Regulation and function of the Clara cell secretory protein

Sammanfattning: The Clara cell secretory protein (CCSP; also known as Polychlorinated Biphenyl- binding protein (PCB-BP) or Uteroglobin) is specifically produced and secreted into the distal airway lumen by the non-ciliated Clara cell. It is a low molecular weight homodimeric protein and binds certain metabolites of PCBs as well as progestins and calcium. Yet, the physiological function of CCSP has not been established. The aims of this thesis have been to use the CCSP gene and its regulation as a model to study mechanisms of lung development and cellular differentiation and to eluci- date the physiological function of CCSP with the aid of structure/function-analyses and targeted gene inactivation in intact animals, the latter also to define its role in PCB-bioaccumulation. Initially, the expression of CCSP during fetal development of rat lung was studied. The protein appears at day 19 of gestation and is preceeded by one day by its mRNA. Levels were increased 2-3 fold by glucocorticoids. To investigate the mechanisms behind the differentiation-dependent CCSP expression, we turned to isolated rat Clara cells. These cells proliferate and dedifferentiate in primary culture and levels of CCSP decrease concommitantly. Proximal elements in the CCSP-promoter exhib- ited a cell-specific activity in primary lung cells and further analysis demonstrated the importance of a C/EBP-related transcription factor interacting with proximal elements to regulate CCSP-expression. These studies establish CCSP as a useful differentiation marker and suggest a role for C/EBP in the differentiation of the terminal airways. To investigate the molecular basis behind CCSPs calcium-binding, we applied a hydrophobic contrast function to the three-dimensional structure of CCSP. One metal binding site per monomer was found and site directed mutagenesis confirmed that the identified region is a functional calcium-binding site. Prompted by the similar- ity of this site to the calcium-binding sites in annexins that mediate binding to phospholipids, we could show CCSP binding to phospholipids in a calcium-de- pendent manner. This suggests that one function of calcium-binding is to associate the protein to phospholipid bilayers. Finally, in an attempt to elucidate the physiological function of CCSP we generated CCSP deficient mice by targeted gene "knock-out". These mice do not accumulate methyl-sulfonyl metabolites of PCBs in lung as wild type mice do. Moreover, they show specific ultrastructural changes within the secretory apparatus of the Clara cells, including a total absence of secretory granules and reduced amounts of rough endoplasmic reticulum. These results establish CCSP as a determinant of PCB bio- accumulation and suggests a role for the protein in the secretory pathway of the Clara cell. Key words: Lung; Clara cells; Uteroglobin; Polychlorinated biphenyls; Rat; Mouse; Hu- man; Fetal development; Glucocorticoids; Cells, cultured; Gene expression regulation; C/ EBP; Calcium; Protein conformation; Computer simulation; Mutagenesis, site-directed; Calcium binding proteins; Phospholipids; Mice, knockout. ISBN 91-628-2342-6