Excessive airway inflammation occurs in asthma, bronchitis and bronchiectasis. Local human bronchial epithelial (HBE) cells express chemoattractant cytokines and hematopoietic growth factors that serve to recruit immune effector neutrophils and lymphocytes, amplifying the airway inflammatory response. Molecular mechanisms regulating HBE cell expression of inflammatory cytokines will be elucidated. Stimulated HBE cells express substantial IL-8 and little IL-2, and stimulated T-cells express substantial IL-2 and little IL-8. This reciprocal expression of IL-8 and IL-2 in HBE and T-cells is regulated by transcription factors NF-kappaB and the CsA-sensitive purine-box regulator. The CsA-sensitive purine-box regulator in HBE and T-cells binds to purine-rich DNA sequences including the IL-8 NF-kappaB site and the NF-AT target site, and mediates sequence-specific transcriptional repression. Cell stimulation that mobilizes calcium triggers the conversion of the purine-box regulator from a repressor into a transcriptional activator: this conversion is most extensive in activated T-cells. The CsA-sensitive purine-box regulator in HBE and T-cells contains subunits, NF45, NF90, Ku70, Ku80, and the DNA-dependent protein kinase, catalytic subunit. The stimulation-induced structural changes in the purine-box regulator subunits which control the functional conversion from a repressor into a transcriptional activator will be identified, using immunoprecipitation, phosphopeptide mapping, and in vitro transcription experiments. The molecular mechanisms through which CsA and FK506 destabilize the purine-box regulator/NF-kappaB repressor and induce constitutive IL-8 secretion, will be elucidated. In related studies, the molecular mechanisms of action of a novel antiinflammatory drug, PG490 (triptolide), derived from a Chinese herbal remedy for arthritis, will be determined. PG490 inhibits NF-kappaB transcriptional activation and inflammatory cytokine gene expression by epithelial cells and T-cells. A signaling enzyme in HBE cells which is specifically inactivated by PG490 and which regulates NF-kappaB transcriptional activation in the nucleus will be isolated and characterized.
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