This project is focused on defining the factors which regulate the differentiation and mucus-secretory properties of intestinal goblet cells. Previous investigations have been limited by the inability to maintain differentiated goblet cells under any conditions in vitro. Recently, however, a human colon cancer cell line, HT29, was found to express the morphological and enzymatic phenotypes characteristic of small intestinal epithelium when grown in glucose-free medium. Furthermore, a clone, designated """"""""N2"""""""", has been isolated from the parent HT29 line, which differentiates into a pure population of goblet cells when grown in glucose-free medium. The HT29 and N2 cell lines will be developed as model systems to study the regulation of goblet cell differentiation and mucus secretion. Morphological, immunochemical and biochemical landmarks of the goblet cell differentiation process will be defined. Extracellular matrix components and permeable substrates will be tested for their influence on the proliferation and differentiation of HT29 and N2 goblet cells. Indirect evidence suggests that T-lymphocytes in parasite-infected rats may secrete a soluble factor which specifically modulates goblet cell growth. T-lymphocytes from Nippostrongylus brasiliensis-infected rats will be exposed to antigen in vitro and the conditioned medium tested for its ability to induce goblet cell proliferation and differentiation in the HT29 and N2 cell lines. The role of calcium in mediating the baseline and accelerated forms of mucus secretion will be established by using morphological, autoradiographic and radioimmune assay techniques to measure the secretory rates in varying concentrations of extracellular calcium or in the presence of a calcium channel blocker (Co2+). Phorbol esters will be used to test the hypothesis that activation of protein kinase C is an essential step in the compound exocytosis of mucous secretory granules. The basic information gained from these studies will provide new insights into the process of goblet cell formation, and will enhance the understanding of how the mucosa maintains its protective coating of mucus.
