Alpha1-proteinase inhibitor (a1PI) is the major antiprotease in human plasma and is responsible for limiting the degradative action of many proteases, including neutrophil elastase in the lung. Severe a1PI deficiency, characteristic of the PiZZ phenotype, is associated with pulmonary emphysema and cirrhosis of the liver. PiZZ a1PI deficiency is characterized by decreased plasma a1PI levels and accumulations of aberrantly glycosylated a1PI in hepatocytes, the site of a1PI synthesis. Presently there is no human liver cell line available which carries and expresses the PiZ gene.
The aims of this proposal are to 1) Construct human liver cell lines bearing the PiZ gene for studies of the structure, secretion, and intracellular distribution of PiZ a1PI. Focus will be on transformation of HepG2 and SK-1 cells with recombinant SV40-based expression vectors. 2) Compare the carbohydrate composition, intracellular accumulation, and rate of secretion of PiZ a1PI with those of PiM a1PI, the normal variant. Having identified translational and post-translational modifications of a1PI and the kinetics of a1PI secretion in both PiM and PiZ-bearing cells, we shall determine the effects of hepatocyte stimulating agents on these characteristics. 3) Determine the effects of hepatocyte stimulating agents on transcriptional control of PiM a1PI. We shall employ a system for directly assessing transcription of the endogenous gene. 4) Identify and establish the mechanisms by which trans-acting factors control transcription of a1PI genes in human cells. Part of this study involves examination of chromosomal organization of the endogenous a1PI gene. These studies will provide an important model system for study of the modulation of PiZ gene expression. Through this work we shall also gain insights into the development of therapies based on pharmacological agents and genetically-engineered cells to protect and treat PiZZ individuals against emphysema associated with a1PI deficiency.
