title: Analysis of alternative promoter usage in expression of HSD11B1 including the development of a transcript-specific quantitative real-time PCR method.
abstract: Overexpression of the microsomal enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, human gene HSD11B1 or SDR26C1) associates with the metabolic syndrome as well as some inflammatory diseases. HSD11B1 expression is known to be highly tissue-specific and driven by two distinct promoters, an aspect which to date has been studied very little. Here, we sought to assess differential promoter usage in various glucocorticoid target tissues and cell lines. As transcription from the distal promoter P1 or the proximal promoter P2 results in transcripts differing in the 5'-UTR, we first used 5'-UTR-specific primers for their detection in semi-quantitative PCR after reverse transcription. We also established a quantitative Real-Time PCR method using 5'-UTR-specific fluorescent probes in combination with 5'-UTR-specific primers for absolute quantification of the two human transcripts in a duplex approach. The combined results demonstrate that transcription from P2 predominated in most tissues and cell lines assessed, including human liver, human lung, human subcutaneous adipose tissue, and the cell lines A549, Caco-2, C2C12 and 3T3-L1. Transcription from P1 predominated in the human tumor cell lines A431 and HT-29 and contributed significantly to overall HSD11B1 expression in human lung. Moreover, HSD11B1 transcription was upregulated in C2C12 and 3T3-L1 cells in the course of differentiation to myotubes and adipocytes, respectively, mostly reflecting increased transcription from P2, although also transcription from P1 gradually increased in the differentiation process. Interestingly, HSD11B1 transcript levels in all tested human tumor cell lines, namely A431, A549, Caco-2, HCT-116, HepG2, HT-29 and PANC-1, were either close to detection limit or undetectable. In conclusion, the results provide the first evidence for tissue- and differentiation state-specific promoter usage in expression of human HSD11B1. Finally, HSD11B1 appears to be frequently downregulated in human tumor cell lines.
authors.affiliation: Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Kiel, Germany.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Staab
authors.forename: Jochen P
authors.initials: JP
authors.lastname: Stegk
authors.forename: Sierk
authors.initials: S
authors.lastname: Haenisch
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Neiss
authors.forename: Katrin
authors.initials: K
authors.lastname: Köbsch
authors.forename: Bettina
authors.initials: B
authors.lastname: Ebert
authors.forename: Ingolf
authors.initials: I
authors.lastname: Cascorbi
authors.forename: Edmund
authors.initials: E
authors.lastname: Maser
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000235:genetics; D041721:3T3-L1 Cells; D020121:5' Untranslated Regions / Q000235:genetics; D000328:Adult; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D050151:Subcutaneous Fat / Q000166:cytology / Q000378:metabolism; D013997:Time Factors
chemical_list: D020121:5' Untranslated Regions; D005456:Fluorescent Dyes; D005938:Glucocorticoids; D012333:RNA, Messenger; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C468386:HSD11B1 protein, human