Browsing by Subject "Glucocorticoid recepor"

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Der Glucocorticoidrezeptor des Schweins: Herstellung und Charakterisierung eines polyklonalen Antiserums, sowie Studien zur Verteilung des GCR im Intestinaltrakt von Ebern und Kastraten
(2002) Gutscher, Monika; Claus, Rolf
Glucocorticoids are well known to be essential for many physiological and developmental processes. Such functions include their effects on carbohydrate and protein metabolism and their regulatory influences on the immune system. In cell regulation they play a dose-dependent key role for differentiation and apoptosis. In rapidly renewing tissues the stringent control of these mechanisms is central to the maintenance of tissue homeostasis. ln the gastrointestinal tract both the adaption to changing nutrients and the presentation with a vast array of different types of antigens, including potential pathogens and harmless dietary antigens requires a granular regulation of cell proliferation, differentiation and cell death. In the pig, the differences in the turn-over rate for instance between skeletal muscle and the gut tissue could be attributed to different GCR concentrations respectively. This explains the tissue specific sensitivty on circulating corticoids. Thus studies on GCR distribution contributes to the clarification of the role of glucocorticoids in the regulation of these mechanisms in the intestinal tract. In the pig, so far receptor detection has been performed by radio ligand binding assays, which only measures steroid unoccupied non-activated receptors in the cytoplasm. Selective GCR antibodies react with both occupied and unoccupied GCR. In addition, antibodies enable celltype specific detection of the GCR in complex tissues by immunocytochemistry. The aim of this investigation was the production of porcine GCR-specific polyclonal antibodies by detailed analysis of the cDNA sequence of the GCR and the recombinant expression of a suitable antigen fragment. A fragment with 2.1 kb of the GCR cDNA (gcr2.1) was sequenced. Based on Blast sequence analysis a GCR antigen fragment for recombinant expression was selected from the modulatory region (GCRmr) and cloned in a T7-expression system as a His-tag fusion protein. After affinity chromatographic und preparative purification The anti-pGCR-antibodies bind the pGCRmr antigen with high affinity, as well as the denatured receptor in western blot analysis. In additon, immunoprecipitation assays demonstrated that cytosolic GCR is recognized regardless of whether it is unoccuppied or occuppied with dexamethasone. Thus, the antiserum is able to bind the native GCR both in its inactivated form as a multiprotein complex in association with HSP90 and in its activated form with shed HSP 90. Our investigations with immunoprecipitation assays support the applicability of the anti-pGCR antiserum in immunohistochemistry. The characterized antibodies were implemented in immunohistochemy for studies of distribution and localization of the GCR in the small bowl and colon of boars and barrows. The intracellular distribution of the GCR was examined by western blot assays. Immunohistochemical studies showed an increased number of immunostained GCR in the colon compared with the small intestine, as has been shown earlier with ligand-binding assays. 32,9 % and 14,5 % of the cells of the lamina propria were GCR immunoreactive in the small intestine of barrows and boars. In the colon 49,3 % and 43,3 % showed immunostaining. Epithelial cells showed a reversed pattern compared to the lamina propria in both groups. The number of GCR immunoreactive cells in barrows and boars decreased from 9,6 % and 9 % in the small intestine to 5,4 and 5,6 % in the colon, respectively. Comparison of both groups ? barrows and boars - revealed significant differences in the number of GCR immunoreactive cells in the lamina propria of the small bowl. Boars showed a decreased GCR expression of 10 % in the duodenum and 30 % in the jejunum. The number of GCR immuostained colonic cells amounts to 36,9 % in the colon ascendens and 49,2% in the colon descendens of boars and 47,5 % and 51 % in barrows. Studies of the subcellular localization by western blot analysis of cytosolplasmic and nuclear proteins demonstrated that in both groups in the ileum a higher amount of GCR was translocated into the nucleus. In the colon the number of cytoplasmic GCR was higher. The different subcellular GCR distribution in the two segments of the intestine can be explained by the increased expression of 11â-hydroxysteroid dehydrogenase 2 in the colon. 11â-HSD 2 inactivates cortisol and thus inhibits receptor activation and thereby translocation to the nucleus.