Molecular Mechanism Of The Induction Of 11β-HSD1 By Interleukin-1β And Cortisol In Human Fetal Lung And Amnion Fibroblasts

Context and Aims:Synchronization of fetal maturation, especially the lung maturation, and initiation of parturition is crucial for the survival of the newborn. Accumulating evidences suggest that increased glucocorticoid level towards the end of gestation triggers both of the events. Due to the unique structure of the fetal adrenal and the existence of placental glucocorticoid barrier, the regeneration of cortisol from cortisone by 11β-hydroxysteroid dehydrogenase type 1 (11(3-HSD1) in the fetal tissue plays a significant role in elevating the local level of glucocorticoids within the tissues. Substantial evidence indicated the presence of 11β-HSD1 in most of the animal fetal lung tissues. Furthermore, 11β-HSD1-/- mice exhibit impaired fetal lung maturation, suggesting the crucial role of 11β-HSD1 in fetal lung maturation. However, the expression of 11β-HSD1 in human fetal lung remains controversial. In addition to the glucocorticoid regeneration by the fetal organ per se, glucocorticoids derived from 11β-HSD1 in the fetal membranes is believed to be another important extra-adrenal source of fetal cortisol in late gestation. Of interest, up-regulation of 11β-HSD1 by proinflammatory cytokines and anti-inflammatory glucocorticoids has been reported in the fetal membranes; however, the molecular mechanism underlying this intriguing up-regulation of 11β-HSD1 by proinflammatory cytokines and glucocorticoids remains largely unknown. In this study, we investigated the expression of 11β-HSD1 in human fetal lung tissues and examined whether the up-regulation of 11β-HSD1 by proinflammatory cytokines and glucocorticoids also holds true in the fetal lung cells. Based on these findings, we investigated the molecular mechanisms underlying the induction of 11 P-HSD1 by Interlukin-1β(IL-1β) and cortisol in human fetal lung fibroblast cell line (HFL-1) and primary human amnion fibroblasts respectively. Elucidation of these mechanisms may help us to understand better the process of human fetal lung maturation.Methods and Results:1. Regular PCR and immunohistochemistry revealed that 11β-HSD1 mRNA was detectable in the fetal lung tissues both at 8 and 4 months of gestation, but 11β-HSD1 protein was only detected in the fetal lung tissue at 8 months of gestation. Positive immunostaining for 11β-HSD1 was found in the epithelium lining the small bronchus, bronchiole and in the fibroblast-enriched mesenchymal tissue of the small bronchus. The detection of 11β-HSD1 mRNA, protein and enzyme activity in HFL-1 cells lends further support for the expression of 11β-HSD1 in human fetal lung. Meanwhile, our findings also indicate that HFL-1 cell is a good model for the further study of the regulation of 11β-HSD1 expression.2. By using real-time PCR, Western blotting and enzyme activity assay, we demonstrated that IL-1βand cortisol induced 11β-HSD1 expression in HFL-1 cells as well as in human amnion fibroblasts.3.Further studies with real-time PCR illustrated that the induction of 11β-HSD1 in HFL-1 cells and human amnion fibroblasts by both IL-1βand cortisol could be blocked by the messenger RNA transcription inhibitor DRB and the protein synthesis inhibitor CHX, suggesting that the induction is dependent on ongoing transcription and synthesis of at least one other protein. Of interest, the induction of 117β-HSD1 in HFL-1 cells and human amnion fibroblasts by both IL-1βand cortisol could also be blocked by transfection of CMV-500-A-C/EBP which expresses dominant negative C/EBP, suggesting that C/EBPs might be involved in the induction of 11β-HSD1 by both IL-1βand cortisol in both HFL-1 cells and human amnion fibroblasts.4. We further studied molecular mechanisms of the induction of 11β-HSD1 by IL-1βin HFL-1 cells. We found that:(1) NF-kB transactivation inhibitor 5HPP-33 could block IL-1β-induced COX-2 expression but not 11β-HSD1 expression, suggesting a unique pathway underlying the induction of 11β-HSD1 by IL-1β, which differs from the classical pro-inflammatory signaling pathway. (2) Reporter gene assay demonstrated that IL-1βstimulated 11β-HSD1 promoter activity, which was attenuated by mutagenesis of a C/EBP binding site in the promoter. (3) Western blotting results illustrated that HFL-1 cells expressed barely detectable C/EBPa but high level of C/EBPβthat could be induced dramatically by IL-1β. (4) C/EBPβwas found bound to the putative C/EBP binding site in 11β-HSD1 promoter upon IL-1βstimulation of HFL-1 cells with ChIP and EMSA. (5) Transfection of pMSV-C/EBPβplasmid over-expressing C/EBPβor CMV-500-A-C/EBP could mimic or antagonize the induction of 11β-HSD1 by IL-1β. The above results indicate that the induction of 11β-HSDl gene expression by IL-1βdepends on the activation of C/EBPβbinding to the CCAAT box in the promoter in HFL-1 cells.5. The molecular mechanisms underlying the induction of 11β-HSD1 by cortisol were further investigated in primary human amnion fibroblasts. Results showed that:(1) The induction of 11β-HSD1 by cortisol could be blocked by GR antagonist RU486, suggesting the induction is a GR-dependent process. (2) Report gene assay demonstrated that the induction of 11β-HSD1 promoter activity by cortisol was attenuated by mutagenesis of a glucocorticoid response element (GRE) and a CCAAT consensus sequence in the promoter, respectively. (3) Western blotting illustrated that human amnion fibroblasts expressed low C/EBPβbut high C/EBPαlevel that can be increased by cortisol. (4) ChIP and EMSA demonstrated that both GRαand C/EBPαcould bind the GRE and C/EBP binding site upon cortisol stimulation of human amnion fibroblasts (5) The induction of 11β-HSD1 by cortisol could be blocked by transfection of CMV-500-A-C/EBP. All these results lead to the conclusions that induction of 11β-HSD1 gene expression by cortisol involves both activation of GRαand C/EBPαbinding to the promoter in human amnion fibroblasts.Conclusions:11β-HSD1 is expressed in human fetal lung tissue at least at later gestation. The induction of 11β-HSD1 by IL-1βand cortisol holds true in HFL-1 cells as well as in primary human amnion fibroblasts. The induction of 11β-HSD1 gene expression by IL-1βdepends on the activation of C/EBPβbinding to the CCAAT box in the promoter in HFL-1 cells, while the induction of 11β-HSD1 gene expression by cortisol involves both activation of GRαand C/EBPαbinding to the promoter in human amnion fibroblasts. Based upon all these findings, we propose that C/EBPs be the key transcription factors involved in the induction of 11β-HSD1 by both IL-1βand cortisol.