Proteinases play a major role in the development of inflammatory lung diseases such as cystic fibrosis, asthma, chronic bronchitis, emphysema and chronic lung disease of prematurity. A better understanding of the regulation of proteinases by inhibitors synthesized by the lung itself could facilitate efforts to develop specific treatments for these diseases. Squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the high molecular weight serine proteinase inhibitor (serpin) family. Although SCCA1 inhibits lysosomal cysteine proteinases, cathepsins (cat)L,S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. SCCA1 and SCCA2 show a tissue restricted expression pattern and are co-localized in the tracheal, bronchial and bronchiolar epithelium. In addition, target proteinases of SCCA1, catS and catK, are expressed by the airway epithelial cells. Another source of these potent elastolytic cysteine proteinases in the lung is alveolar macrophages. Based on the in vitro inhibitory profiles and distribution patterns of SCCA1 and SCCA2 in the airways, the investigators hypothesize that these two serpins protect the airways against proteinase mediated injury. The objective of this proposal is to test this hypothesis using in vitro cell culture and in vivo transgenic animal models.
The specific aims of the proposed project are to: 1) characterize the deleterious effects of exogenous and endogenous cysteine proteinases catS and catK on bronchial epithelial cells in vitro, 2) determine whether SCCA1 and/or SCCA2 can protect cultured bronchial epithelial cells from the proteinase-mediated injury and isolate the target proteinases, 3) determine whether targeted expression of SCCA1 and SCCA2 can protect the airways from proteinase-mediated injury. The experimental design involves use of cell cultures in conjunction with stable transfections to overexpress SCCA1 and SCCA2. Barrier function of the airway epithelium will be studied by permeability and transepithelial resistance measurements as well as structural analysis. Affinity chromatography and co-immunoprecipation will be used to identify the target proteinases of SCCA1 and SCCA2 in proteinase-mediated injury in vivo. The rat clara cell 10kD protein promoter (CC10) will be used to target expression of SCCA1 and SCCA2 genes to the lung. Animals will be examined to determine the extent of protection against proteinase-mediated lung injury following exposure to acrolein. These studies should enhance our understanding of the mechanisms of proteinase mediated injury and whether the locally synthesized inhibitors such as SCCA1 and SCCA2 can prevent this type of damage.
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