The mammalian small intestine is lined by a continuously renewable population of simple columnar cells which arise in the crypts and acquire a differentiated phenotype as they migrate to the villus compartment. The enterocytes, which comprise 95% of the differentiated villus cells, possess a brush-border at their luminal surface containing the enzymes and transporters essential for the digestion and absorption of dietary nutrients. Villus atrophy occurs in a variety of disease states and is associated with diarrhea, malabsorption, and impairment in gut barrier function. An understanding of the molecular mechanisms underlying Villus atrophy could lead to important therapies designed to maintain both the structural and functional integrity of the gastrointestinal tract. It has generally been assumed that the physiological derangements associated with villus atrophy are due to a decrease in the number of absorptive epithelial cells; that is, the enterocytes are unchanged, there are just less of them. However, data from four independent rat models of villus atrophy/hyperplasia demonstrate that enterocyte phenotype changes dramatically as a function of epithelial growth state. It is clear from these preliminary studies that the process of crypt-villus differentiation is closely linked to the proliferative state of the epithelium. Intestinal alkaline phosphatase (IAP) is a brush-border enzyme expressed exclusively in differentiated enterocytes. IAP is unique among the various markers of enterocyte differentiation in that its expression is regulated by developmental, dietary, and hormonal factors in vivo, and that it undergoes regulated expression in vitro (HT-29 cells). Thus, IAP will be used as a tool to elucidate the molecular mechanisms underlying two distinct but related processes: (1) the crypt-villus axis of enterocyte differentiation, and (2) the alterations in enterocyte phenotype that accompany changes in epithelial growth state. Functional studies on the endogenous IAP gene, as well as transfection experiments using IAP reporter constructs, will allow for the identification of those elements within the IAP gene that are transcriptionally active and which mediate its regulation within the contexts of both enterocyte growth and differentiation. DNase l footprinting and mobility shift assays will then be performed in order to identify and characterize the specific sites of DNA-protein interactions which are responsible for IAP regulation. Finally, we plan to identify new genes whose expression is altered as a function of intestinal atrophy/hyperplasia, to more fully understand the genetic programs which underlie the changes in enterocyte phenotype. These studies will bring together the molecular mechanisms which regulate the crypt-villus axis of differentiation with those regulating intestinal epithelial proliferation, and, as such, could lead to a unifying model with which to understand the processes of enterocyte growth and differentiation.
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