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Endocrinology Vol. 147, No. 6 2879-2885
Copyright © 2006 by The Endocrine Society

A Novel Promoter for the 11ß-Hydroxysteroid Dehydrogenase Type 1 Gene Is Active in Lung and Is C/EBP{alpha} Independent

Charlotte Bruley, Val Lyons, Alan G. F. Worsley, Margaret D. Wilde, Gretchen D. Darlington, Nik M. Morton, Jonathan R. Seckl and Karen E. Chapman

Endocrinology Unit (C.B., V.L., A.G.F.W., N.M.M., J.R.S., K.E.C.), Centre for Cardiovascular Sciences, Queen’s Institute for Medical Research, University of Edinburgh, Edinburgh EH16 4TJ, Scotland, United Kingdom; and Huffington Center on Aging (M.D.W., G.D.D.), N805, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Karen E. Chapman, Endocrinology Unit, Centre for Cardiovascular Sciences, Queen’s Institute for Medical Research, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: Karen.Chapman{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) increases intracellular glucocorticoid action by converting inactive to active glucocorticoids (cortisol, corticosterone) within cells. It is highly expressed in glucocorticoid target tissues including liver and lung, and at modest levels in adipose tissue and brain. A selective increase in adipose 11ß-HSD1 expression occurs in obese humans and rodents and is likely to be of pathogenic importance in the metabolic syndrome. Here we have used 5' rapid amplificaiton of cDNA ends (RACE) to identify a novel promoter, P1, of the gene encoding 11ß-HSD1. P1 is located 23 kb 5' to the previously described promoter, P2. Both promoters are active in liver, lung, adipose tissue, and brain. However, P1 (encoding exon 1A) predominates in lung and P2 (encoding exon 1B) predominates in liver, adipose tissue, and brain. Adipose tissue of obese leptin-deficient C57BL/6J-Lepob mice showed higher expression only of the P2-associated exon 1B-containing 11ß-HSD1 mRNA variant. In contrast to P2, which is CAAAT/enhancer binding protein (C/EBP)-{alpha} inducible in transiently transfected cells, the P1 promoter was unaffected by C/EBP{alpha} in transfected cells. Consistent with these findings, mice lacking C/EBP{alpha} had normal 11ß-HSD1 mRNA levels in lung but showed a dramatic reduction in levels of 11ß-HSD1 mRNA in liver and brown adipose tissue. These results therefore demonstrate tissue-specific differential regulation of 11ß-HSD1 mRNA through alternate promoter usage and suggest that increased adipose 11ß-HSD1 expression in obesity is due to a selective increase in activity of the C/EBP{alpha}-regulated P2 promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN VIVO, 11ß-HYDROXYSTEROID dehydrogenase type 1 (11ß-HSD1) catalyzes the reduction of the inactive glucocorticoids, cortisone, and 11-dehydrocorticosterone, to their active counterparts, cortisol and corticosterone (1, 2). 11ß-HSD1 thus locally (within cells) increases ligand supply to glucocorticoid receptors, thereby elevating local glucocorticoid action (1, 2). The predominant reductase direction of 11ß-HSD1 in intact cells is conferred by virtue of its close proximity within the endoplasmic reticulum to hexose-6-phosphate dehydrogenase that supplies reduced cofactor, driving the reductase activity of 11ß-HSD1 (3, 4).

11ß-HSD1 is widely expressed, with high levels in liver and lung and moderate levels in adipose tissue and brain (5, 6, 7). In obese humans and in certain rodent models of obesity, 11ß-HSD1 expression is markedly increased specifically in adipose tissue, yet hepatic expression is either reduced or unchanged (8, 9, 10). This has led to the proposal that increased 11ß-HSD1 expression in adipose tissue raises intraadipose glucocorticoid action that contributes to, or is causal in, the pathogenesis of the metabolic syndrome of visceral (abdominal) obesity, insulin resistance/type 2 diabetes, hypertension, dyslipidaemia, and increased risk of cardiovascular disease (11, 12). In support of this hypothesis, transgenic overexpression of 11ß-HSD1 in adipose tissue recapitulates the essential features of metabolic syndrome in mice (10, 13). In contrast, 11ß-HSD1-deficient mice exhibit a cardioprotective metabolic phenotype (14), particularly on a high-fat diet (15, 16). Similarly, hepatic insulin sensitivity is improved by selective inhibition of 11ß-HSD1 in diabetic mice (17, 18) and by the nonselective 11ß-HSD inhibitor, carbenoxolone in humans (19, 20).

In contrast to the metabolic role of 11ß-HSD1 in liver and adipose tissue, 11ß-HSD1 has been implicated in xenobiotic detoxification (21) and surfactant production (22) in lung, the other major site of expression. The carbonyl reductase activity of 11ß-HSD1 is important in detoxification of the tobacco-specific carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (23, 24). Lungs of 11ß-HSD1-deficient fetal mice appear immature, with lower levels of surfactant compared with those of control mice (22).

Given the diversity of roles played by 11ß-HSD1 and its dysregulation in adipose in obesity, it is important to understand how the Hsd11b1 gene (encoding 11ß-HSD1) is regulated. 11ß-HSD1 mRNA levels are potently regulated by cytokines and hormones, with proinflammatory IL-1 and TNF-{alpha} increasing 11ß-HSD1 mRNA levels in several cell types (25, 26, 27, 28, 29, 30), whereas agonists for nuclear receptors—estrogen receptor, peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma}, and liver X receptor {alpha}—decrease 11ß-HSD1 expression in vivo (31, 32, 33, 34), although the latter are likely to be indirect effects (32, 33, 34, 35). In liver, C/EBP transcription factors exert a major direct control over Hsd11b1 gene transcription in vivo and in vitro, binding to several sites on the promoter (36). Of the C/EBP isoforms expressed in liver and adipose, C/EBP{alpha} is a potent activator of Hsd11b1. In the absence of C/EBP{alpha}, C/EBPß is a weak activator. However, if coexpressed with C/EBP{alpha} it acts as a relative repressor, antagonizing the effect of C/EBP{alpha} (36). C/EBPs are widely expressed, frequently transduce hormonal signals, are key factors regulating insulin sensitivity (37, 38) and are likely candidate intermediaries in hormonal/cytokine regulation of 11ß-HSD1.

Although a single Hsd11b1 transcription start site predominates in liver and hippocampus, in kidney transcription additionally initiates at another two sites in rat; 264 bp further 5' as well as within the first intron (39). The latter start site transcribes an 11ß-HSD1 mRNA encoding a nonfunctional protein (40, 41). Furthermore, although two different cDNAs cloned from mouse liver (5, 42) encode identical proteins, the cDNA sequences differ in the 5' untranslated region (UTR), suggesting alternate transcription starts. To resolve this uncertainty and to investigate the basis for adipose dysregulation of 11ß-HSD1 in obesity, we have determined the transcription start site of Hsd11b1 in adipose tissue and found evidence of a novel promoter. Here we report tissue specificity of the novel promoter and its independence of C/EBP{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male (8–10 wk) C57BL/6J and ob/ob mice (Harlan Olac, Oxon, UK) were given rodent chow and water ad libitum and housed under standard conditions on a 12-h light, 12-h dark cycle (lights on at 0700 h) at a temperature of 21 ± 1 C. Mice homozygous for a deletion in the gene encoding C/EBP{alpha} were generated as previously described (43). All animal experimentation was conducted in strict accord with accepted standards of humane animal care after prior approval by local ethical committees.

RNA extraction and analysis
Tissues were frozen on dry ice and RNA extracted after homogenization in Trizol (Invitrogen, Paisley, Scotland, UK). RNA was resuspended in deionized formamide for ribonuclease (RNase) protection assays and in RNase-free water for PCR and Northern analysis. Northern blotting was carried out on total RNA isolated from newborn homozygous C/EBP{alpha}-deficient mice or their heterozygous or wild-type control littermates as previously described (36) using a mouse 11ß-HSD1 cDNA probe encoding nucleotides 158–618 (5).

5'-RACE PCR
5'-RACE was carried out on mouse mesenteric fat RNA using a FirstChoice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer’s instructions. This RACE procedure is specific for capped RNAs and therefore only amplifies from full-length mRNA. First-strand synthesis of cDNA was carried out on 2 µg total RNA in a 20-µl reaction using the outer RNA adapter primer supplied with the kit and 3'-primer, 5'-CCATAGTGCCAGCAATGTAGTGAG-3', according to the manufacturer’s protocol. A total of 1.5 µl of the PCR was used in a nested PCR with 3' primer 5'-TTTCTCTTCCAATCCTTGCTGG-3' and the inner RNA adapter primer supplied with the kit. PCR products were cloned, without further purification, into pGEM-Teasy (Promega, Madison, WI), and sequenced using a T7 primer.

RNase protection assays
RNase protection assays were carried out using a HybSpeed RPA kit (Ambion). Probes were synthesized from NcoI-linearized plasmids using [{alpha}-32P]-GTP (3000 Ci/mmol; Amersham International, Buckinghamshire, UK) and SP6 phage polymerase to transcribe cRNA. The exon 1A template consisted of a subcloned RT-PCR product from lung RNA (primers 5'-GGATGAGACAGAAGGATAGAGA-3' and 5'-TCAAGGCAGCGAGACACTAC-3'). The exon 1B template was a subcloned 5'-RACE product.

RNA was coprecipitated with 5–10 x 105 cpm 32P-labeled cRNA probe, resuspended in 20 µl hybridization buffer (supplied with the kit) at 95 C and incubated at 68 C for 1 h. Reactions were incubated with RNase A/T1 (1/100 dilution) for 30 min at 37 C. RNA products were separated on a 4% polyacrylamide gel containing 7 M urea and visualized using autoradiography or a Fuji FLA 2000 phosphorimager (Raytek Scientific Ltd., Sheffield, UK).

Transfections
pGL3-P2(–1799/+49) was generated by subcloning a HindIII fragment encoding –1799 to +49 of the P2 promoter of Hsd11b1 from pr11ß1(–1799/+49) (44) into the promoterless vector, pGL3-Basic (Promega). pGL3-P1(–2928/+89), pGL3-P1(–2127/+89) and pGL3-P1(–895/+89) were created in pGL3-Basic by subcloning PCR products obtained from a bacterial artificial chromosome DNA template (RP23–451H2) using the following primers: common 3' primer, 5'-CCCAAGCTTCAGATAAGTGCGGCTCCTT-3' and 5' primers, (–895) 5'-GAAGATCTGTGCCTATATATGTGTGTCTGC-3', (–2127) 5'-GAAGATCTATGAGATCCCTTGCTGCT-3', (–2928) 5'-GAAGATCTGATTCTTGCCCTCATTT-3'; restriction sites used in cloning are underlined. Numbering is with respect to the most 5' transcription start site identified. pMSV-C/EBP{alpha} was a gift from S. L. McKnight and W.-C. Yeh.

HepG2 cells were maintained and transfected as previously described (36). 5 x 105 cells seeded per 60-mm dish were transfected using the calcium phosphate procedure with 5 µg test plasmid, 1 µg pRSV-LacZ (as internal control), 1 µg pMSV-C/EBP{alpha} or pMSV vector, made to a total of 10 µg with pGEM3 (Promega). A549 cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. For transfection, 1.5 x 105 cells were seeded per well of a six-well plate and transfected the next day using lipofectamine (Invitrogen) with 250 ng test plasmid, 250 ng pRSV-LacZ and 50 ng pMSV-C/EBP{alpha} or pMSV vector.

Statistics
Data were analyzed using ANOVA. Significance was set at P < 0.05. Values are mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5'-RACE shows alternate promoter usage in adipose tissue
To determine the predominant transcription start site of the Hsd11b1 gene in adipose tissue, 5'-RACE analysis was carried out on mouse mesenteric fat RNA. Sequencing of 16 independent clones revealed two variant 11ß-HSD1 mRNAs with differing 5'UTR (Fig. 1Go). One class, represented by 13 clones, revealed a cluster of transcription initiation sites which mapped to genomic sequence approximately 100 nucleotides 5' to the translation start, here designated P2 (Fig. 1BGo). This transcription start is homologous to that identified in rat liver (39). One additional clone clearly contained the same 5'UTR (designated exon 1B) but initiated further 3' to the majority of the clones. The other class, represented by two clones, contain the same exon 1 sequence (designated exon 1A) as a cDNA previously isolated from a mouse liver cDNA library (42). In these clones, transcription initiates from a putative promoter, P1, which lies approximately 23 kb 5' of the translation start (Fig. 1AGo). Splicing from exon 1A occurred to exon 2, before the translation start. Thus, both variant mRNAs encode the same protein but differ in the 5'UTR and the promoter, P1 or P2, used to initiate transcription.


Figure 1
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  		FIG. 1. 5'-RACE identification of transcription starts for the mouse Hsd11b1 gene. Sequence analysis of 16 5'-RACE clones indicated two promoters for the Hsd11b1 gene, 23 kb apart. The transcription start of two clones (A) mapped to a genomic region approximately 23 kb upstream of the cluster of transcription start sites contained within the remaining 14 5'-RACE clones (B). A, Sequence of mouse genomic DNA surrounding the P1 transcription start. Sequence in lower case indicates nontranscribed genomic DNA, and upper case indicates sequences identified here as transcribed (designated exon 1A). Numbering is according to the February 2005 release of the mouse genome sequence of chromosome 1 (ensembl). The 5' end of the 2 RACE clones are indicated by * below the sequence. Splicing occurred from a splice donor site at the 3' end of exon 1A into the downstream exon 1 (the common region is here designated exon 2), 26 nucleotides 5' to the translation start. The 5' end of a previously reported cDNA (42 ) isolated from a liver cDNA library is indicated ({Delta}), as is the 5' end of the reference sequence in ensembl ({diamond}). B, DNA sequence surrounding the P2 transcription start. Sequence in upper case indicates sequences identified here as transcribed (designated exon 1B-exon 2). The 5' end of the 14 RACE clones are indicated by * below the sequence, with numbers referring to the number of clones initiating at the indicated nucleotide. The position at which splicing occurred from exon 1A is indicated below the sequence (^), which is preceded by an AG splice acceptor site. C, Representation of exon 1A and exon 1B relative to exon 2 containing the translation start. Exon 1B and exon 2 are contiguous. Open boxes represent exon sequences, a thick black line indicates genomic DNA and promoters (P1 and P2) are indicated by bent arrows.

 
Variant 11ß-HSD1 mRNAs are tissue specific
RT-PCR, using 5' primers specific for exon 1A or 1B and a common 3' primer in exon 4, showed that both 11ß-HSD1 mRNA variants are present in liver, lung, and brain, as well as adipose tissue. No RT-PCR product was obtained with testis that, in the mouse, does not express 11ß-HSD1 (5) (data not shown). To investigate the relative abundance of each of the variant 11ß-HSD1 mRNAs, RNase protection analysis was carried out on RNA from lung, liver, brain, brown adipose tissue (BAT), and white adipose tissue. In addition, as 11ß-HSD1 mRNA is increased in adipose tissue of leptin-deficient obese ob/ob mice (10), we also examined RNA isolated from obese ob/ob mouse adipose tissue. Two cRNA probes were used, each complementary to a specific exon 1 as well as the common region of the transcripts, from exon 2. Thus, each probe gives two products; a long fragment protected from RNase by the 11ß-HSD1 mRNA variant containing the corresponding exon 1-exon 2 sequence and a shorter fragment produced from 11ß-HSD1 mRNA containing exon 2 but lacking the target exon 1 sequence.

RNase protection assays clearly showed that although exon 1B-containing 11ß-HSD1 mRNA predominates in liver, brain, BAT, and white adipose depots, the majority of 11ß-HSD1 mRNA in lung contains exon 1A. RNase protection assays using an exon 1A-specific probe showed a protected fragment of 363 nucleotides (corresponding to 98 nucleotides of exon 1A and 265 nucleotides of exons 2–4) in lung (Fig. 2AGo). In contrast, very little, if any, exon 1A-containing transcript was detectable in RNA from liver, brain, BAT, or white adipose depots (Fig. 2AGo). Interestingly, although a strong 11ß-HSD1 signal was apparent in adipose tissue of ob/ob mice, there was no increase in exon 1A-containing 11ß-HSD1 mRNA, ruling out an increase in activity of promoter P1 in obesity. In contrast, when a cRNA probe specific for exon 1B was used, a protected 243 nucleotide fragment corresponding to exon 1B-exon 2/3 was produced with RNA from liver, brain, BAT, and white adipose tissue, with much lower levels apparent in lung (Fig. 2BGo).


Figure 2
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  		FIG. 2. Tissue-specific expression of 11ß-HSD1 mRNA variants. RNase protection assays using cRNA probes complementary to exon 1A-exon 2–4 (A) or to exon 1B-exon 2/3 (B) were carried out on RNA isolated from tissues of leptin-deficient obese ob/ob (o/o), heterozygous (o/+) or control mice (+/+): liver (L), lung (Lu), brain (Br), epididymal adipose (Epi), mesenteric adipose (Mes), sc adipose (Sc) and BAT (Ba). A, Protection of the 461 nucleotide exon 1A-exon 2–4 probe by transcripts starting at P1 produces a 363 nucleotide fragment (exon 1A-exon 2–4) whereas protection of the probe by transcripts initiated from P2 produces a 265 nucleotide fragment (exons 2–4). A minor band at approximately 320 nucleotides results from partially digested probe. Assays contained 20 µg total RNA except for lanes 4 (50 µg) and lanes 1, 6, 7, and 11 (each 10 µg). B, Protection of the 341 nucleotide exon 1B-exon 2/3 probe by transcripts starting at P2 produces a 243 nucleotide fragment (exon 1B-exon 2/3), whereas protection of the probe by transcripts initiated from P1 produces a 176 nucleotide fragment (exons 2/3). Due to a fold in the dried gel, the 243 nucleotide fragment appears higher in lanes 7 and 8. Assays contained 10 µg total RNA except for lanes 3 (50 µg) and 4 (15 µg).

 
In contrast to the proximal (P2) promoter, the distal (P1) promoter is C/EBP{alpha}-independent in vitro and in vivo
To determine whether the exon 1A sequence present in the cDNA is associated with a promoter, plasmids encoding the putative P1 promoter including most of the untranslated exon 1A were transfected into HepG2 (human hepatoma), JEG3 (human placental trophoblast), or A549 (human lung alveolar) cells. Promoter activity was compared with that of a plasmid encoding approximately 1800 bp of the P2 promoter, which we have previously shown to be C/EBP-regulated (36).

Whereas the construct containing 2.9kb of P1 promoter sequence was inactive, deletion of 800 bp to –2127 unmasked strong promoter activity in all cell lines (Fig. 3Go), suggesting the presence of a repressor element between –2938 and –2127. Further deletion to –895 reduced promoter activity in all cell lines (Fig. 3Go). Cotransfection of C/EBP{alpha} had no significant effect on the activity of either of the active P1 promoter constructs, P1(–2127/+89) or P1(–895/+89) (Fig. 3Go), whereas it increased the minimal basal level activity of the P2 promoter in HepG2 cells by 7-fold and by 18-fold in A549 cells (Fig. 3Go). In JEG3 cells, the P2 promoter was neither basally active nor C/EBP{alpha}-inducible (Fig. 3CGo), indicating that additional factors, missing in JEG3 cells, are required for Hsd11b1 P2 promoter activity and/or C/EBP{alpha} activity in this cell line.


Figure 3
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  		FIG. 3. In contrast to the P2 promoter of the Hsd11b1 gene, the P1 promoter is not regulated by C/EBP{alpha}. HepG2 hepatoma (A), A549 lung (B), and JEG3 placental (C) cells were cotransfected with an expression plasmid encoding C/EBP{alpha} (hatched bars) or empty vector (black bars) together with luciferase reporter plasmids; pGL3-basic (vector), P2(–1799/+49) encoding –1799 to +49 of the P2 promoter of rat Hsd11b1 [numbering with respect to the transcription start (39 )], P1(–2938/+89), P1(–2127/+89) or P1(–895/+89) encoding, respectively, –2938, –2127 or –895 to +89 of the P1 promoter of mouse Hsd11b1 [numbering with respect to the longest cDNA identified (42 )]. Values are mean ± SEM of at least three transfection experiments, each carried out independently in triplicate. *, Significantly different to corresponding vector control; P < 0.05.

 
The lack of C/EBP{alpha} effect upon P1 promoter activity predicts that 11ß-HSD1 mRNA transcription from this promoter should be C/EBP{alpha} independent. In contrast, we have shown transcription from the P2 promoter is C/EBP{alpha}-dependent in liver in vivo (36). Because lung RNA predominantly contains exon 1A initiating from the distal promoter, we have compared levels of 11ß-HSD1 mRNA in RNA isolated from lung of C/EBP{alpha}–/– mice and their control littermates. C/EBP{alpha}–/– mice had a dramatic reduction in the levels of 11ß-HSD1 mRNA in their livers, whereas the levels in lung were completely unaffected (Fig. 4Go). C/EBP{alpha}–/– mice lack white adipose tissue but retain BAT (43, 45). Consistent with the predominance of P2 in adipose tissue, C/EBP{alpha}–/– mice had reduced levels of 11ß-HSD1 mRNA in their BAT compared with normal or heterozygous littermates (Fig. 4Go).


Figure 4
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  		FIG. 4. The P1 promoter of Hsd11b1 is C/EBP{alpha}-independent in vivo. Northern analysis of 11ß-HSD1 mRNA in liver, lung, and BAT of C/EBP{alpha}–/– (–/–), C/EBP{alpha}+/– (+/–) and control (+/+) mice. A, Lower panel is a longer exposure of the same autoradiograph as shown in the upper panel, to show detection of 11ß-HSD1 mRNA in BAT. The larger hybridizing band visible in lung and control liver RNA is likely to arise from alternate polyadenylation of 11ß-HSD1 mRNA. B, Ethidium bromide-stained gel to show loading of RNA. Lanes contained 25 µg total RNA except for lanes 13 (22 µg), 14 (18 µg), 15 (26 µg), 16 (18 µg), 17 (15 µg), and 18 (12 µg).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we have identified two promoters that actively transcribe the mouse Hsd11b1 gene. Transcription from P2 predominates in liver, adipose tissue, and brain, although in these tissues a significant minority of 11ß-HSD1 mRNA initiates from P1, consistent with the previous isolation of an exon 1A-containing cDNA from liver (42). The converse is true in lung, where the predominant species of 11ß-HSD1 mRNA contains exon 1A.

Transcription from P2 initiates at a discrete cluster of sites, homologous to the predominant transcription start site used in rat liver (39) and proceeds through exons 1B and 2, which are contiguous. P1 is located approximately 23 kb 5' to exon 2, and encodes a 5'UTR similar to that previously described in a mouse liver cDNA (42). Some minor sequence discrepancies at the exon 1-exon 2 boundary are likely to result from errors in the previous report (42). No 5'-RACE clones were identified corresponding to the alternate transcription start sites previously identified in rat kidney (39). Neither the novel exon 1A nor exon 1B contains an additional ATG codon, suggesting that the alternate promoter usage does not alter the encoded protein. It is possible, however, that the 5'UTR may influence translation efficiency.

As in liver, 11ß-HSD1 expression in BAT is dependent upon C/EBP{alpha}, consistent with the predominance of P2 in adipose tissue. C/EBP{alpha} is required for lipid accumulation in white adipose tissue (37, 43) and C/EBP{alpha}-deficient mice have a dramatic reduction in white adipose tissue (43), so it was not possible to test whether in white adipose tissue 11ß-HSD1 expression is similarly dependent upon C/EBP{alpha}, although we predict, from the RNase protection data (showing similar predominance of P2 in both white and BAT), that it will be. The residual 11ß-HSD1 mRNA in liver of C/EBP{alpha}-deficient mice may represent the minority of 11ß-HSD1 transcripts that originate from P1. Alternatively, it could be due to the action of transcription factors other than C/EBP{alpha} (e.g. C/EBPß) at the P2 promoter. Expression of 11ß-HSD1 in lung is C/EBP{alpha} independent. The C/EBP{alpha} independence of P1 and the C/EBP{alpha} dependence of P2 was borne out in two cell lines, HepG2 hepatoma cells and A549 lung alveolar cells. In contrast, in JEG3 cells, the Hsd11b1 gene P2 promoter was completely inactive, irrespective of the presence of C/EBP{alpha}, although it is C/EBP{alpha} regulated in HeLa and MCF7 cells (Lyons, V., and K. E. Chapman, unpublished data). The interaction of C/EBP{alpha} with other cell-specific factors is clearly important in determining activity of P2. The largest P1 reporter plasmid was inactive in all three transfected cell lines. The significant basal activity of P2 and lack of P1 activity in A549 cells was unexpected for A549 cells, which are derived from human lung (46). However, A549 cells resemble type II alveolar cells (46), which express 11ß-HSD1 at 10-fold lower level than lung interstitial fibroblasts (47). This 10:1 (fibroblast:alveolar cell) ratio is similar to the exon 1A:exon 1B ratio in 11ß-HSD1 mRNA in lung. These data raise the possibility that the C/EBP{alpha}-dependent 11ß-HSD1 P2 promoter is active in the surfactant producing type II alveolar epithelial cells, whereas the C/EBP{alpha}-independent P1 promoter is active in the interstitial fibroblasts, accounting for the majority of 11ß-HSD1 mRNA expression in lung. Consistent with this, C/EBP{alpha} is highly expressed in type II alveolar cells (48) and C/EBP{alpha}-deficient mice show hyperproliferation of type II pneumocytes and disturbed alveolar architecture (49).

Compared with liver and lung, adipose tissue expression of 11ß-HSD1 is modest in lean mice but is dramatically increased in obese mice in which plasma glucocorticoid levels are maintained or elevated (10). Although we were able to detect exon 1A-containing 11ß-HSD1 mRNA in adipose tissue of obese ob/ob mice, clearly the majority contained exon 1B, and we are able to rule out increased P1 promoter activity as a cause of the increased adipose 11ß-HSD1 expression. Similarly, obese Zucker rats (which have a mutated leptin receptor) have increased adipose 11ß-HSD1 (8). Levels of C/EBP{alpha} are severalfold elevated in adipose tissue but not in liver of obese Zucker rats (50). If C/EBP{alpha} is similarly elevated specifically in adipose of ob/ob mice, this might explain the selective increase in adipose of Hsd11b1 gene P2 activity and suggests that the increased adipose 11ß-HSD1 in Zucker rats reflects increased P2 activity. Adipose C/EBP{alpha} levels are increased in human obesity (51), suggesting that this may be the underlying mechanism for the increased adipose 11ß-HSD1 expression in human obesity.

The upstream exon 1A is conserved in humans. Although not specifically detailed in the literature, a placental cDNA has been identified that is clearly homologous to exon 1A-containing 11ß-HSD1 mRNA (RefSeq NM_181755). The genomic sequence of exon 1A, including the splice junction and the 3' flanking region, is well conserved among mice, humans, and rats, although the extended region between exon 1A and exon 1B is considerably less well conserved. In human liver, transcription of the HSD11B1 gene predominantly starts at the identical P2 transcription start site (RefSeq NM_005525; http://dbtss.hgc.jp/index.html) to that in rat liver and mouse adipose encoding an mRNA containing exon 1B. The P1 promoter and exon 1A have not yet been documented in rat, but because the genomic region encoding exon 1A is highly conserved between rat and mouse, it is extremely likely that P1 is also used in rat to transcribe 11ß-HSD1 in lung and possibly elsewhere.

In contrast to P2, which initiates transcription at a tight cluster of start sites, there does not appear to be a single predominant transcription start site for P1. Rather, transcription initiates at staggered start sites within a highly purine-rich region. The region is not contained within a CpG island, in which typically, transcription initiates from staggered transcription starts. Intriguingly, the P1 promoter, although some 23 kb 5' to exon 2, is only 8 kb downstream of the neighboring gene, G0s2, that is transcribed in the same direction as Hsd11b1. The proximity suggests the two genes may interact or be coregulated in some way. The protein encoded by G0s2 is small (14 kDa) and of unknown function (52). G0S2 is highly expressed in adipose tissue, is induced during adipogenesis, and is a PPAR target gene (53). Furthermore, like 11ß-HSD1, the protein is localized within the endoplasmic reticulum (53). The relationship between the two neighboring genes will be important to dissect and is likely to provide important information on the regulation of 11ß-HSD1.


   Acknowledgments
 
We thank members of the Endocrinology Unit for helpful discussions and Lynne Ramage for technical help.


   Footnotes
 
This work was supported by a Wellcome Trust Programme grant. N.M.M. is supported by a Wellcome Trust Intermediate Fellowship.

Disclosure Statement: C.B., V.L., A.G.F.W., M.D.W., G.D.D., N.M.M., and K.E.C. have nothing to declare. J.R.S. has consulted for Incyte, Vitae Pharmaceuticals, Ipsen, and Janssen.

First Published Online March 16, 2006

Abbreviations: BAT, Brown adipose tissue; C/EBP-{alpha}, CAAAT/enhancer binding protein; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; PPAR, peroxisome proliferator-activated receptor; RACE, rapid amplificaiton of cDNA ends; RNase, ribonuclease; UTR, untranslated region.

Received December 20, 2005.

Accepted for publication March 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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