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Regulated expression by PPARa and unique localizationof 17b-hydroxysteroid dehydrogenase type 11 proteinin mouse intestine and liverYasuhide Yokoi*, Yuka Horiguchi*, Makoto Araki and Kiyoto MotojimaDepartment of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo, JapanPeroxisome proliferator-activated receptor-a (PPARa)is one of the members of the nuclear hormone receptorsuperfamily, and functions as a ligand-dependent tran-scription factor [1]. It is now well accepted thatPPARa is particularly important in lipid catabolism inthe liver, upregulating the expression of a variety ofKeywordshydroxysteroid dehydrogenase;immunocytochemistry; intestine; lipiddroplet; proliferator-activated receptorCorrespondenceK. Motojima, Department of Biochemistry,Meiji Pharmaceutical University, 2-522-1Noshio, Kiyose, Tokyo 204-8588, JapanFax: +81 42 495 8474Tel: +81 42 495 8474E-mail: motojima@my-pharm.ac.jp*These authors contributed equally to thiswork(Received 30 May 2007, revised 21 July2007, accepted 24 July 2007)doi:10.1111/j.1742-4658.2007.06005.x17b-Hydroxysteroid dehydrogenase type 11 (17b-HSD11) is a member ofthe short-chain dehydrogenase⁄ reductase family involved in the activationand inactivation of sex steroid hormones. We recently identified17b-HSD11 as a gene that is efficiently regulated by peroxisome prolifera-tor-activated receptor-a PPARa in the intestine and the liver [Motojima K(2004) Eur J Biochem 271, 4141–4146]. In this study, we characterized17b-HSD11 at the protein level to obtain information about its physiologicrole in the intestine and liver. For this purpose, specific antibodies against17b-HSD11 were obtained. Western blotting analysis showed that adminis-tration of a peroxisome proliferator-activated receptor-a agonist induced17b-HSD11 protein in the jejunum but not in the colon, and to a muchhigher extent than in the liver of mice. A subcellular localization studyusing Chinese hamster ovary cells and green fluorescent protein-tagged17b-HSD11 showed that it was mostly localized in the endoplasmic reticu-lum under normal conditions, whereas it was concentrated on lipid dropletswhen they were induced. A pulse-chase experiment suggested that17b-HSD11 was redistributed to the lipid droplets via the endoplasmicreticulum. Immunohistochemical analysis using tissue sections showed that17b-HSD11 was induced mostly in intestinal epithelia and hepatocytes,with heterogeneous localization both in the cytoplasm and in vesicularstructures. A subcellular fractionation study of liver homogenates con-firmed that 17b-HSD11 was localized mostly in the endoplasmic reticulumwhen mice were fed a normal diet, but was distributed in both the endo-plasmic reticulum and the lipid droplets of which formation was inducedby feeding a diet containing a proliferator-activated receptor-a agonist.Taken together, these data indicate that 17b-HSD11 localizes both in theendoplasmic reticulum and in lipid droplets, depending on physiologic con-ditions, and that lipid droplet 17b-HSD11 is not merely an endoplasmicreticulum contaminant or a nonphysiologically associated protein in thecultured cells, but a bona fide protein component of the membranes ofboth intracellular compartments.AbbreviationsACSL3, long-chain acyl-CoA synthetase 3; ADRP, adipose differentiation-related protein; 17b-HSD11, 17b-hydroxysteroid dehydrogenasetype 11; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FABP, fatty acid-binding protein; GFP, green fluorescent protein; L-FABP,liver-type FABP; LD, lipid droplet; PNS, postnuclear supernatant; PPARa, proliferator-activated receptor-a; TIP47, tail-interacting protein 47.FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4837genes that encode proteins involved in fatty acid trans-port, b-oxidation, and lipoprotein metabolism [2,3].However, PPARa is expressed not only in the liver butalso in other organs [4]. We have been interested in theextrahepatic functions of PPARa, and recently identi-fied the gene encoding 17b-hydroxysteroid dehydro-genase type 11 (17b-HSD11) as a gene that is veryeffectively PPARa-regulated in the intestine [5]. 17b-HSD11 is a member of the short-chain dehydroge-nase ⁄ reductase superfamily (family member 8), and itinterconverts 17b-OH ⁄ 17-oxosteroids [6]. Thus, theactivities of the androgen metabolite pair androster-one ⁄ 3,17-androstanediol and abundant expression insteroidogenic tissues such as placenta and gonads havebeen described [7]. 17b-HSD11 also has a proteindomain of glucose ⁄ ribitol dehydrogenase, and its widesubstrate specificities have been reported [8,9]. Thus,high-level induction of 17b-HSD11 in the intestine andliver by PPARa may have a different physiologic sig-nificance from that in sex hormone metabolism.Elucidation of the subcellular distribution of 17b-HSD11 will be important in uncovering other func-tions, but this has not yet been firmly demonstrated.Chai et al. [7] first suggested that human 17b-HSD11was localized in the cytoplasm, on the basis of obser-vations made using 17b-HSD11 tagged with greenfluorescent protein (GFP) at the N-terminus. Usingproteomics analysis, Fujimoto et al. [10] identified 17b-HSD11 as one of the major lipid droplet (LD)-associ-ated proteins in human hepatoma cells when formationof LDs was artificially induced by incubating the cellswith an excess amount of fatty acids. It might be nec-essary to carefully examine these previous observationsto clarify the subcellular distribution of 17b-HSD11from another point of view, because in both cases,experimental conditions did not reflect in vivo condi-tions. Addition of an artificial tag sequence at theN-terminus of 17b-HSD11 might have disturbed themechanism of intracellular localization and forcedinduction of LDs in the cells, and might have causedabortive association of proteins on their surfaces.In this study, we characterized mouse 17b-HSD11at the protein level, both in cultured cells and in thetissues of mice, to elucidate its subcellular distributionunder various conditions. For this purpose, we pre-pared specific antibodies against mouse 17b-HSD11 todetect endogenous protein: 17b-HSD11 tagged withGFP at the C-terminus to efficiently detect the pro-tein, and Halo-tagged 17b-HSD11 to follow its redis-tribution in a pulse-chase experiment. We found that17b-HSD11 is localized both on the endoplasmic retic-ulum (ER) and on LDs, depending on physiologicconditions.Results17b-HSD11 protein is greatly induced in theintestine and liver by a PPARa agonistTo confirm our previous observation at the mRNAlevel that 17b-HSD11 was greatly induced in the intes-tine and liver of mice by administration of a PPARaagonist, Wy-14 643 [5], the changes in protein levelswere examined by western blotting. For this purpose,we prepared antibodies in rabbits against both the syn-thetic peptide corresponding to Ser95 to Glu109 andthe recombinant protein corresponding to Ile19 toLys298 expressed in Escherichia coli. The postnuclearfractions were prepared from the tissues of mice fed acontrol diet or one containing Wy-14 643, and the pro-teins were separated by SDS ⁄ PAGE for western blot-ting analysis. Both antibodies specifically recognizedthe same protein on the SDS ⁄ PAGE gels having amolecular mass of 34 kDa (Fig. 1A), and a representa-tive result obtained using postnuclear fractions of theintestinal mucosa and liver and antibodies to recombi-nant 17b-HSD11 is shown in Fig. 1B. The amounts of17b-HSD11 protein were markedly induced in both themouse intestine and liver by administration of thePPARa agonist. The induction ratios in both tissueswere greater than 20, and the induced protein level inthe intestinal mucosa was about five times higher thanthat in the liver when normalized with the amounts ofproteins analyzed. These results on a protein level areconsistent with our previous findings on mRNA levels[5]. Thus, 17b-HSD11 is a unique protein whoseexpression level is regulated much more efficiently byPPARa and its ligand in the intestine than in the liver.17b-HSD11 is distributed only in duodenum andjejunumTo examine the distribution of the induced 17b-HSD11 along the gut, proteins from portions of stom-ach, duodenum, jejunum, ileum, cecum and colon ofmice fed a diet containing Wy-14 643 were preparedand analyzed by western blotting using antibodies to17b-HSD11 and liver fatty acid-binding protein(L-FABP) [11]. 17b-HSD11 was detected only in duo-denum and jejunum, with a similar expression patternto that of L-FABP (Fig. 2B). Absence of these pro-teins in stomach, colon and cecum and restrictedexpression at the site of fatty acid absorption suggesttheir roles in lipid absorption and metabolism at theprimary site, the small intestine [12]. L-FABP proteinlevels in the small intestine were higher in the duode-num than in the jejunum, whereas 17b-HSD11 levels17-b-Hydroxysteroid dehydrogenase type 11 Y. Yokoi et al.4838 FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBSwere almost the same in both sites, with a drop at theboundary between the two sites, showing a biphasicexpression pattern (Fig. 2C).17b-HSD11 colocalized with an ER marker proteinin transfected Chinese hamster ovary (CHO) cellsWe next investigated the intracellular location of 17b-HSD11 in a model cell system. To efficiently detect17b-HSD11 in the cell, a plasmid vector expressingchimeric 17b-HSD11 with GFP or a Myc-tag at theC-terminus was constructed and transfected into CHOcells. Confocal fluorescence microscopy examinationdetecting indirect immunofluorescence from antibodiesspecific for the ER marker protein calnexin [13] andMyc-tag showed a strong overlap of the signals(Fig. 3), indicating that 17b-HSD11 is predominantlylocalized in the ER. A similar reticular pattern wasobserved, consistent with ER localization of 17b-HSD11, when it was detected by the indirect immuno-fluorescence method using a specific antibody againstthe protein. These microscopic observations suggestedthat 17b-HSD11 is associated with the ER in the trans-fected CHO cells under normal cell culture conditions.45691012378stomachjejunumcolonileumduodenumcaecumAB123456789101’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’17β-HSD1117β-HSD11L-FABPL-FABPCFig. 2. Expression of 17 b-HSD11 protein in the gastrointestinaltract. (A) The gastrointestinal tract of mice [29] fed a diet containingWy-14 643 for 5 days was divided into 10 parts, and the portionsindicated by circles were homogenized to obtain total proteins. (B)Equal amounts of proteins (20 lg) were separated by SDS ⁄ PAGE,and expression levels of 17b-HSD11 and L-FABP were analyzed bywestern blotting using the specific antibodies. (C) The duodenumand jejunum [corresponding to parts 3–6 in (A)] of another mousethat had been treated in the same way were further divided into 10portions, and the expression levels of 17b-HSD11 and L-FABP ineach portion (1¢ to 10¢) were analyzed by western blotting.75375025(kDa)75M++Peptide RecombinantWy14,643Western blot50CBB-stain3725IntestineLiver+ ++ +Wy14,64317β-HSD11CBB-stainBAFig. 1. Effects of Wy14 643 on protein expression levels of17b-HSD11 in the liver and intestine of mice. (A) Specificity of theantibodies to peptide (Peptide) and recombinant protein (Recombi-nant) against 17b-HSD11. The PNS proteins of the liver from micefed a normal diet (–) or one containing a PPARa ligand, Wy-14 643(–), were separated by SDS ⁄ PAGE and analyzed by western blot-ting. After analysis, the transferred membrane was stained withCoomassie Brilliant Blue R250 to ensure equal protein loading. (B)Comparison of the induction levels of 17b-HSD11 in the liver andintestine. The same amounts (20 lg) of proteins from the PNS frac-tions of liver and small intestinal mucosa were analyzed by westernblotting using antibodies to recombinant protein. The transferredmembrane was stained for equal protein loading.Y. Yokoi et al. 17-b-Hydroxysteroid dehydrogenase type 11FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4839This result is inconsistent with that of Chai et al. [7],who used N-terminally tagged protein, and the ERprotein might have been mistargeted to the cytoplasm.In regard to this, it is of interest to point out the recentpublication by Fujimoto et al. [10], showing that17b-HSD11 was identified as the third most abundantprotein after adipose differentiation-related protein(ADRP) and long-chain acyl-CoA synthetase 3(ACSL3) among 17 major proteins associated with theLDs formed in the human hepatocyte cell line HuH7.17b-HSD11 is associated with LDs in CHO cellsTo examine the possibility that 17b-HSD11 expressedin CHO cells is also associated with LDs, as demon-strated by Fujimoto et al. [10], we first established theconditions to induce formation of LDs in CHO cellsby incubating with various concentrations of fattyacids. In the cells incubated with 200 lm oleic acid for24–48 h, droplet structures were formed that could beefficiently stained with Oil Red O (Fig. 4A, upper).Higher concentrations of fatty acids accelerated theformation of the LDs, but the toxic effects of fattyacids on the cells became evident. Therefore, the cellscultured under normal conditions were transientlytransfected with the 17b-HSD11-GFP expression plas-mid, and were then cultured in a medium supple-mented with or without 200 lm oleic acid to induceformation of the LDs. A reticular pattern of GFPsignals was observed in the cytoplasm of the cells innormal medium, as in Fig. 3, but the pattern under-went a marked change to largely localized signalsaround the LDs upon their formation in the transfect-ed cells (Fig. 4A, lower, and Fig. 4B). A large increasein the signals around the droplets and a significantdecrease in the reticular signals suggested that eitherpre-existing 17b-HSD11-GFP (Fig. 4A) and 17b-HSD11-Myc (Fig. 4B) on the ER moved out to theLDs, or that only newly synthesized 17b-HSD11 wascotranslationally localized and stabilized around theLDs, although other mechanisms could not beexcluded.17b-HSD11 on the ER moves to the LDsTo examine whether pre-existing 17b-HSD11 on theER stays there or moves out to the LDs upon theirformation, we pulse-labeled 17b-HSD11 on the ERusing a Halo-tag system [14], and detected the changesin distribution of the labeled protein before and afterthe formation of the LDs. To complete pulse-labelingof the Halo-tagged 17b-HSD11 on the ER, the cellswere exposed to binding dye for 15 min, and were thenwashed and induced for LD formation by changingthe culture medium to one containing oleic acid. Inthis way, the pre-existing 17b-HSD11 could be clearlydistinguished, by the fluorescent dye covalently boundto the attached Halo-tag, from the newly synthesizedunlabeled Halo-tag-containing 17b-HSD11. As shownin Fig. 5, 17b-HSD11 showed a reticular pattern in thecytoplasm before induction of LD formation, as inFig. 3, but most of the labeled 17b-HSD11 was foundaround the LDs after LD formation. This result clearlyindicated that pre-existing 17b-HSD11 on the ERmoved to the LDs, although whether it moved duringor after the formation of the LDs was not clarified inthis study. It is also noteworthy that the reticularsignals were significantly decreased upon formation ofPI CalnexinHSD-MycMerged(Calnexin + HSD)HSD-MycMerged (PI + HSD)Fig. 3. Subcellular localization of 17b-HSD11 in CHO cells. CHO cellsstably expressing the myc-tagged 17b-HSD11at the C-terminus werefixed and stained with monoclonal antibody to myc and thefluorescein isothiocyanate-labeled anti-mouse IgG. The cells werealso counterstained with propidium iodide (top left) for nuclei andwith anti-calnexin (top right) for ER localization, using Arexa 594-labeled secondary antibodies (red). Areas of colocalization appear asyellow in the merged images (merged). The bar represents 10 lm.17-b-Hydroxysteroid dehydrogenase type 11 Y. Yokoi et al.4840 FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBSthe LDs. This could be achieved by a mechanismwhereby either most of the pre-existing 17b-HSD11moved to the LDs, or a limited amount of 17b-HSD11moved to the droplets but only 17b-HSD11 aroundthe LDs was stabilized.Induced 17b-HSD11 protein localized bothin the cytoplasm and in the vesicular structuresin mouse tissuesAll of the above observations on the subcellularlocalization of 17b-HSD11 were obtained with amodel system using overexpressed mouse 17b-HSD11with tag sequences, in most cases in a heterologousCHO cell line. It was therefore important to examinewhether essential aspects of the above conclusionscould be verified in mouse tissues. For this purpose,we performed immunohistochemical analyses usingtissue sections and specific antibodies to localizethe basal and induced 17b-HSD11 protein in theintestine and liver of mice fed a normal diet or onecontaining a PPARa agonist. As shown in Fig. 6,17b-HSD11 was clearly not detected in either theintestine or liver sections prepared from mice fed anormal diet. On the other hand, feeding a PPARaagonist for 5 days induced the expression of the pro-tein in both tissues to such an extent that it waseasily detected by enzyme-linked immunohistochemis-try. In the small intestine, the cytoplasm or the retic-ular structure of microvillous epithelia was heavilystained with the antibodies only after treatment ofmice with a PPARa agonist, and only a few micro-vesicular structures were detected that were sur-rounded by 17b-HSD11 (Fig. 6A). These patterns areconsistent with the previous observation of substan-tial induction of the enzyme by the compound(Fig. 1) and also with the assumption that 17b-HSD11 is involved in lipid absorption. Substantialinduction of 17b-HSD11 was also confirmed in theliver, and the parenchymal cells were heavily stained.However, 17b-HSD11 was concentrated on vesicularstructures (Fig. 6B), suggesting that most of the 17b-HSD11 molecules are associated with LDs in theliver.ControlOleic acidOil Red OHSD-GFPCalnexinPIHSD-MycHSD-MycMerged (PI + HSD) Merged (Calnexin + HSD)BAFig. 4. Localization of 17b-HSD11 around the LDs. (A) Induction ofLD formation and distribution of 17b-HSD11 in CHO cells. The cellswere incubated with or without 200 lM oleic acid for 48 h, andstained with Oil Red O. Distributions of 17b-HSD11 in parallel cellsexpressing GFP-tagged 17b-HSD11 were visualized by confocalmicroscopy. (B) Subcellular distribution of 17b-HSD11 in the LD-containing CHO cells. The myc-HSD11-expressing cells wereinduced for LD formation, and counterstained with propidium iodide(top left) for nuclei and with anti-calnexin (top right) for ER localiza-tion, using Arexa 594-labeled secondary antibodies (red). Areas ofcolocalization appear as yellow in the merged images (merged).The bar represents 10 lm.Y. Yokoi et al. 17-b-Hydroxysteroid dehydrogenase type 11FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 484117b-HSD11 was fractionated with both the ERand LDs by subcellular fractionation of the liverFinally, we performed a subcellular fractionation studyto confirm biochemically the above results obtainedwith microscopic studies. To confirm the dual associa-tion of 17b-HSD11 with the ER and with the LDsin the tissues and to determine the distribution ratiobetween the two compartments, we performed subcel-lular fractionation of the liver homogenate followed bywestern blot analysis, because the method of subcellu-lar fractionation has been established to be optimal forthe liver but not for the intestine. The liver homogen-ates from mice fed with a normal diet or one contain-ing the PPARa agonist Wy-14 643 were fractionatedby the differential centrifugation method, and the pro-teins in each fraction were separated on SDS⁄ PAGEgels for western blot analysis (Fig. 7). The free LDswere recovered in the top fraction in the tubes aftercentrifugation, as expected, but were contaminatedwith the adjacent cytoplasmic proteins by our manualseparation, as evidenced by significant contaminationby L-FABP proteins. However, a significant amountof 17b-HSD11 was detected in the top fraction of theliver homogenate of mice fed a diet containingWy-14 643 but not a normal diet, in accordance withthe microscopic observations above (Fig. 4). In con-trast, the distribution of an ER protein, calnexin, didnot change at all after treatment with the compound,again in accordance with segregated transfer of 17b-HSD11 from the ER to the droplets (Fig. 5). From aquantitative viewpoint, however, the recovery of 17b-HSD11 (10–20%) in the top fraction after centrifuga-tion seemed to be quite low when compared withobservations from the microscopic images (Fig. 6B).This may be due to a strong fluorescent signal fromthe concentrated 17b-HSD11 on the LDs in contrastto a weak signal from the dispersed 17b-HSD11 in theER, and ⁄ or because all of the LDs are not free frommembranous structures in the cell. A difference in cellBefore LD formation After LD formationHalo-tag LigandDiAcFAMAnti-17ββ-HSD11mergedFig. 5. Redistribution of 17b-HSD11 fromthe ER to LDs. 17b-HSD11 with Halo-tag-expressing vector was transiently transfect-ed into CHO cells and incubated with Halo-tag ligand for 15 min after protein expres-sion for 24 h. The pulse-labeled 17b-HSD11was visualized for its distribution in the cellsbefore formation of LDs or chased in themedium containing 150 lM oleic acid toinduce formation of LDs. The chased 17b-HSD11 was analyzed for its redistribution inthe cells with LDs. Bars represent 10 lm.17-b-Hydroxysteroid dehydrogenase type 11 Y. Yokoi et al.4842 FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBSand tissue types should also be considered. In princi-ple, the microscopic observations on the distributionof 17b-HSD11 under various conditions were essen-tially confirmed by another biochemical analysis. Weconclude that 17b-HSD11 is localized both on the ERand on LDs, depending on physiologic conditions.Discussion17b-HSD11 is greatly induced in the small intestinalmucosa by a PPARa agonist, Wy-14 643. This induc-tion is more effective than in the liver parenchymalcells when normalized by the amounts of total pro-teins in the postnuclear supernatant (PNS) fractions(Fig. 1). The 17b-HSD11 gene is an unique gene,because most PPARa-regulated genes respond effi-ciently to the PPARa ligand in the liver but far less inextrahepatic tissues. The intestine-type FABP gene isanother exception, because it responds to the PPARaligand better in the intestine than in the liver, althoughthe induction ratio in the intestine is not as high asthat of 17b-HSD11. It is noteworthy that none ofthese promoter sequences responds to a PPARa ligandin a typical reporter gene assay system, even withcotransfection of PPARa and RXRa expression vec-tors [15]. Although it has not been finally demon-strated that the 17b-HSD11 gene is directly regulatedby PPARa and its ligand in the intestine, because ofmethodologic difficulties, involvement of a tissue-spe-cific factor, in addition to PPARa and RXRa, is likelyto be essential for the induced expression of thesegenes [16]. The peroxisomal genes that contributedto the discovery of PPARa and establishment ofthe PPARa ⁄ RXRa activation model are not typicalPPARa-responsive genes in a physiologic sense,because substantial transcriptional activation of theperoxisomal genes by a PPARa ligand is only observedin the rodent liver, and not in many other tissues or inhuman tissues [16]. Understanding how PPARais involved in the transcriptional activation of theControl Wy14,6435050400400Wy14,643Control5050400400ABFig. 6. Immunohistochemical staining of 17b-HSD11 in mouse tissues. The intestine (A) and liver (B) tissue sections from mice fed a con-trol diet (control) or one containing Wy-14 643 (Wy14 643) for 5 days were incubated with affinity-purified rabbit antibodies to recombinant17b-HSD11 or preimmune rabbit IgG (not shown), followed by goat anti-rabbit-horseradish peroxidase and the chromogenic substrate 2,4-diaminobutyric acid. The brown reaction product produced by the 2,4-diaminobutyric acid reaction denotes positively stained cells. The tis-sue sections were counterstained using hematoxylin.Y. Yokoi et al. 17-b-Hydroxysteroid dehydrogenase type 11FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 484317b-HSD11 gene in the intestine may be of help inunderstanding the physiologic role of PPARa.The subcellular distribution of 17b-HSD11 was firstdescribed by Chai et al. [7] as being in the cytoplasm.However, our conclusion drawn from this study is that17b-HSD11 is an ER protein (Fig. 3) and translocatesto LDs when their formation is induced (Figs 4 and 5).Chai et al. used a chimeric construct of 17b-HSD11with a tag sequence at the N-terminus, whereas wemade a construct with a tag at the C-terminus, because17b-HSD11 has a hydrophobic N-terminal sequence [5]that seemed to be important for targeting the protein.Our preliminary deletion analysis suggests that thisregion is essential for its association with the LDs(detailed analyses are in progress in our laboratory),and deletion or masking of this sequence in theconstructs of Chai et al. might have led to the proteinbeing in the wrong location. We examined the subcellu-lar distribution of 17b-HSD11 in the cells containingthe LDs (Fig. 4), and confirmed its association withthe LDs. Recently, Fujimoto et al. [10] identified17b-HSD11 as the third most abundant protein afterADRP and ACSL3 among 17 major proteins associatedwith the LDs formed in the human hepatoma cell lineHuH7. However, their analyses of its subcellular distri-bution by western blot and immunocytochemistryshowed that most of the 17b-HSD11 was localized inthe LD-enriched fraction, whereas another protein iden-tified as LD-associated, ACSL3, was equally localizedon the LDs and particulate fractions, including the ERmembranes. Their result showing an LD-restricteddistribution is not consistent with our result showingdual localization on both the LDs and the ER. Thesediscrepancies can be explained by different cell typesused in the experiments, possible differences in thematuration stages of LD formation, and the structureof the LD itself. Our histocytochemical analysis of theintestine and liver of mice fed Wy-14 643 showed thatthe induced expression of 17b-HSD11 and formation ofLDs are not coupled, at least in the intestine (Fig. 6Aversus Fig. 6B). The physiologic role of 17b-HSD11 inthe intestine may be different from that in the liver.The mechanism of LD formation has not been fullyelucidated, but it is thought to involve budding off ofneutral lipid accumulations surrounded by a phospho-lipid monolayer containing proteins from the ERmembranes [17–19]. More than 17 proteins have beenidentified as being associated with LDs [10,20,21],including perilipin, ADRP, and the tail-interactingprotein, TIP47, collectively known as the PAT proteinfamily [22]. These PAT proteins lack long runs ofhydrophobic amino acid residues that would corre-spond to signal peptides or transmembrane domains,and the targeting signals for LDs seem to be quitediverse even among PAT members, suggesting multiplemodes of association with LDs [23]. The studies onother proteins are very limited, and 17b-HSD11 has along hydrophobic region at the N-terminus that is notcleaved off, as directly shown by its sequence analysis[5]. The N-terminal hydrophobic sequence seems to beessential for localization of 17b-HSD11 on the ER.Our pulse-chase experiment suggests that 17b-HSD11is localized on the LDs via the ER. Thus, the N-termi-nal sequence may be required for its localization onthe ER, but may not be sufficient for its relocalizationon the LDs. It is probable that 17b-HSD11 has asequence that will be recognized by other componentsfor localization to ER and LDs. Our present studyusing both cultured cells and animal tissues showedthat 17b-HSD11 is a bona fide LD-associated protein.Among the LD-associated proteins so far character-ized, few have been characterized for their functionson the LDs. The most widely distributed LD proteinsare perilipin, ADRP and TIP47, and they help to sup-port the basic structure and function of the LDs, asWy14,643ControlPNSMitoMicroCytoTopPNSMitoMicroCytoTop17β-HSD11CalnexinL-FABPCBB-stain(100)(100)49491111323288(%)(%)((100)100)585899882525protein recoveryprotein recoveryFig. 7. Subcellular fractionation studies on the localization of 17b-HSD11 in mouse liver. The livers of mice fed a control diet (–) orone containing Wy-14 643 for 5 days were homogenized to obtainPNS fractions. The PNS was fractionated into subcellular fractions,mitochondrial (Mito), microsome (Micro), cytosol (Cyto) and LD-richfraction (Top), by a differential centrifugation method. The proteinscorresponding to amounts of recovered protein in each fraction(shown beneath each lane as a percentage of PNS) were separatedon SDS ⁄ PAGE and analyzed by western blotting using the specificantibodies against 17b-HSD11, calnexin and L-FABP.17-b-Hydroxysteroid dehydrogenase type 11 Y. Yokoi et al.4844 FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBSsupported in recent work on ADRP-deficient mice,which display impairment of LD formation and resis-tance to diet-induced fatty liver [24]. The rapid degra-dation of ADRP by a proteasome-mediated pathwayduring LD regression was also reported [25]. ACSL3was recently identified as a major commonly LD-asso-ciated protein [10], and its essential role in LD forma-tion, through involvement in local synthesis of neutrallipids, was also reported [26,27]. In contrast to theseLD proteins, 17b-HSD11 may not play such an essentialrole in LD formation and function, because of its uniquetissue distribution [7] and regulated expression [5].We recently found a new PAT protein, myocardialLD protein, which is highly enriched in the heart, andits precise function is not known, although we sug-gested its involvement in the metabolic response to fast-ing [23]. Tissue-specific association of these accessoryproteins with the LDs may play a key role in wholebody energy homeostasis, including dietary lipid metab-olism, especially in the case of 17b-HSD11, which isuniquely induced in the small intestine and liver. Fur-ther studies will be required to clarify these possibilities.Experimental proceduresAnimals and treatmentsAll procedures involving animals were approved by theMeiji Pharmaceutical University Committee for Ethics ofExperimentation and Animal Care. Male C57BL miceabout 6 weeks of age were maintained under a 12 hlight ⁄ 12 h dark cycle with free access to food and water.After being fed a diet containing the PPARa agonistWy14 643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]ace-tic acid} (Tokyo-Kasei, Tokyo, Japan) at 0.05% (w ⁄ w) or anormal laboratory diet, the mice were killed by cervicaldislocation, and portions of the intestine and liver wereremoved for sample preparation.Antibody production and western blottingAntibodies to 17b-HSD11 were raised in rabbits using asynthetic peptide corresponding to amino acids 95–109(accession number Q9EQ06) or recombinant glutathioneS-transferase fusion 17b-HSD11 protein as antigen. Bothantibodies were affinity purified using antigens, and usedfor western blotting and immunohistochemistry. Westernblotting was performed by resolving proteins onSDS ⁄ PAGE gels and transferring them to poly(vinylidenedifluoride) membranes as previously described [11]. Peroxi-dase-conjugated secondary antibody and an enhancedchemiluminescent kit (Super Signal West Pico, Pierce, Rich-mond, IL, USA) were used. Rabbit anti-mouse calnexinserum was purchased from Stressgen (Victoria, Canada).Plasmid constructionFull-length cDNA clone for mouse 17b-HSD11 containing40 nucleotides of the 5¢-noncoding sequence (GenBankaccession number NM053262) was obtained by PCR usingprimers 5¢-GGgaattcGTTTAGGACCGGGAACGAGAGC(added EcoRI site in lower case) and 5¢-GGCctcgagTCAATCGGCTTTCAGGGAACC (with XhoI site). The DNAfragment was digested with the enzymes and inserted intothe EcoRI–XhoI site of a plasmid vector. The correctsequence was confirmed, and the plasmid DNA was usedfor further plasmid construction. To obtain GST-fused17b-HSD11 protein for antibody production, a DNA frag-ment corresponding to amino acids 19–298 was amplifiedby PCR using primers 5¢-GGCCCgaattcATTGAGTCTCTTGTCAAGC (added EcoRI site in lower case) and 5¢-GGCctcgagTCAATCGGCTTTCAGGGAACC (with XhoIsite) and cloned into the pGEX4T-1 plasmid vector. Toobtain an expression plasmid for 17b-HSD11 with GFP atthe C-terminus, a DNA fragment was amplified by PCRusing primers 5¢-GGgaattcGTTTAGGACCGGGAACGAGAGC (added EcoRI site in lower case) and 5¢-gcctcgagCTTGTCTTTGTACCCAACAAC (with XhoI site)and cloned into pCGFP2 or pCMVtag5A. To obtain anexpression plasmid for 17b-HSD11 with a Halo-tag at theC-terminus, a DNA fragment was amplified by PCR withprimers 5¢-AAAgctagcGTTTAGGACCGGGAAC (addedNhe site in lower case) and 5¢-atcCTTGTCTTTGTACCCAACAACTGCATC-3 (with EcoR site) and cloned intoHalo-tag pHT2 (Promega, Madison, WI, USA). All of theconstructed plasmid DNAs were subjected to sequencinganalysis for confirmation of mutation-free amplification.Cell culture and DNA transfectionCHO-K1 cells were maintained in F-12 nutrient mixturemedium (Gibco, Grand Island, NY, USA) supplementedwith 10% (v ⁄ v) fetal bovine serum at 37 °C in a humidifiedatmosphere of air ⁄ CO2(5%). Transient transfection ofCHO-K1 cells was carried out using Lipofectamine Reagent(Invitrogen, Carlsbad, CA, USA) according to the manu-facturer’s instructions. Briefly, 0.4 lg per well of plasmidDNA was incubated with 0.8 lL per well of PlusReagent,1.2 lL per well of Lipofectamine Reagent and 250 lLofserum free F-12 medium, and the 60% confluent cells ina 24-well plate were exposed to this preincubated DNA–lipofectin complex. After exposure for 3 h, the cells werecultured in F-12 medium supplemented with 10% fetalbovine serum.Fluorescence imagingStably or transiently transfected CHO cells cultured onpoly-lysine-coated coverslips were washed with NaCl ⁄ Piand then fixed with 4% paraformaldehyde for 10 min atY. Yokoi et al. 17-b-Hydroxysteroid dehydrogenase type 11FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4845room temperature. After being washed with NaCl ⁄ Pi, cellswere permeabilized with 0.1% Triton X-100 in NaCl ⁄ Pifor30 min at room temperature. The cells, preincubated with5% BSA in NaCl ⁄ Pifor 30 min at room temperature toblock nonspecific binding, were then incubated with theantibodies for 30 min at room temperature. After beingrinsed with wash buffer (0.2% Triton X-100 in NaCl ⁄ Pi),the cells were incubated with secondary goat anti-(rabbitIgG) or anti-(mouse IgG) conjugated with fluorescein iso-thiocyanate (MP Biochemicals, Aurora, OH, USA) or withAlexa Fluor 594-labeled goat anti-(rabbit IgG) (Invitrogen).After being washed with wash buffer and rinsed withNaCl ⁄ Pi, the cells were fixed in MOWIOL (Sigma-Aldrich,St Louis, MO, USA), and the specimens were subjected toconfocal fluorescence microscopy with a Fluoview FV500microscope (Olympus, Tokyo, Japan).Pulse-chase experiment using the Halo-tag systemCells transiently expressing 17b-HSD11-Halo-tag on cover-slides were labeled with 1 lm Halo-tag diAcFAM ligandfor 15 min according to the manufacturer’s instructions(Promega) after protein expression for 24 h. Unboundligand was removed by washing the cells with NaCl ⁄ Pi,and the cells were incubated for 1 h in the F-12 mediumcontaining 10% fetal bovine serum. Some cells on slideswere fixed and mounted for microscopic observation of‘pulse-labeled’ cells. Other cells were incubated for a fur-ther 24 h in the F-12 medium supplemented with 10%fetal bovine serum and 0.15 mm oleate to induce formationof LDs and then processed as ‘chased cells’ for micro-scopic observation.ImmunohistochemistryTissue samples from mice fed a control diet or one contain-ing 0.05% Wy-14 643 were fixed in a formaldehyde-basedfixing solution (Genostaff, Inc., Tokyo, Japan) overnight at4 °C, embedded in paraffin, and cut into 6-lm-thick sec-tions. Deparaffinized and rehydrated slides were subjectedto microwave antigen retrieval by boiling for 10 min in10 mm citric acid buffer (pH 6.0), and then treating with3% H2O2in methanol for 15 min at room temperature.Slides were washed in Tris-buffered saline (NaCl ⁄ Tris), andthen blocked with Dako Protein Block (Dako X0909) for10 min at room temperature. Affinity-purified rabbit anti-17b-HSD11 recombinant serum or preimmune rabbit IgG(0.5 lgÆmL)1) were incubated with sections overnight at4 °C. Slides were washed in NaCl ⁄ Tris with Triton X-100(NaCl ⁄ TrisT) and then with NaCl ⁄ Tris, followed by bio-tinylated goat anti-(rabbit IgG) for 30 min at room tem-perature. After washing with NaCl ⁄ TrisT and NaCl ⁄Tris, binding was detected by developing with diam-inobenzidine. The tissue sections were counterstained usinghematoxylin.Subcellular fractionationSubcellular fractionation was performed as described previ-ously [28], with modifications. The livers were homogenizedwith a Teflon-glass homogenizer at 1200 r.p.m. in fivevolumes of ice-cold homogenization buffer [0.25 m sucrose,1mm EDTA, 0.1% ethanol, 0.1% protease inhibitor mix(Wako, Tokyo, Japan), 10 mm Tris ⁄ HCl, pH 7.4]. A PNSfraction was prepared by centrifugation for 5 min at 800 g(Kubota 6500 with AG-508R rotor; Kubota, Tokyo,Japan). The supernatant was recentrifuged for 20 min at25 400 g (Kubota 6500 with AG-508R rotor). The pelletwas suspended in five volumes of ice-cold homogenizationbuffer (‘Mito’ fraction). The supernatant was recentrifugedfor 60 min at 100 000 g (CB80WX centrifuge with RPS40rotor; Hitachi Koki, Tokyo, Japan) to obtain the superna-tant (‘Cyto’ fraction). The top white fraction was separatedas the ‘Top’ fraction. The pellet was suspended in five vol-umes of ice-cold homogenization buffer (‘Micro’ fraction).AcknowledgementsWe thank Dr K. Higashi and the Motojima laboratorymembers for helpful discussions. This work was sup-ported in part by the Meiyaku Open Research Projectand grants-in-aid for Scientific Research from theJapan Society for the Promotion of Science.References1 Desvergne B & Wahli W (1999) Peroxisome prolifera-tor-activated receptors: nuclear control of metabolism.Endocr Rev 20, 649–688.2 Kersten S, Desvergne B & Wahli W (2000) Roles ofPPARs in health and disease. 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Regulated expression by PPARa and unique localization of 17b-hydroxysteroid dehydrogenase type 11 protein in mouse intestine and liver Yasuhide. the mouse intestine and liver by administration of the PPARa agonist. The induction ratios in both tissueswere greater than 20, and the induced protein
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