17 b -Hydroxysteroid dehydrogenase type 11 is a major peroxisome proliferator-activated receptor a-regulated gene in mouse intestine Kiyoto Motojima Department of Biochemistry, Meiji Pha
Trang 117 b -Hydroxysteroid dehydrogenase type 11 is a major peroxisome proliferator-activated receptor a-regulated gene in mouse intestine Kiyoto Motojima
Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan
In order to study the role of peroxisome
proliferator-acti-vated receptor a in mouse intestine, its agonist-induced
proteins were identified by peptide mass fingerprinting
fol-lowed by Northern blot analysis using their cDNAs One of
the most remarkably induced proteins was identified as
17b-hydroxysterol dehydrogenase type 11 Its very rapid
induction by various agonists was most efficient in intestine
and then in liver These findings together with recently
reported results showing the enzyme family’s wide substrate
spectrum, including not only glucocorticoids and sex ster-oids but also bile acids, fatty acids and branched chain amino acids, suggest new roles for both peroxisome proliferator-activated receptor a and 17b-hydroxysterol dehydrogenase type 11 in lipid metabolism and/or detoxification in the intestine
Keywords: PPAR; intestine; hydroxysteroid dehydrogenase; lipid metabolism
Peroxisome proliferator-activated receptors [PPARa, b(d),
and c] are members of the nuclear hormone receptor
superfamily and function as ligand-dependent transcription
factors, playing crucial roles in several processes including
energy metabolism, cellular differentiation, and
inflamma-tion [1,2] It is now well accepted that PPARa is particularly
important in lipid catabolism in the liver by upregulating
the expression of a variety of genes that encode proteins
involved in fatty acid transport [3], a-oxidation and
lipoprotein metabolism [4,5] However, it is important to
point out that these studies have been mostly carried out
using rodent models and strong synthetic PPARa agonists
PPARa was originally cloned from a mouse cDNA
library to explain a rodent-specific response called
per-oxisome proliferation to a variety of synthetic compounds
[6] The amount of PPARa in the mouse liver is 10 times
higher than that in human liver, and fibrates, hypolipidemic
drugs and PPARa agonists do not cause peroxisome
proliferation and a large induction of proteins involved in
lipid metabolism in human liver [7,8] Thus there is a
possibility that our knowledge on the role of PPARa in lipid
metabolism is biased against its extra-hepatic functions
In this study, I examined the PPARa agonist-induced
proteins in the intestine, another important organ for lipid
metabolism expressing a fairly large amount of PPARa in
mouse and human, to obtain new insight into the roles of
PPARa A major change in the intestine at the protein level,
namely a rapid induction of 17b-hydroxycholestrol
dehy-drogenase type 11 (17b-HSD-11) by PPARa ligand, was identified
Materials and methods Animals and treatment Normal male C57BL and PPARa-null mice [9] were kept under a 12-h light–dark cycle and provided with food and water ad libitum Mice were fed either a control diet or
a diet containing a drug at the concentration (%, w/w) and for the number of days or weeks indicated in the figure legends All animal procedures were approved by the Meiji Pharmaceutical University Committee for Ethics of Experi-mentation and Animal Care
Preparation of postnuclear fractions and SDS/PAGE analysis
The livers and intestinal mucosa from wild (C57BL) or PPARa-null mice fed a control or a diet containing Wy14 643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]acetic acid} purchased from Tokyo-Kasei, Tokyo, Japan) for
5 days were homogenized in five volumes of sucrose buffer [0.25M sucrose, 1 mM EDTA, 0.1% (v/v) ethanol, the protease inhibitor mixture (Wako, Tokyo, Japan), pH 7.4]
by Potter–Elvehjem homogenizer [10] The homogenates
were centrifuged for 13 min at 2000 g and the supernatant,
postnuclear fraction was obtained Total proteins in the intestinal postnuclear fraction of wild-type mice fed a diet containing 0.05% Wy14 643 were separated by SDS/PAGE (12% gel) as described previously [10]
Identification of Wy14643-induced proteins The proteins separated by SDS/PAGE were electric-ally transferred to a nylon membrane (Immobilon, Millipore, MA, USA) and stained with Coomassie blue The proteins of interest were excised from the membrane,
Correspondence toDepartment of Biochemistry, Meiji Pharmaceutical
University, 2-522-1 Noshio, Kiyose, Tokyo 204–8588, Japan.
Tel./Fax: +81 424 958474; E-mail: motojima@my-pharm.ac.jp
Abbreviations: FABP, fatty acid binding protein; HSD,
hydroxy-steroid dehydrogenase; PMF, peptide mass fingerprinting; PPAR,
peroxisome proliferator-activated receptor.
(Received 20 July 2004, revised 24 August 2004,
accepted 31 August 2004)
Trang 2carboxymethylated and digested with endoproteinase
Lys-C The resultant peptides were subsequently analyzed
by MALDI-TOF mass spectrometry The spectra were used
to identify the proteins, using theMS-FIT search program
[11] Each protein band contained two or more proteins
and the protein mass fingerprinting alone could not identify
the proteins of interest without ambiguity
RNA preparation and Northern blotting
Total RNA was prepared from the mouse tissues and
cultured cells by the acid guanidinium isothiocyanate/
phenol/chloroform extraction method [12] Northern
blot-ting analysis was carried out essentially as described
previously using Express Hyb hybridization solution
(Clon-tech, CA, USA) [13] The cDNAs used for probes were
described previously [3,14] or obtained by the cloning of
PCR products of cDNA synthesized from poly(A+) RNA
isolated from the liver of Wy14,543-fed mice The
synthe-ticoligonucleotides used to amplify the respective cDNA
sequences were 5¢- GGGAATTCGTTTAGGACCGGGA
ACGAGAGC-3¢ and 5¢-CCCTCGAGCGAAATCCCTG
CAAGCACCTGT-3¢ for 17b-HSD-11 (corresponding to
nucleotide numbers 62–860 of the published sequence with
additional nucleotides for restriction enzyme digestion
underlined; GenBank accession number AK049355);
5¢-GGGAATTCGACGGGCGTGTGGTGTTGGTCA-3¢ and 5¢-GGCTCGAGGAAGTGGCTTATACAGCTC
CAA-3¢ for 17b-HSD-4 (corresponding to nucleotide
num-bers 43–1273 of the published sequence with additional
nucleotides underlined; GenBank accession number
NM008292) The PCR products were digested with EcoRI
and XhoI, cloned into a plasmid vector, and sequenced for
identification
Cell culture and DNA transfection
Fao cells (a subclone of rat hepatoma HIIE cells) were
cultured in Ham’s F-12 medium, and CV-1 cells (monkey
kidney-derived cells) were cultured in minimal essential
medium under the conditions described previously [13]
A PPARa ligand Wy14 643 was added to the medium at
a final concentration of 50 lM (Fao cells) or 100 lM
(CV-1 cells) A 1.8 kb DNA fragment containing possible enhancer sequences in the 17b-HSD-11 gene promoter was amplified by PCR using the mouse genomic DNA and the oligonucleotide primers The entire fragment and digested enhancer truncated fragment were cloned into the enhancer vector pGL3 (Promega, WI, USA) Transfection was performed in 24-well plates with SuperFect (Qiagen, CA, USA) using the Dual Luciferase assay system (Promega) according to the manufacturer’s protocol [13]
Results and Discussion SDS/PAGE analysis of the proteins whose expression levels were regulated by PPARa and its ligand
To detect the protein bands whose expression levels were markedly altered by administration of a PPARa ligand, postnuclear fractions of the liver and intestine were prepared from mice fed a PPARa ligand for 5 days The result of one-dimensional SDS/PAGE analysis of these proteins from wild (C57BL) or PPARa-null mice fed a control or Wy14,643-containing diet is shown in Fig 1 As already reported [14,15], several proteins including peroxisomal enzymes were largely induced in the liver of wild mice by feeding Wy14 643 and it was difficult to assign uncharacterized new protein bands on one-dimensional gels In contrast, the number of affected protein bands was limited in the intestine of wild mice and we chose three protein bands as uniquely increased
in the intestine by Wy14 643 in a PPARa-dependent manner These include proteins having molecular masses of 32, 78 and
80 kDa as shown by arrows in Fig 1B
Peptide mass fingerprinting (PMF) analysis
of the PPARa-regulated proteins in the intestine Among the three proteins bands, I further selected P32 and P78 for PMF analysis, because they seemed to be expressed
Fig 1 Effects of Wy14 643 on protein expression in the liver and intestine of control and PPARa-null mice Wild-type (+/+) and PPARa-null mice (–/–) were fed either a control (–) diet or one containing 0.05% Wy14 643 (+) for 5 days The proteins in postnuclear fractions from the liver and intestine were analyzed by SDS/PAGE (10%, w/v) followed by staining with Coomassie brilliant blue (A) The portions (indicated by boxes) of the gel containing the induced proteins (indicated by arrows) in the intestine are shown enlarged (B).
Trang 3more in the intestine than in the liver and isolated from
other major protein bands on the one-dimensional gel After
enzymatic digestion using endoproteinase Lys-C and
ana-lysis of the resulting peptides by MALDI-TOF mass
spectrometry, the masses of 12 among 29 peptides derived
from P32 were consistent with those calculated from
the peptide sequences from 17b-HSD-11 (gi|16716597|
ref|NP_444492.1|), and the masses of 12 peptides matched
those from annexin IV (gi|7304889|ref|NP_038499.1|) The
masses of 18 among 79 peptides derived from P80 were
consistent with those calculated from the peptide sequences
from 17b-HSD-4 (gi|1706397|sp.|P51660|) The peptides
from P80 also contained 32 peptides from Ezrin
(gi|32363497|sp.|P26040|) and 18 peptides from P450
oxidoreductase (gi|6679421|ref|NP_032924.1|)
Among these proteins, 17b-HSD-4 is known as a
peroxisomal enzyme and induction of several peroxisomal
enzymes in the liver has been extensively studied [16,17]
17b-HSD-4 is not a major protein in the liver, but it could be
identified as a distinct protein band on the one dimensional
gel of all the proteins in the post nuclear fraction probably
because of the absence of abundant liver-specific proteins in
the intestine Furthermore, the increase of 17b-HSD-4
caused by the PPARa ligand in the liver as protein amount
was not remarkable when compared with that of mRNA,
and Corton et al suggested the possibility of
post-trans-lational regulation of the protein levels in the liver [16]
It was noteworthy that two types of 17b-HSDs were identified as the most remarkably increased proteins in the intestine by PPARa ligand in total protein mixture of the postnuclear fraction of the mouse intestine 17b-HSD-4 is a multifunctional protein involved in not only inactivation of estradiol but also successive steps of a-oxidation of long-and branched-chain fatty acids in peroxisomes In contrast, 17b-HSD-11 is a new member of the 17b-HSD family [18] Mouse 17b-HSD-11 was found from a large set of full-length cDNAs by sequence homology and functional annotation [19] and human 17b-HSD-11 was identified in expressed sequence tag databases with conserved domains
of the family members [20] and therefore its function has not been fully characterized yet [18] 17b-HSD-11 has no peroxisomal targeting signal at the C-terminus but a possible hydrophobic signal sequence at the N-terminus The N-terminal sequence was expected to be cleavable by signal peptidase (SignalP, http://www.cbs.dtu.dk) but the exact N-terminal sequence was detected during PMF analysis of P32 protein as shown in Table 1 Thus 17b-HSD-11 should be a membrane protein and our preliminary data using green fluorescence protein linked
to the C-terminus of the protein suggested association with the endoplasmic reticulum In contrast, Chai et al [20] recently reported that the myc-tagged human 17b-HSD-11
at the N-terminus localized in the cytoplasm The tagged myc sequence should have abolished the function of the
Table 1 Summary of PMF analysis of P32 and P80 The residue numbers for P32 are from the peptide sequence of 17b-HSD-11 and those for P80 are from that of 17b-HSD-4.
Observed m/z Theoretical MH+ Delta Residues Peptide sequence Modification P32
766.45 766.47 )0.02 283–288 K)HRINVK
898.5 898.47 0.03 289–296 K)FDAVVGYK
1000.6 1000.58 0.02 63–70 K)LVLWDINK
1692.9 1692.87 0.03 140–153 K)TFEVNVLAHFWTTK
2527.6 2527.58 0.02 3–24 K)YLLDLILLLPLLIVFSIESLVK
2577.26 2577.24 0.02 83–105 K)LGAQAHPFVVDCSQREEIYSAAK
2630.44 2630.43 0.01 33–58 K)SVAGEIVLITGAGHGIGRLTAYEFAK
2951.45 2951.48 )0.03 162–189 K)NNHGHIVTVASAAGHTVVPFLLAYCSSK
2984.54 2984.5 0.04 228–254 K)NPSTNLGPTLEPEEVVEHLMHGILTEK 0Met–ox P80
873.54 873.52 0.02 636–643 K)SVGREVVK
1001.61 1001.61 0 636–644 K)SVGREVVKK
1229.67 1229.64 0.03 579–588 K)EGNRIHFQTK
1315.73 1315.68 0.05 645–655 K)ANAVFEWHITK
152.7 1352.63 0.07 69–81 K)AVANYDVEAGEK
1597.98 1597.97 0.01 247–259 K)LRWERTLGAIVRK
1728.92 1728.97 )0.05 169–184 K)LGILGLCNTLAIEGRK
1985.99 1985.94 0.05 384–402 K)SMMNGGLAEVPGLSFNFAK 1Met–ox
2820.47 2820.4 0.07 302–330 K)VDSEGISPNRTSHAAPAATSGFVGAVGHK
2969.45 2969.42 )0.03 141–168 K)QNYGRILMTSSASGIYGNFGQANYSAAK 0Met–ox
Trang 4N-terminal leader sequence Table 1 summarizes
assign-ment of the peptide masses to 17b-HSD-11 and 17b-HSD-4
Northern blot analysis of 17b-HSD-11 mRNA
To confirm that intestinal expression of 17b-HSD-11 is
regulated by PPARa and Wy14,643, we analyzed the effect
of the drug on the levels of 17b-HSD-11 and 17b-HSD-4
mRNAs in the liver and intestine of wild-type or
PPARa-null mice (Fig 2) Two types 17b-HSD mRNAs were
largely induced in both the liver and intestine in a
PPARa-and ligPPARa-and-dependent manner Their inductions were more
outstanding than those of two typical PPARa-target gene
transcripts, liver-type fatty acid binding protein (L-FABP)
and intestine-type fatty acid binding protein (I-FABP)
mRNAs [3,21] When compared to each other, some
preference of 17b-HSD-11 for intestine and of 17b-HSD-4
for liver was observed Thus it was confirmed that
17b-HSD-11 is a new protein whose expression is regulated by
PPARa and its ligand in the intestine
17b-HSD-11 mRNA was efficiently induced by various
types of PPARa activators in addition to a potent
Wy14 643 (Fig 3) The time course of the induction was
very rapid not only in the liver but also in intestine (Fig 4)
and the rapid induction of the mRNA by Wy14 643 was
reproduced in the cultured hepatoma Fao cells (Fig 5)
Almost a maximal level of induction was achieved in both
tissues within a day, making a sharp contrast to the cases of
typical PPARa-target genes so far studied [3,13,21]
Tran-scription of the peroxisomal hydratase-dehydrogenase
(HD) and L-FABP genes is activated by PPARa within a
few hours and the mRNAs reach their maximal levels in a
day in the liver but not in intestine [13] The levels of their
mRNAs in intestine slowly increase during a few days of
feeding a diet containing Wy14 643 This slow time course
of induction is also the case for the intestine-specific
PPARa-target gene I-FABP [21] Thus the induction of
two 17b-HSD mRNAs by a PPAR ligand in the intestine is
much more efficient than that of typical PPARa-target genes
Promoter structure of the17b-HSD-11 gene and transcriptional regulation
All the above data strongly suggest that expression of the 17b-HSD-11 gene is directly regulated by PPARa and its ligand So the genome database was searched for the
Fig 2 Influence of PPARa and Wy14 643
on the expression levels of 17b-HSD-11 and 17b-HSD-4 mRNAs Wild-type (+/+) and PPARa-null mice (–/–) were fed either a con-trol (–) diet or one containing 0.05% (w/v) Wy14 643 for 5 days Total RNA from indi-vidual livers and intestines (5 lg) was analyzed
by Northern blotting using cDNAs for 17b-HSD-11, 17b-HSD-4, 17b-HSD-10, liver-type fatty acid binding protein (L-FABP), intes-tine-type fatty acid binding protein (I-FABP) and ribosomal S14 protein (S14, loading control).
Fig 3 PPARa activator specificity for 17b-HSD-11 mRNA induction
in the liver Wild-type mice were fed either a control diet or one containing 0.05% (w/v) Wy14,643, 0.5% (w/v) clofibrate, 2% (w/v) di(2-ethylhexyl)adipate (DEHA), or 2% (w/v) di(2-ethylhexyl)phtha-late (DEHP) for 5 days Total RNA isodi(2-ethylhexyl)phtha-lated from individual livers was subjected to Northern blot analysis using the cDNA probes for 17b-HSD-11, L-FABP, a2u-globulin (a2u), and apolipoprotein E (apoE, loading control).
Trang 5promoter sequence and PPAR binding site The mouse
17b-HSD-11 gene is located in cytoband E4 on
chromo-some 5 and the sequence has been published (accession
number, AL714024) Searching for a typical PPRE by
TRANSSEARCH program (http://www.cbrc.jp/research/db/
TFSEARCHJ.html) in the region between 3 kb upstream
and 2 kb downstream of the estimated transcriptional
start site from the 5¢ end of the reported cDNA [19]
showed no significantly similar motif The cloned
pro-moter sequence up to)1800 bp from the transcriptional
start site did not respond to a PPARa ligand Wy14 643
in the reporter gene assay (data not shown) Although an
essential role of PPARa in the ligand-dependent
tran-scriptional activation of the 17b-HSD-11 gene in the
intestine became clear, whether another region of the
gene is necessary or an unknown mechanism is operating
for the activation could not be clarified by the
conven-tional methods used in this study The molecular
mechanism of the unusual induction of the 17b-HSD-11
gene in the intestine is clearly important and we are
trying to solve this problem in our laboratory hoping to
uncover a new role of PPARa in this organ
Possible roles of 17b-HSD-11 in the intestine Mouse 17b-HSD-11 may reside not in the extracellular space as previously predicted but in the cell probably on the membrane as noted above Supporting this prospect, Chai
et al [20] recently reported that human normal liver parenchymal cells and epithelium of the endomerium and small intestine, as well as steroidogenic cells, were immuno-histochemically stained by anti-human 17b-HSD-11 Ig They also suggested that 17b-HSD-11 was localized to cytoplasm in the cell, but it should be associated with the endoplasmic reticulum (see above)
17b-HSD-11 belongs to the short-chain dehydrogenase/ reductase superfamily (SDR family member 8) and also has a protein domain of glucose/ribitol dehydrogenase (Mouse Genome Informatics, http://www.informatics jax.org) Recent studies on the specificities of several types of 17b-HSDs have revealed their wide substrate spectrum, including not only glucocorticoids and sex steroids but also bile acids, fatty acids and branched chain amino acids [18,22] Thus 17b-HSDs in the epithelium of the intestine may metabolize potentially toxic compounds included in the diet to protect the organism, as suggested by Chai et al [20] More work is required to address the in vivo function of 17b-HSD-11 and physiological significance of its rapid and marked induction by PPARa ligands in the intestine
Acknowledgements
The author thanks Dr A Iwamatsu (Central Laboratories for Key Technology, Kirin Brewery Co Ltd, Yokohama, Japan) for PMF analysis.
References
1 Gelman, L & Auwerx, J (1999) Peroxisome proliferator-activated receptors: mediators of a fast food impact on gene regulation Curr Opin Clin Nutr Metab Care 2, 307–312.
2 Hihi, A.K., Michalik, L & Wahli, W (2002) PPARs: transcrip-tional effectors of fatty acids and their derivatives Cell Mol Life Sci 59, 790–798.
3 Motojima, K., Passilly, P., Peters, J.M., Gonzalez, F.J & Latruffe,
N (1998) Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor a and c activators in a tissue- and inducer-specific manner J Biol Chem.
273, 16710–16714.
Fig 4 Time course of induction of
17b-HSD-11 and 17b-HSD-4 mRNAs in the liver and
intestine (A) Wild-type mice were fed either a
control diet or one containing 0.05%
Wy14 643 for 1–5 days as indicated (B)
Wild-type mice were fed either a control diet or one
containing 0.5% (w/v) clofibrate (Clofib.) or
0.1% (w/v) troglitazone (Trogli.) for 2 or
8 weeks as indicated Total RNA isolated
from individual livers and intestine was
sub-jected to Northern blot analysis using cDNAs
as described in the legends of Figs 2 and 3.
Fig 5 Time course of 17b-HSD-11 and 17b-HSD-4 mRNA induction
by Wy14 643 in hepatoma Fao cells Wy14 643 was added to the
medium of rat hepatoma Fao cells in the confluent stage at time 0 and
the cells were collected at the time indicated for total RNA isolation
and Northern blotting analysis using the cDNA probes as described in
the legends of Figs 2 and 3.
Trang 64 Gelman, L., Fruchart, J.C & Auwerx, J (1999) An update on the
mechanisms of action of the peroxisome proliferator-activated
receptors (PPARs) and their roles in inflammation and cancer.
Cell Mol Life Sci 55, 932–943.
5 Staels, B., Dallongeville, J., Auwerx, J., Schoonjans, K.,
Leitersdorf, E & Fruchart, J.C (1998) Mechanism of action
of fibrates on lipid and lipoprotein metabolism Circulation 98,
2088–2093.
6 Issemann, I & Green, S (1990) Activation of a member of the
steroid hormone receptor superfamily by peroxisome
pro-liferators Nature 347, 645–650.
7 Palmer, C.N., Hsu, M.H., Griffin, K.J., Raucy, J.L & Johnson,
E.F (1998) Peroxisome proliferator activated receptor-alpha
expression in human liver Mol Pharmacol 53, 14–22.
8 Holden, P.R & Tugwood, J.D (1999) Peroxisome
proliferator-activated receptor alpha: role in rodent liver cancer and species
differences J Mol Endocrinol 22, 1–8.
9 Lee, S.S., Pineau, T., Drago, J., Lee, E.J., Owens, J.W., Kroetz,
D.L., Fernandez-Salguero, P.M., Westphal, H & Gonzalez, F.J.
(1995) Targeted disruption of the alpha isoform of the peroxisome
proliferator-activated receptor gene in mice results in abolishment
of the pleiotropic effects of peroxisome proliferators Mol Cell.
Biol 15, 3012–3022.
10 Motojima, K & Goto, S (1994) Histidyl phosphorylation and
dephosphorylation of P36 in rat liver extract J Biol Chem 269,
9030–9037.
11 Clauser, K.R., Baker, P & Burlingame, A.L (1999) Role of
accurate mass measurement (+/– 10 ppm) in protein identification
strategies employing MS or MS/MS and database searching Anal.
Chem 71, 2871–2882.
12 Chomczynski, P & Sacchi, N (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction Anal Biochem 162, 156–159.
13 Sato, O., Kuriki, C., Fukui, Y & Motojima, K (2002) Dual
promoter structure of mouse and human fatty acid translocase/
CD36 genes and unique transcriptional activation by peroxisome
proliferator-activated receptor a and c ligands J Biol Chem 277,
15703–15711.
14 Fukui, Y., Masui, S., Osada, S., Umesono, K & Motojima, K (2000) A new thiazolidinedione, NC-2100, which is a weak PPAR-c activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice Diabetes 49, 759–767.
15 Motojima, K., Ohmori, A., Takino, Y & Goto, S (1993) Increase
in the amount of elongation factor 2 in rat liver by peroxisome proliferators J Biochem (Tokyo) 114, 779–785.
16 Corton, J.C., Bocos, C., Moreno, E.S., Merritt, A., Marsman, D.S., Sausen, P.J., Cattley, R.C & Gustafsson, J.A (1996) Rat 17 b-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferator-inducible gene Mol Pharmacol 50, 1157–1166.
17 Fan, L.Q., Cattley, R.C & Corton, J.C (1998) Tissue-specific induction of 17 b-hydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor a J Endocrinol 158, 237–246.
18 Mindnich, R., Moller, G & Adamski, J (2004) The role of 17b-hydroxysteroid dehydrogenases Mol Cell Endocrinol 218, 7–20.
19 Okazaki, Y., Furuno, M., Kasukawa, T., Adachi, J., Bono, H., Kondo, S., Nikaido, I., Osato, N., Saito, R., Suzuki, H et al (2002) Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs Nature 420, 563–573.
20 Chai, Z., Brereton, P., Suzuki, T., Sasano, H., Obeyesekere, V., Escher, G., Saffery, R., Fuller, P., Enriquez, C & Krozowski, Z (2003) 17b-Hydroxysteroid dehydrogenase type XI localizes to human steroidogenic cells Endocrinology 144, 2084–2091.
21 Motojima, K (2000) Differential effects of PPARa activators on induction of ectopic expression of tissue-specific fatty acid binding protein genes in the mouse liver Int J Biochem Cell Biol 32, 1085–1092.
22 Shafqat, N., Marschall, H.U., Filling, C., Nordling, E., Wu, X.Q., Bjork, L., Thyberg, J., Martensson, E., Salim, S., Jornvall, H & Oppermann, U (2003) Expanded substrate screenings of human and Drosophila type 10 17b-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hor-mone metabolism: characterization of multifunctional 3a/7a/7b/ 17b/20b/21-HSD Biochem J 376, 49–60.