Glucocorticoids are steroid hormones that have potent anti-inflammatory actions. Endogenous glucocorticoid action is modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyses the interconversion of active glucocorticoids (cortisol, corticosterone) and intrinsically inert forms (cortisone, 11-dehydrocorticosterone). There are 2 isozymes; 11β-HSD type 1 regenerates active glucocorticoids in vivo whereas 11β-HSD type 2 inactivates glucocorticoids. Although 11β-HSD1 is highly expressed in the lung, its role there has been little explored. In this study, the expression and localization of 11β-HSD1 mRNA in lung was confirmed by in situ hybridization. Immunohistochemical staining of mouse lung localized 11β-HSD1 to the cytoplasm of fusiform cells in alveolar walls, in a multivesicular pattern characteristic of interstitial fibroblasts. A lung fibrosis model of inflammation was used to test the role and regulation of 11β-HSD1. The results suggest that levels of 11β-HSD1 mRNA and enzyme were not changed during bleomycin-induced lung inflammation. However, 11β-HSD1-deficient mice showed a more severe inflammatory response than congenic wild-type controls, with greater inflammatory cell infiltration into the lung, and increased levels of HO-1 and iNOS mRNA 14 days following bleomycin installation into lung. Picrosirius red staining of lung sections suggested more collagen deposition in 11β-HSD1-deficient mice than in wild-type controls during the course of the lung inflammatory response. Moreover, whereas naïve 11β-HSD1-deficient mice had significantly lower collagen content in lung (84% of WT levels, p<0.05). 28d after bleomycin there was no significant difference between genotypes (KO having 94% of WT levels, p=0.42) confirming more collagen production in 11β-HSD1-deficient mice following bleomycin. Fibroblasts are critical in the regulation of inflammatory responses and are essential in the model of bleomycin-induced lung injury. Lung fibroblasts may have a different transcriptional regulation of 11β-HSD1 compared to other tissues. In the majority of tissues, 11β-HSD1 can be transcribed from 2 promoters; the P1 promoter is the main promoter used in lung, with other tissues mainly using the P2 promoter. To address the relevance of the P1 promoter in lung and to identify the cell type using the P1 promoter, mouse lungs were collagenase-digested to isolate primary fibroblast and epithelial cells. Isolated lung fibroblasts highly expressed 11β-HSD1, predominantly from the P1 promoter. During passage, primary lung fibroblasts switched promoter usage from P1 to P2. In fibroblast primary culture, treatment with TGF-β for 72h markedly decreased 11β-HSD1 expression to 38% of untreated levels, an effect which was reversed by SB431542, a TGF-β receptor antagonist. Whilst TGF-β reduced levels of mRNA initiating at the P2 promoter, initiation from the P1 promoter was completely repressed. Treatment with TGF-β receptor antagonist increased levels of P1-initiated 11β-HSD1 mRNA by 6.6-fold compared to untreated cells. These data suggest that the switch in 11β-HSD1 promoter usage may be regulated by TGF-β during an inflammatory response. Furthermore, as the P1 and P2 promoters are differentially regulated (e.g. by C/EBPβ, a cytokine-responsive transcription factor), the promoter switch may place 11β-HSD1 under a different transcriptional regulation during inflammation. Taken together, these results suggest that 11β-HSD1 deficiency worsens lung inflammation and results in greater lung fibrosis. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the inflammatory response and shape fibroblast function, limiting subsequent collagen production and fibrosis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563078 |
| Date | January 2010 |
| Creators | Yang, Fu |
| Contributors | Chapman, Karen |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4828 |
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