A and Whitsett JA. Tumor necrosis factor-a decreases surfactant protein B mRNA in murine lung. Am J Physiol 270(Lung Cell Mol Physiol 14): L714-721, 1996.

Pryhuber GS, Khalak R and Zhao Q. Regulation of surfactant protein A and B by TNF-a and phorbol ester independent of NFkB. Am J Physiol (Lung Cell Mol Physiol). 8(2): L 289-L 295, 1998.

Pryhuber GS . Regulation and Function of Pulmonary Surfactant Protein B. Molecular Genetics and Metabolism, 64, 217-228, 1998 (Invited Review).

Khalak R, Huyck H and Pryhuber GS. Antagonistic Effects of Pyrrolidine Dithiocarbamate (PDTC) and N-Acetyl-l-Cysteine on Surfactant Protein Expression. Exp Lung Research, Submitted, 1998.

Pryhuber, GS, Reichlen, G and Huyck H. Regulation of Tumor Necrosis Factor Receptor Associated Factor 1 (TRAF-1) in Pulmonary Epithelial Cells by TNF-a and Phorbol Ester. Am J Resp Cell Mol Biol, Submitted, 1998.