A culture system of canine fundic mucosal cells that proliferate in response to growth factors (GF) of physiologic relevance has been developed. We hypothesize that mucosal growth regulation, a process integral to the maintenance of mucosal integrity and to healing, entails a complex interplay between GF delivered by endocrine, neurocrine and autocrine/paracrine routes. We propose a reductionist approach to elucidate cellular mechanisms underlying growth regulation. Conditions permitting culture of dividing cells in serum-free, GF-free medium (Ro) were developed. The epithelial nature of the replicating cells will be confirmed and cell proliferation monitored by [3H]-thymidine (THM) incorporation. Differentiated cell markers (pepsinogen, H+/K+-ATPase, and mucin) will be detected by immunohistochemistry and in situ hybridization and dividing cells identified by [3H]-THM autoradiography and incorporation of bromodeoxyuridine immunohistochemistry. We will extend preliminary studies which indicate that most dividing cells are undifferentiated, while a few contain pepsinogen. In Ro, gastrin and bombesin stimulate growth. Receptors for these peptides will be characterized and localized to specific cell types using radioligand techniques. Analogues and antagonists will be used to establish receptor specificity. We will differentiate effects of GF on undifferentiated dividing cells vs. chief cell precursors to localize the site of their mitogenic action. Growth in Ro is dependent upon plating cell density, suggesting autocrine and/or paracrine growth control We will pursue preliminary data indicating that transforming GFalpha (TGFalpha) and insulin-like GF-I(IGF-I) are involved in autocrine/paracrine regulation assessing the following: (1) production of GF in the replicating cells themselves (GF content by radioimmunoassay, GF mRNA expression, GF mRNA localization by in situ hybridization), (2) localization and characterization of GF receptors (3) release of GF into medium, (4) inhibition of growth in Ro by immunoabsorption with specific antibodies, and (5) regulation of expression of GF mRNA by potential growth modulators. Localization of regulatory events to a specific cell type will entail cell separation techniques and/or a combination of the following: (1) bromodeoxyuridine immunohistochemistry of [3H]-THM autoradiography to identify dividing cells, (2) immunohistochemistry or in situ hybridization for GF content and expression, and (3) immunohistochemistry or in situ hybridization for differentiative cell markers. We will also confirm findings that TGFBeta and somatostatin inhibit mucosal cell proliferation and use immunoneutralization to determine if TGFBeta exerts autocrine growth control. From these studies we will develop a new model system to evaluate the regulation of mucosal growth at a cellular level. These mucosal cells replicate rapidly and are very responsive to physiologic concentrations of GF, suggesting that this reductionist approach may allow elucidation of basic mechanisms relevant to growth control.
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