DISTINCT CHANGES IN INTESTINAL EPITHELIAL GENE EXPRESSION UPON METHOTREXATE TREATMENT

Abstract

Introduction. Structure, function and differentiation of the intestinal epithelium are severely affected in patients receiving cytostatic drugs. Especially in pediatric patients, major side effects like malabsorption and malnutrition are of great concern. Little is known about effects of cytostatics on gene expression in the intestinal epithelium and about the mechanisms that mediate the mucosal damage and regeneration. Aim. In order to get insight in the functional capacities of intestinal epithelium during tissue damage and subsequent regeneration, we focussed on rats treated with methotrexate (MTX) as a model to investigate quantitavely the changes in epithelium specific gene expression. Methods. Rats were injected i.v. with a single dose of MTX (30 mg/kg). Up to 10 days after treatment rats were sacrificed and jejunal segments were removed and processed for histological and biochemical analysis. Gene expression of cell type specific differentiation markers was quantified at the level of mRNA, protein and enzyme activity using Northern blots, Western blots and enzyme assays, respectively. Results. Histologically, mucosal damage resulted in villus shortening at day 1 that increased to almost complete villus atrophy at day 4. During day 1 to day 4, enterocyte markers carbamoyl phosphate synthese (CPS) and sucrase-isomaltase (SI) decreased one after another to undetectable levels, whereas the enterocyte marker intestinal fatty acid binding protein (iFABP) increased to 120% of control values. In these tissue samples, expression of the mucin MUC2, a goblet cell marker, increased 1.5-fold. Furthermore, expression of chromogranin A (chromA), a marker for differrentiated entero-endocrine cells, decreased 2.5-fold during villus shortening. Expression of lysozyme, a marker for Paneth cells, increased 3-fold at day 2. The crypt villus architecture was regenerated at day 6. During regeneration, expression of all differentiation markers returned to normal values. Expression patterns of mRNA, protein and enzyme activity of each marker gene were parallel during the course of the experiment. Conclusions. Our results indicate a distinct effects of MTX on gene expression in different intestinal epithelial cell types. Decreased expression of CPS and SI together with an increased expression of iFABP after MTX treatment, suggests selective changes in enterocyte metabolism during damage and regeneration. Furthermore, goblet cells and Paneth cells contribute to increased synthesis of the cytoprotective molecules, MUC2 and lysozyme, during epithelial damage. Since effects of MTX parallelled at the different levels of expression, changes in epithelial gene expression seem to be mainly regulated at pretranslational level.