Regulated expression by PPARa and unique localization of 17b-hydroxysteroid dehydrogenase type 11 protein in mouse intestine and liver Yasuhide Yokoi*, Yuka Horiguchi*, Makoto Araki and Kiyoto Motojima Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan Keywords hydroxysteroid dehydrogenase; immunocytochemistry; intestine; lipid droplet; proliferator-activated receptor Correspondence K Motojima, Department of Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan Fax: +81 42 495 8474 Tel: +81 42 495 8474 E-mail: motojima@my-pharm.ac.jp *These authors contributed equally to this work (Received 30 May 2007, revised 21 July 2007, accepted 24 July 2007) doi:10.1111/j.1742-4658.2007.06005.x 17b-Hydroxysteroid dehydrogenase type 11 (17b-HSD11) is a member of the short-chain dehydrogenase ⁄ reductase family involved in the activation and inactivation of sex steroid hormones We recently identified 17b-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-a PPARa in the intestine and the liver [Motojima K (2004) Eur J Biochem 271, 4141–4146] In this study, we characterized 17b-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver For this purpose, specific antibodies against 17b-HSD11 were obtained Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-a agonist induced 17b-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17b-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced A pulse-chase experiment suggested that 17b-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum Immunohistochemical analysis using tissue sections showed that 17b-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures A subcellular fractionation study of liver homogenates confirmed that 17b-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-a agonist Taken together, these data indicate that 17b-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17b-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments Peroxisome proliferator-activated receptor-a (PPARa) is one of the members of the nuclear hormone receptor superfamily, and functions as a ligand-dependent tran- scription factor [1] It is now well accepted that PPARa is particularly important in lipid catabolism in the liver, upregulating the expression of a variety of Abbreviations ACSL3, long-chain acyl-CoA synthetase 3; ADRP, adipose differentiation-related protein; 17b-HSD11, 17b-hydroxysteroid dehydrogenase type 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 4837 17-b-Hydroxysteroid dehydrogenase type 11 Y Yokoi et al genes that encode proteins involved in fatty acid transport, b-oxidation, and lipoprotein metabolism [2,3] However, PPARa is expressed not only in the liver but also in other organs [4] We have been interested in the extrahepatic functions of PPARa, and recently identified the gene encoding 17b-hydroxysteroid dehydrogenase type 11 (17b-HSD11) as a gene that is very effectively PPARa-regulated in the intestine [5] 17bHSD11 is a member of the short-chain dehydrogenase ⁄ reductase superfamily (family member 8), and it interconverts 17b-OH ⁄ 17-oxosteroids [6] Thus, the activities of the androgen metabolite pair androsterone ⁄ 3,17-androstanediol and abundant expression in steroidogenic tissues such as placenta and gonads have been described [7] 17b-HSD11 also has a protein domain of glucose ⁄ ribitol dehydrogenase, and its wide substrate specificities have been reported [8,9] Thus, high-level induction of 17b-HSD11 in the intestine and liver by PPARa may have a different physiologic significance from that in sex hormone metabolism Elucidation of the subcellular distribution of 17bHSD11 will be important in uncovering other functions, but this has not yet been firmly demonstrated Chai et al [7] first suggested that human 17b-HSD11 was localized in the cytoplasm, on the basis of observations made using 17b-HSD11 tagged with green fluorescent protein (GFP) at the N-terminus Using proteomics analysis, Fujimoto et al [10] identified 17bHSD11 as one of the major lipid droplet (LD)-associated proteins in human hepatoma cells when formation of LDs was artificially induced by incubating the cells with an excess amount of fatty acids It might be necessary to carefully examine these previous observations to clarify the subcellular distribution of 17b-HSD11 from another point of view, because in both cases, experimental conditions did not reflect in vivo conditions Addition of an artificial tag sequence at the N-terminus of 17b-HSD11 might have disturbed the mechanism of intracellular localization and forced induction of LDs in the cells, and might have caused abortive association of proteins on their surfaces In this study, we characterized mouse 17b-HSD11 at the protein level, both in cultured cells and in the tissues of mice, to elucidate its subcellular distribution under various conditions For this purpose, we prepared specific antibodies against mouse 17b-HSD11 to detect endogenous protein: 17b-HSD11 tagged with GFP at the C-terminus to efficiently detect the protein, and Halo-tagged 17b-HSD11 to follow its redistribution in a pulse-chase experiment We found that 17b-HSD11 is localized both on the endoplasmic reticulum (ER) and on LDs, depending on physiologic conditions 4838 Results 17b-HSD11 protein is greatly induced in the intestine and liver by a PPARa agonist To confirm our previous observation at the mRNA level that 17b-HSD11 was greatly induced in the intestine and liver of mice by administration of a PPARa agonist, Wy-14 643 [5], the changes in protein levels were examined by western blotting For this purpose, we prepared antibodies in rabbits against both the synthetic peptide corresponding to Ser95 to Glu109 and the recombinant protein corresponding to Ile19 to Lys298 expressed in Escherichia coli The postnuclear fractions were prepared from the tissues of mice fed a control diet or one containing Wy-14 643, and the proteins were separated by SDS ⁄ PAGE for western blotting analysis Both antibodies specifically recognized the same protein on the SDS ⁄ PAGE gels having a molecular mass of 34 kDa (Fig 1A), and a representative result obtained using postnuclear fractions of the intestinal mucosa and liver and antibodies to recombinant 17b-HSD11 is shown in Fig 1B The amounts of 17b-HSD11 protein were markedly induced in both the mouse intestine and liver by administration of the PPARa agonist The induction ratios in both tissues were greater than 20, and the induced protein level in the intestinal mucosa was about five times higher than that in the liver when normalized with the amounts of proteins analyzed These results on a protein level are consistent with our previous findings on mRNA levels [5] Thus, 17b-HSD11 is a unique protein whose expression level is regulated much more efficiently by PPARa and its ligand in the intestine than in the liver 17b-HSD11 is distributed only in duodenum and jejunum To examine the distribution of the induced 17bHSD11 along the gut, proteins from portions of stomach, duodenum, jejunum, ileum, cecum and colon of mice fed a diet containing Wy-14 643 were prepared and analyzed by western blotting using antibodies to 17b-HSD11 and liver fatty acid-binding protein (L-FABP) [11] 17b-HSD11 was detected only in duodenum and jejunum, with a similar expression pattern to that of L-FABP (Fig 2B) Absence of these proteins in stomach, colon and cecum and restricted expression at the site of fatty acid absorption suggest their roles in lipid absorption and metabolism at the primary site, the small intestine [12] L-FABP protein levels in the small intestine were higher in the duodenum than in the jejunum, whereas 17b-HSD11 levels FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Yokoi et al A Wy14,643 17-b-Hydroxysteroid dehydrogenase type 11 Peptide Recombinant + M + (kDa) 75 stomach 50 Western blot jejunum A ileum duodenum colon 10 37 B caecum 10 17β-HSD11 25 75 L-FABP 50 CBB-stain 37 C 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 17β-HSD11 L-FABP 25 B Wy14,643 Liver + + Intestine + + 17β-HSD11 CBB-stain Fig Expression of 17b-HSD11 protein in the gastrointestinal tract (A) The gastrointestinal tract of mice [29] fed a diet containing Wy-14 643 for days was divided into 10 parts, and the portions indicated 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 by western blotting using the specific antibodies (C) The duodenum and jejunum [corresponding to parts 3–6 in (A)] of another mouse that had been treated in the same way were further divided into 10 portions, and the expression levels of 17b-HSD11 and L-FABP in each portion (1¢ to 10¢) were analyzed by western blotting 17b-HSD11 colocalized with an ER marker protein in transfected Chinese hamster ovary (CHO) cells Fig Effects of Wy14 643 on protein expression levels of 17b-HSD11 in the liver and intestine of mice (A) Specificity of the antibodies to peptide (Peptide) and recombinant protein (Recombinant) against 17b-HSD11 The PNS proteins of the liver from mice fed a normal diet (–) or one containing a PPARa ligand, Wy-14 643 (–), were separated by SDS ⁄ PAGE and analyzed by western blotting After analysis, the transferred membrane was stained with Coomassie Brilliant Blue R250 to ensure equal protein loading (B) Comparison of the induction levels of 17b-HSD11 in the liver and intestine The same amounts (20 lg) of proteins from the PNS fractions of liver and small intestinal mucosa were analyzed by western blotting using antibodies to recombinant protein The transferred membrane was stained for equal protein loading were almost the same in both sites, with a drop at the boundary between the two sites, showing a biphasic expression pattern (Fig 2C) We next investigated the intracellular location of 17bHSD11 in a model cell system To efficiently detect 17b-HSD11 in the cell, a plasmid vector expressing chimeric 17b-HSD11 with GFP or a Myc-tag at the C-terminus was constructed and transfected into CHO cells Confocal fluorescence microscopy examination detecting indirect immunofluorescence from antibodies specific for the ER marker protein calnexin [13] and Myc-tag showed a strong overlap of the signals (Fig 3), indicating that 17b-HSD11 is predominantly localized in the ER A similar reticular pattern was observed, consistent with ER localization of 17bHSD11, when it was detected by the indirect immunofluorescence method using a specific antibody against the protein These microscopic observations suggested that 17b-HSD11 is associated with the ER in the transfected CHO cells under normal cell culture conditions FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4839 17-b-Hydroxysteroid dehydrogenase type 11 Y Yokoi et al PI Calnexin HSD-Myc HSD-Myc Merged (PI + HSD) Merged(Calnexin + HSD) Fig Subcellular localization of 17b-HSD11 in CHO cells CHO cells stably expressing the myc-tagged 17b-HSD11at the C-terminus were fixed and stained with monoclonal antibody to myc and the fluorescein isothiocyanate-labeled anti-mouse IgG The cells were also counterstained with propidium iodide (top left) for nuclei and with anti-calnexin (top right) for ER localization, using Arexa 594labeled secondary antibodies (red) Areas of colocalization appear as yellow in the merged images (merged) The bar represents 10 lm This result is inconsistent with that of Chai et al [7], who used N-terminally tagged protein, and the ER protein might have been mistargeted to the cytoplasm In regard to this, it is of interest to point out the recent publication by Fujimoto et al [10], showing that 17b-HSD11 was identified as the third most abundant protein after adipose differentiation-related protein (ADRP) and long-chain acyl-CoA synthetase (ACSL3) among 17 major proteins associated with the LDs formed in the human hepatocyte cell line HuH7 17b-HSD11 is associated with LDs in CHO cells To examine the possibility that 17b-HSD11 expressed in CHO cells is also associated with LDs, as demon4840 strated by Fujimoto et al [10], we first established the conditions to induce formation of LDs in CHO cells by incubating with various concentrations of fatty acids In the cells incubated with 200 lm oleic acid for 24–48 h, droplet structures were formed that could be efficiently stained with Oil Red O (Fig 4A, upper) Higher concentrations of fatty acids accelerated the formation of the LDs, but the toxic effects of fatty acids on the cells became evident Therefore, the cells cultured under normal conditions were transiently transfected with the 17b-HSD11-GFP expression plasmid, and were then cultured in a medium supplemented with or without 200 lm oleic acid to induce formation of the LDs A reticular pattern of GFP signals was observed in the cytoplasm of the cells in normal medium, as in Fig 3, but the pattern underwent a marked change to largely localized signals around the LDs upon their formation in the transfected cells (Fig 4A, lower, and Fig 4B) A large increase in the signals around the droplets and a significant decrease in the reticular signals suggested that either pre-existing 17b-HSD11-GFP (Fig 4A) and 17bHSD11-Myc (Fig 4B) on the ER moved out to the LDs, or that only newly synthesized 17b-HSD11 was cotranslationally localized and stabilized around the LDs, although other mechanisms could not be excluded 17b-HSD11 on the ER moves to the LDs To examine whether pre-existing 17b-HSD11 on the ER stays there or moves out to the LDs upon their formation, we pulse-labeled 17b-HSD11 on the ER using a Halo-tag system [14], and detected the changes in distribution of the labeled protein before and after the formation of the LDs To complete pulse-labeling of the Halo-tagged 17b-HSD11 on the ER, the cells were exposed to binding dye for 15 min, and were then washed and induced for LD formation by changing the culture medium to one containing oleic acid In this way, the pre-existing 17b-HSD11 could be clearly distinguished, by the fluorescent dye covalently bound to the attached Halo-tag, from the newly synthesized unlabeled Halo-tag-containing 17b-HSD11 As shown in Fig 5, 17b-HSD11 showed a reticular pattern in the cytoplasm before induction of LD formation, as in Fig 3, but most of the labeled 17b-HSD11 was found around the LDs after LD formation This result clearly indicated that pre-existing 17b-HSD11 on the ER moved to the LDs, although whether it moved during or after the formation of the LDs was not clarified in this study It is also noteworthy that the reticular signals were significantly decreased upon formation of FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Yokoi et al Fig Localization of 17b-HSD11 around the LDs (A) Induction of LD formation and distribution of 17b-HSD11 in CHO cells The cells were incubated with or without 200 lM oleic acid for 48 h, and stained with Oil Red O Distributions of 17b-HSD11 in parallel cells expressing GFP-tagged 17b-HSD11 were visualized by confocal microscopy (B) Subcellular distribution of 17b-HSD11 in the LDcontaining CHO cells The myc-HSD11-expressing cells were induced for LD formation, and counterstained with propidium iodide (top left) for nuclei and with anti-calnexin (top right) for ER localization, using Arexa 594-labeled secondary antibodies (red) Areas of colocalization appear as yellow in the merged images (merged) The bar represents 10 lm 17-b-Hydroxysteroid dehydrogenase type 11 A Control Oleic acid Oil Red O HSD-GFP the LDs This could be achieved by a mechanism whereby either most of the pre-existing 17b-HSD11 moved to the LDs, or a limited amount of 17b-HSD11 moved to the droplets but only 17b-HSD11 around the LDs was stabilized B PI Calnexin HSD-Myc HSD-Myc Induced 17b-HSD11 protein localized both in the cytoplasm and in the vesicular structures in mouse tissues All of the above observations on the subcellular localization of 17b-HSD11 were obtained with a model system using overexpressed mouse 17b-HSD11 with tag sequences, in most cases in a heterologous CHO cell line It was therefore important to examine whether essential aspects of the above conclusions could be verified in mouse tissues For this purpose, we performed immunohistochemical analyses using tissue sections and specific antibodies to localize the basal and induced 17b-HSD11 protein in the intestine and liver of mice fed a normal diet or one containing a PPARa agonist As shown in Fig 6, 17b-HSD11 was clearly not detected in either the intestine or liver sections prepared from mice fed a normal diet On the other hand, feeding a PPARa agonist for days induced the expression of the protein in both tissues to such an extent that it was easily detected by enzyme-linked immunohistochemistry In the small intestine, the cytoplasm or the reticular structure of microvillous epithelia was heavily stained with the antibodies only after treatment of mice with a PPARa agonist, and only a few microvesicular structures were detected that were surrounded by 17b-HSD11 (Fig 6A) These patterns are consistent with the previous observation of substantial induction of the enzyme by the compound (Fig 1) and also with the assumption that 17bHSD11 is involved in lipid absorption Substantial induction of 17b-HSD11 was also confirmed in the Merged (PI + HSD) Merged (Calnexin + HSD) liver, and the parenchymal cells were heavily stained However, 17b-HSD11 was concentrated on vesicular structures (Fig 6B), suggesting that most of the 17bHSD11 molecules are associated with LDs in the liver FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4841 17-b-Hydroxysteroid dehydrogenase type 11 Before LD formation Y Yokoi et al After LD formation Halo-tag Ligand DiAcFAM Antiβ 17β-HSD11 Fig Redistribution of 17b-HSD11 from the ER to LDs 17b-HSD11 with Halo-tagexpressing vector was transiently transfected into CHO cells and incubated with Halotag ligand for 15 after protein expression for 24 h The pulse-labeled 17b-HSD11 was visualized for its distribution in the cells before formation of LDs or chased in the medium containing 150 lM oleic acid to induce formation of LDs The chased 17bHSD11 was analyzed for its redistribution in the cells with LDs Bars represent 10 lm merged 17b-HSD11 was fractionated with both the ER and LDs by subcellular fractionation of the liver Finally, we performed a subcellular fractionation study to confirm biochemically the above results obtained with microscopic studies To confirm the dual association of 17b-HSD11 with the ER and with the LDs in the tissues and to determine the distribution ratio between the two compartments, we performed subcellular fractionation of the liver homogenate followed by western blot analysis, because the method of subcellular fractionation has been established to be optimal for the liver but not for the intestine The liver homogenates from mice fed with a normal diet or one containing the PPARa agonist Wy-14 643 were fractionated by the differential centrifugation method, and the proteins in each fraction were separated on SDS ⁄ PAGE gels for western blot analysis (Fig 7) The free LDs were recovered in the top fraction in the tubes after centrifugation, as expected, but were contaminated 4842 with the adjacent cytoplasmic proteins by our manual separation, as evidenced by significant contamination by L-FABP proteins However, a significant amount of 17b-HSD11 was detected in the top fraction of the liver homogenate of mice fed a diet containing Wy-14 643 but not a normal diet, in accordance with the microscopic observations above (Fig 4) In contrast, the distribution of an ER protein, calnexin, did not change at all after treatment with the compound, again in accordance with segregated transfer of 17bHSD11 from the ER to the droplets (Fig 5) From a quantitative viewpoint, however, the recovery of 17bHSD11 (10–20%) in the top fraction after centrifugation seemed to be quite low when compared with observations from the microscopic images (Fig 6B) This may be due to a strong fluorescent signal from the concentrated 17b-HSD11 on the LDs in contrast to a weak signal from the dispersed 17b-HSD11 in the ER, and ⁄ or because all of the LDs are not free from membranous structures in the cell A difference in cell FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Yokoi et al A 17-b-Hydroxysteroid dehydrogenase type 11 Control Wy14,643 Control B Wy14,643 50 50 50 50 400 400 400 400 Fig Immunohistochemical staining of 17b-HSD11 in mouse tissues The intestine (A) and liver (B) tissue sections from mice fed a control diet (control) or one containing Wy-14 643 (Wy14 643) for days were incubated with affinity-purified rabbit antibodies to recombinant 17b-HSD11 or preimmune rabbit IgG (not shown), followed by goat anti-rabbit-horseradish peroxidase and the chromogenic substrate 2,4diaminobutyric acid The brown reaction product produced by the 2,4-diaminobutyric acid reaction denotes positively stained cells The tissue sections were counterstained using hematoxylin and tissue types should also be considered In principle, the microscopic observations on the distribution of 17b-HSD11 under various conditions were essentially confirmed by another biochemical analysis We conclude that 17b-HSD11 is localized both on the ER and on LDs, depending on physiologic conditions Discussion 17b-HSD11 is greatly induced in the small intestinal mucosa by a PPARa agonist, Wy-14 643 This induction is more effective than in the liver parenchymal cells when normalized by the amounts of total proteins in the postnuclear supernatant (PNS) fractions (Fig 1) The 17b-HSD11 gene is an unique gene, because most PPARa-regulated genes respond efficiently to the PPARa ligand in the liver but far less in extrahepatic tissues The intestine-type FABP gene is another exception, because it responds to the PPARa ligand better in the intestine than in the liver, although the induction ratio in the intestine is not as high as that of 17b-HSD11 It is noteworthy that none of these promoter sequences responds to a PPARa ligand in a typical reporter gene assay system, even with cotransfection of PPARa and RXRa expression vectors [15] Although it has not been finally demonstrated that the 17b-HSD11 gene is directly regulated by PPARa and its ligand in the intestine, because of methodologic difficulties, involvement of a tissue-specific factor, in addition to PPARa and RXRa, is likely to be essential for the induced expression of these genes [16] The peroxisomal genes that contributed to the discovery of PPARa and establishment of the PPARa ⁄ RXRa activation model are not typical PPARa-responsive genes in a physiologic sense, because substantial transcriptional activation of the peroxisomal genes by a PPARa ligand is only observed in the rodent liver, and not in many other tissues or in human tissues [16] Understanding how PPARa is involved in the transcriptional activation of the FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4843 17-b-Hydroxysteroid dehydrogenase type 11 Top Top Cyto Cyto 32 Micro Micro 11 PNS Mito protein recovery (100) 49 Mito Wy14,643 Control PNS Y Yokoi et al 25 (%) 17β-HSD11 Calnexin L-FABP CBB-stain (100) 58 Fig Subcellular fractionation studies on the localization of 17bHSD11 in mouse liver The livers of mice fed a control diet (–) or one containing Wy-14 643 for days were homogenized to obtain PNS fractions The PNS was fractionated into subcellular fractions, mitochondrial (Mito), microsome (Micro), cytosol (Cyto) and LD-rich fraction (Top), by a differential centrifugation method The proteins corresponding to amounts of recovered protein in each fraction (shown beneath each lane as a percentage of PNS) were separated on SDS ⁄ PAGE and analyzed by western blotting using the specific antibodies against 17b-HSD11, calnexin and L-FABP 17b-HSD11 gene in the intestine may be of help in understanding the physiologic role of PPARa The subcellular distribution of 17b-HSD11 was first described by Chai et al [7] as being in the cytoplasm However, our conclusion drawn from this study is that 17b-HSD11 is an ER protein (Fig 3) and translocates to LDs when their formation is induced (Figs and 5) Chai et al used a chimeric construct of 17b-HSD11 with a tag sequence at the N-terminus, whereas we made a construct with a tag at the C-terminus, because 17b-HSD11 has a hydrophobic N-terminal sequence [5] that seemed to be important for targeting the protein Our preliminary deletion analysis suggests that this region is essential for its association with the LDs (detailed analyses are in progress in our laboratory), and deletion or masking of this sequence in the constructs of Chai et al might have led to the protein being in the wrong location We examined the subcellular distribution of 17b-HSD11 in the cells containing the LDs (Fig 4), and confirmed its association with the LDs Recently, Fujimoto et al [10] identified 17b-HSD11 as the third most abundant protein after 4844 ADRP and ACSL3 among 17 major proteins associated with the LDs formed in the human hepatoma cell line HuH7 However, their analyses of its subcellular distribution by western blot and immunocytochemistry showed that most of the 17b-HSD11 was localized in the LD-enriched fraction, whereas another protein identified as LD-associated, ACSL3, was equally localized on the LDs and particulate fractions, including the ER membranes Their result showing an LD-restricted distribution is not consistent with our result showing dual localization on both the LDs and the ER These discrepancies can be explained by different cell types used in the experiments, possible differences in the maturation stages of LD formation, and the structure of the LD itself Our histocytochemical analysis of the intestine and liver of mice fed Wy-14 643 showed that the induced expression of 17b-HSD11 and formation of LDs are not coupled, at least in the intestine (Fig 6A versus Fig 6B) The physiologic role of 17b-HSD11 in the intestine may be different from that in the liver The mechanism of LD formation has not been fully elucidated, but it is thought to involve budding off of neutral lipid accumulations surrounded by a phospholipid monolayer containing proteins from the ER membranes [17–19] More than 17 proteins have been identified as being associated with LDs [10,20,21], including perilipin, ADRP, and the tail-interacting protein, TIP47, collectively known as the PAT protein family [22] These PAT proteins lack long runs of hydrophobic amino acid residues that would correspond to signal peptides or transmembrane domains, and the targeting signals for LDs seem to be quite diverse even among PAT members, suggesting multiple modes of association with LDs [23] The studies on other proteins are very limited, and 17b-HSD11 has a long hydrophobic region at the N-terminus that is not cleaved off, as directly shown by its sequence analysis [5] The N-terminal hydrophobic sequence seems to be essential for localization of 17b-HSD11 on the ER Our pulse-chase experiment suggests that 17b-HSD11 is localized on the LDs via the ER Thus, the N-terminal sequence may be required for its localization on the ER, but may not be sufficient for its relocalization on the LDs It is probable that 17b-HSD11 has a sequence that will be recognized by other components for localization to ER and LDs Our present study using both cultured cells and animal tissues showed that 17b-HSD11 is a bona fide LD-associated protein Among the LD-associated proteins so far characterized, few have been characterized for their functions on the LDs The most widely distributed LD proteins are perilipin, ADRP and TIP47, and they help to support the basic structure and function of the LDs, as FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Yokoi et al supported in recent work on ADRP-deficient mice, which display impairment of LD formation and resistance to diet-induced fatty liver [24] The rapid degradation of ADRP by a proteasome-mediated pathway during LD regression was also reported [25] ACSL3 was recently identified as a major commonly LD-associated protein [10], and its essential role in LD formation, through involvement in local synthesis of neutral lipids, was also reported [26,27] In contrast to these LD proteins, 17b-HSD11 may not play such an essential role in LD formation and function, because of its unique tissue distribution [7] and regulated expression [5] We recently found a new PAT protein, myocardial LD protein, which is highly enriched in the heart, and its precise function is not known, although we suggested its involvement in the metabolic response to fasting [23] Tissue-specific association of these accessory proteins with the LDs may play a key role in whole body energy homeostasis, including dietary lipid metabolism, especially in the case of 17b-HSD11, which is uniquely induced in the small intestine and liver Further studies will be required to clarify these possibilities Experimental procedures Animals and treatments All procedures involving animals were approved by the Meiji Pharmaceutical University Committee for Ethics of Experimentation and Animal Care Male C57BL mice about weeks of age were maintained under a 12 h light ⁄ 12 h dark cycle with free access to food and water After being fed a diet containing the PPARa agonist Wy14 643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]acetic acid} (Tokyo-Kasei, Tokyo, Japan) at 0.05% (w ⁄ w) or a normal laboratory diet, the mice were killed by cervical dislocation, and portions of the intestine and liver were removed for sample preparation Antibody production and western blotting Antibodies to 17b-HSD11 were raised in rabbits using a synthetic peptide corresponding to amino acids 95–109 (accession number Q9EQ06) or recombinant glutathione S-transferase fusion 17b-HSD11 protein as antigen Both antibodies were affinity purified using antigens, and used for western blotting and immunohistochemistry Western blotting was performed by resolving proteins on SDS ⁄ PAGE gels and transferring them to poly(vinylidene difluoride) membranes as previously described [11] Peroxidase-conjugated secondary antibody and an enhanced chemiluminescent kit (Super Signal West Pico, Pierce, Richmond, IL, USA) were used Rabbit anti-mouse calnexin serum was purchased from Stressgen (Victoria, Canada) 17-b-Hydroxysteroid dehydrogenase type 11 Plasmid construction Full-length cDNA clone for mouse 17b-HSD11 containing 40 nucleotides of the 5¢-noncoding sequence (GenBank accession number NM053262) was obtained by PCR using primers 5¢-GGgaattcGTTTAGGACCGGGAACGAGAGC (added EcoRI site in lower case) and 5¢-GGCctcgagTCA ATCGGCTTTCAGGGAACC (with XhoI site) The DNA fragment was digested with the enzymes and inserted into the EcoRI–XhoI site of a plasmid vector The correct sequence was confirmed, and the plasmid DNA was used for further plasmid construction To obtain GST-fused 17b-HSD11 protein for antibody production, a DNA fragment corresponding to amino acids 19–298 was amplified by PCR using primers 5¢-GGCCCgaattcATTGAGTCTCTT GTCAAGC (added EcoRI site in lower case) and 5¢-GG CctcgagTCAATCGGCTTTCAGGGAACC (with XhoI site) and cloned into the pGEX4T-1 plasmid vector To obtain an expression plasmid for 17b-HSD11 with GFP at the C-terminus, a DNA fragment was amplified by PCR using primers 5¢-GGgaattcGTTTAGGACCGGGAACGA GAGC (added EcoRI site in lower case) and 5¢-gcct cgagCTTGTCTTTGTACCCAACAAC (with XhoI site) and cloned into pCGFP2 or pCMVtag5A To obtain an expression plasmid for 17b-HSD11 with a Halo-tag at the C-terminus, a DNA fragment was amplified by PCR with primers 5¢-AAAgctagcGTTTAGGACCGGGAAC (added Nhe site in lower case) and 5¢-atcCTTGTCTTTGTACCCA ACAACTGCATC-3 (with EcoR site) and cloned into Halo-tag pHT2 (Promega, Madison, WI, USA) All of the constructed plasmid DNAs were subjected to sequencing analysis for confirmation of mutation-free amplification Cell culture and DNA transfection CHO-K1 cells were maintained in F-12 nutrient mixture medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v ⁄ v) fetal bovine serum at 37 °C in a humidified atmosphere of air ⁄ CO2 (5%) Transient transfection of CHO-K1 cells was carried out using Lipofectamine Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions Briefly, 0.4 lg per well of plasmid DNA was incubated with 0.8 lL per well of PlusReagent, 1.2 lL per well of Lipofectamine Reagent and 250 lL of serum free F-12 medium, and the 60% confluent cells in a 24-well plate were exposed to this preincubated DNA– lipofectin complex After exposure for h, the cells were cultured in F-12 medium supplemented with 10% fetal bovine serum Fluorescence imaging Stably or transiently transfected CHO cells cultured on poly-lysine-coated coverslips were washed with NaCl ⁄ Pi and then fixed with 4% paraformaldehyde for 10 at FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4845 17-b-Hydroxysteroid dehydrogenase type 11 Y Yokoi et al room temperature After being washed with NaCl ⁄ Pi, cells were permeabilized with 0.1% Triton X-100 in NaCl ⁄ Pi for 30 at room temperature The cells, preincubated with 5% BSA in NaCl ⁄ Pi for 30 at room temperature to block nonspecific binding, were then incubated with the antibodies for 30 at room temperature After being rinsed with wash buffer (0.2% Triton X-100 in NaCl ⁄ Pi), the cells were incubated with secondary goat anti-(rabbit IgG) or anti-(mouse IgG) conjugated with fluorescein isothiocyanate (MP Biochemicals, Aurora, OH, USA) or with Alexa Fluor 594-labeled goat anti-(rabbit IgG) (Invitrogen) After being washed with wash buffer and rinsed with NaCl ⁄ Pi, the cells were fixed in MOWIOL (Sigma-Aldrich, St Louis, MO, USA), and the specimens were subjected to confocal fluorescence microscopy with a Fluoview FV500 microscope (Olympus, Tokyo, Japan) Pulse-chase experiment using the Halo-tag system Cells transiently expressing 17b-HSD11-Halo-tag on coverslides were labeled with lm Halo-tag diAcFAM ligand for 15 according to the manufacturer’s instructions (Promega) after protein expression for 24 h Unbound ligand was removed by washing the cells with NaCl ⁄ Pi, and the cells were incubated for h in the F-12 medium containing 10% fetal bovine serum Some cells on slides were fixed and mounted for microscopic observation of ‘pulse-labeled’ cells Other cells were incubated for a further 24 h in the F-12 medium supplemented with 10% fetal bovine serum and 0.15 mm oleate to induce formation of LDs and then processed as ‘chased cells’ for microscopic observation Immunohistochemistry Tissue samples from mice fed a control diet or one containing 0.05% Wy-14 643 were fixed in a formaldehyde-based fixing solution (Genostaff, Inc., Tokyo, Japan) overnight at °C, embedded in paraffin, and cut into 6-lm-thick sections Deparaffinized and rehydrated slides were subjected to microwave antigen retrieval by boiling for 10 in 10 mm citric acid buffer (pH 6.0), and then treating with 3% H2O2 in methanol for 15 at room temperature Slides were washed in Tris-buffered saline (NaCl ⁄ Tris), and then blocked with Dako Protein Block (Dako X0909) for 10 at room temperature Affinity-purified rabbit anti17b-HSD11 recombinant serum or preimmune rabbit IgG (0.5 lgỈmL)1) were incubated with sections overnight at °C Slides were washed in NaCl ⁄ Tris with Triton X-100 (NaCl ⁄ TrisT) and then with NaCl ⁄ Tris, followed by biotinylated goat anti-(rabbit IgG) for 30 at room temperature After washing with NaCl ⁄ TrisT and NaCl ⁄ Tris, binding was detected by developing with diaminobenzidine The tissue sections were counterstained using hematoxylin 4846 Subcellular fractionation Subcellular fractionation was performed as described previously [28], with modifications The livers were homogenized with a Teflon-glass homogenizer at 1200 r.p.m in five volumes of ice-cold homogenization buffer [0.25 m sucrose, mm EDTA, 0.1% ethanol, 0.1% protease inhibitor mix (Wako, Tokyo, Japan), 10 mm Tris ⁄ HCl, pH 7.4] A PNS fraction was prepared by centrifugation for at 800 g (Kubota 6500 with AG-508R rotor; Kubota, Tokyo, Japan) The supernatant was recentrifuged for 20 at 25 400 g (Kubota 6500 with AG-508R rotor) The pellet was suspended in five volumes of ice-cold homogenization buffer 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T, Yamashita H & Hayakawa T (2001) A Color Atlas of Sectional Anatomy of the Mouse Adthree Publishing, Tokyo FEBS Journal 274 (2007) 4837–4847 ª 2007 The Authors Journal compilation ª 2007 FEBS 4847 ... amounts of 17b-HSD11 protein were markedly induced in both the mouse intestine and liver by administration of the PPARa agonist The induction ratios in both tissues were greater than 20, and the induced... that 17b-HSD11 was greatly induced in the intestine and liver of mice by administration of a PPARa agonist, Wy-14 643 [5], the changes in protein levels were examined by western blotting For this... induction levels of 17b-HSD11 in the liver and intestine The same amounts (20 lg) of proteins from the PNS fractions of liver and small intestinal mucosa were analyzed by western blotting using antibodies