The goal of the proposed research is to define the physiological regulation and functional significance of vitamin K-dependent gamma-carboxylation of glutamic acid residues (Gla) in protein substrates of rat alveolar type II cells, a modification which confers calcium binding properties to these proteins. Type II cell microsomes have a high carboxylase specific activity compared with other non-hepatic tissues, yet little is known of the identity or function of the 6-8 major endogenous substrates present in these cells. The synthesis and carboxylation of all substrates will be studied using 2-dimensional separation of protein isoforms and microsequencing will be performed on each substrate to establish possible identity or homology to known proteins. Specific antibodies and cDNA probes will made to help determine Gla protein function and to study regulation at the mRNA level, respectively. Exogenous peptide substrates will be used in well-defined in vitro assays of cellular microsomes to identify sequence recognition determinants for the type II cell carboxylase(s). These peptides are designed from both hepatic precursors and from one of the type II cell substrates, Matrix Gla Protein (MGP). Culture of type II cells induces both carboxylase enzyme activity and several specific endogenous substrates, a process which can also be carefully regulated by the vitamin K status of the medium. These Gla proteins, which are expressed at different times during culture, act as markers of the type II to type I cell transition, and their identification and function will lead to a better understanding of this phenotypic change in vitro and in vivo. For one substrate, MGP, mRNA levels increase 6-fold from days 3-6 in primary culture in parallel with greatly enhanced rates of MGP secretion. Studies of the surfactant protein SP-A and its mRNA, cellular morphology, protein and DNA synthesis and cytokeratin expression will be performed in parallel as additional indices of type II cell phenotype. Because type II cell phenotype is strongly influenced by the extracellular matrix (ECM), we will study the regulation of MGP and other substrates at the transcriptional and protein level as affected by the ECM and by glucocorticoid hormones. In these studies, the laminin-rich ECM (Matrigel) and a fibronectin-rich type II cell-derived matrix (M6) will be used to distinguish the influence of ECM proteins and cellular shape on Gla protein metabolism. Multiple approaches will be used to investigate the probable role of MGP in cell-ECM interaction. Our results will begin to define the significance of all type II cell vitamin K-dependent proteins and will form a solid basis for future investigations into the pulmonary consequences of acute and chronic anticoagulant therapy.
| Rannels, S R (1996) Impaired surfactant synthesis in fetal type II lung cells from gsd/gsd rats. Exp Lung Res 22:213-29 |
| Dunsmore, S E; Rannels, S R; Grove, R N et al. (1995) Adrenal hormone regulation of extracellular matrix synthesis by type II cells. Am J Physiol 268:L885-93 |
| Rannels, S R; Cancela, M L; Wolpert, E B et al. (1993) Matrix Gla protein mRNA expression in cultured type II pneumocytes. Am J Physiol 265:L270-8 |
| Eckenhoff, R G; Rannels, S R; Fisher, A B (1991) Secretory granule calcium loss after isolation of rat alveolar type II cells. Am J Physiol 260:L129-35 |
