Milan Kment

11β‐hydroxysteroid dehydrogenase 1 and 2 expression in colon from patients with ulcerative colitis

Background and Aim:  11β‐hydroxysteroid dehydrogenase (11βHSD) is an enzyme responsible for the interconversion of active 11β‐hydroxysteroids (cortisol) into biologically inactive 11‐oxosteroids (cortisone). The isoform 11βHSD1 operates predominantly as a reductase converting cortisone to cortisol, whereas 11βHSD2 catalyzes oxidation of cortisol to cortisone. This mechanism of peripheral metabolism of glucocorticoids has been suggested to be involved in increasing the availability of anti‐ inflammatory glucocorticoids as a response to inflammatory stimuli. The aim of this study therefore was to investigate the impact of inflammatory bowel disease on the expression of colonic 11βHSD1 and 11βHSD2.

Local metabolism of glucocorticoids and its role in rat adjuvant arthritis.

11beta-Hydroxysteroid dehydrogenase 1 (11HSD1) regulates local glucocorticoid activity and plays an important role in various diseases. Here, we studied whether arthritis modulates 11HSD1, what is the role of pro-inflammatory cytokines in this process and whether altered local metabolism of glucocorticoids may contribute to the feedback regulation of inflammation. Adjuvant arthritis increased synovial 11HSD1 mRNA and 11-reductase activity but treatments with tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) antagonists etanercept and anakinra reduced 11HSD1 upregulation. Treatment with carbenoxolone, an 11HSD inhibitor, increased expression of TNF-alpha, cyclooxygenase 2, and osteopontin mRNA without any changes in the plasma levels of corticosterone. Similar changes were observed when arthritic rats were treated with RU486, an antagonist of GR. This study suggests that arthritis upregulates synovial 11HSD1, this upregulation is controlled by TNF-alpha and IL-1beta and that the increased supply of local corticosterone might contribute to feedback regulation of inflammation.

Enhanced expression of proproliferative and antiapoptotic genes in ulcerative colitis-associated neoplasia

Background: Inflammatory bowel diseases including long‐standing ulcerative colitis (UC) have an increased risk of evolving into colorectal cancer (CRC). The overexpression of some proproliferative and antiapoptotic genes, such as survivin, telomerase catalytic subunit (hTERT), integrin‐linked kinase (ILK), and regulatory factors c‐MYB and Tcf‐4, has been implicated in the development and progression of several human malignancies including CRC. Methods: In this study we analyzed the expression alterations of these markers and proinflammatory enzymes cyclooxygenase 2 (COX‐2) and inducible nitric oxide synthase (iNOS) during the transition of colonic mucosa from chronic inflammation to epithelial neoplasia in biopsies of UC patients using quantitative real‐time polymerase chain reaction and immunohistochemistry; additionally, we compared the expression profiles of this gene panel in samples of patients with CRC after tumor resection and in human tumor xenografts of SW620 malignant colonic cells. Results: The transcript levels of survivin, c‐MYB, COX‐2, iNOS, and Tcf‐4 showed a statistically significant increase during neoplastic transformation of UC patient colonic mucosa, whereas hTERT and ILK were not elevated. In contrast, the specimens of CRC showed upregulated expression of not only survivin, c‐MYB, Tcf‐4, COX‐2, and iNOS but also hTERT. A similar expression profile was observed in human tumor xenografts in which all transcripts with the exception of c‐MYB were upregulated. Conclusions: These results suggest that telomerase and ILK activation occurs during the later stages of carcinoma progression, whereas upregulation of survivin, c‐MYB, and Tcf‐4 is a feature of the early stage of development of neoplasia, and thus, they might serve as early indicators for UC‐associated colorectal carcinogenesis. (Inflamm Bowel Dis 2010)

Expression profiles of proliferative and antiapoptotic genes in sporadic and colitis-related mouse colon cancer models

Elevated levels of survivin, telomerase catalytic subunit (TERT), integrin‐linked kinase (ILK), cyclooxygenase 2 (COX‐2), inducible nitric oxide synthase (iNOS) and the regulatory factors c‐MYB and Tcf‐4 are often found in human cancers including colorectal cancer (CRC) and have been implicated in the development and progression of tumorigenesis. The aim of this study was to determine the expression of these genes in mouse models of sporadic and colitis‐associated CRC. To address these issues, we used qRT‐PCR approach to determine changes in gene expression patterns of neoplastic cells (high‐grade dysplasia/intramucosal carcinoma) and surrounding normal epithelial cells in A/J and ICR mouse strains using laser microdissection. Both strains were injected with azoxymethane and ICR mice were also given drinking water that contained 2% dextran sodium sulphate. In both sporadic (A/J mice) and colitis‐associated (ICR mice) models of CRC, the levels of TERT mRNA, COX‐2 mRNA and Tcf‐4 mRNA were higher in neoplastic cells than in surrounding normal epithelial cells. In contrast, survivin mRNA was upregulated only in neoplastic cells from A/J mice and ILK mRNA was upregulated only in neoplastic cells from ICR mice. However, the expression of iNOS mRNA was similar in normal and neoplastic cells in both models and c‐MYB mRNA was actually downregulated in neoplastic cells compared with normal cells in both models. These findings suggest that the genetic background and/or the molecular mechanisms of tumorigenesis associated with genotoxic insults and colonic inflammation influence the gene expression of mTERT, COX‐2, Tcf‐4, c‐MYB, ILK and survivin in colon epithelial neoplasia.

Inflammation regulates 11β-hydroxysteroid dehydrogenase type 1 differentially in specific compartments of the gut mucosal immune system

&NA; The bioavailability of glucocorticoids is modulated by enzyme 11&bgr;‐hydroxysteroid dehydrogenase type 1 (11HSD1), which catalyzes the conversion of inactive 11‐oxo‐glucocorticoids to active 11‐hydroxy‐glucocorticoids cortisol and corticosterone and is regulated by pro‐inflammatory cytokines. Our aim was to assess the effect of colitis on the expression of 11HSD1 in specific microanatomical compartments of the mucosal immune system. Using qRT‐PCR we quantified the expression of 11HSD1 and cytokines in the colon, mesenteric lymph nodes (MLN) and spleen of mice with colitis. Microsamples of the MLN cortex, paracortex and medulla, colonic crypt epithelium (CCE), lamina propria and isolated intestinal lymphoid follicles (ILF) were harvested by laser microdissection, whereas splenic and MLN lymphocytes by flow cytometry. Colitis increased 11HSD1 in the CCE, ILF, and MLN cortex but not in the lamina propria and the MLN paracortex and medulla. Expression of IL‐4, IL‐21 and TNF&agr; was increased in both the cortex of MLN and ILF, whereas IL‐1&bgr; and IL‐10 were only increased in the follicles. No positive effect was observed in the case of IFN&ggr; and TGF&bgr;. 11HSD1 was positively correlated with TNF&agr; and less strongly with IL‐21, IL‐1&bgr;, and IL‐4. Colitis also upregulated the 11HSD1 expression of T cells in the spleen and MLN. The study demonstrates the stimulatory effect of inflammation on local glucocorticoid metabolism only in particular compartments of the mucosal immune system. The correlation between cytokines and 11HSD1 in the ILF and MLN cortex indicates that pro‐inflammatory cytokines may amplify glucocorticoid signals in inductive compartments of the mucosal immune system. HighlightsInflammation upregulates 11HSD1 in gut immune system and in mesenteric lymph nodes.This effect varies among specific anatomical compartments of immune system.TNF&agr; is the crucial cytokine for upregulation of 11HSD1 during inflammation.Amplification of glucocorticoid signals during inflammation in not homogenous.

Expression of 11ß-hydroxysteroid dehydrogenase type 2 is deregulated in colon carcinoma

Although the effects of glucocorticoids on proliferation, differentiation and apoptosis are well known, and steroid hormones have been identified to play a role in pathogenesis and the development of various cancers, limited data are available regarding the relationship between the local metabolism of glucocorticoids and colorectal adenocarcinoma (CRC) formation. Glucocorticoid metabolism is determined by 11β-hydroxysteroid dehydrogenases type 1 and 2 (11HSD1, 11HSD2), which increase the local concentration of cortisol due to the reduction of cortisone, or decrease this concentration due to the oxidation of cortisol. The objective of this study was to evaluate the extent of 11HSD1 and 11HSD2 mRNA in pre-malignant colorectal polyps and in CRC. The specimens were retrieved from patients by endoscopic or surgical resection and the expression of 11HSD1 and 11HSD2 was measured by real-time PCR. The polyps were of the following histological types: hyperplastic polyps and adenomas with low- or high-grade dysplasia. The neoplastic tissue of CRC obtained during tumor surgery was also studied. It was found that 11HSD2 was not only downregulated in CRC but already in the early stages of neoplastic transformation (adenoma with low-grade dysplasia). In contrast, the level of 11HSD1 was significantly increased in CRC but not in pre-malignant polyps. The results demonstrate that the downregulation of 11HSD2 gene expression is a typical feature of the development of colorectal polypous lesions and their transformation into CRC.