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 reticu-lum under n
Trang 1of 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
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
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 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 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 adminis-tration 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 reticu-lum 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 con-firmed that 17b-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endo-plasmic 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 con-ditions, 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
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.
Trang 2genes 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 but
also in other organs [4] We have been interested in the
extrahepatic functions of PPARa, and recently
identi-fied the gene encoding 17b-hydroxysteroid
dehydro-genase type 11 (17b-HSD11) as a gene that is very
effectively PPARa-regulated in the intestine [5]
17b-HSD11 is a member of the short-chain
dehydroge-nase⁄ reductase superfamily (family member 8), and it
interconverts 17b-OH⁄ 17-oxosteroids [6] Thus, the
activities of the androgen metabolite pair
androster-one⁄ 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
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-HSD11
was localized in the cytoplasm, on the basis of
obser-vations made using 17b-HSD11 tagged with green
fluorescent protein (GFP) at the N-terminus Using
proteomics analysis, Fujimoto et al [10] identified
17b-HSD11 as one of the major lipid droplet
(LD)-associ-ated 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
nec-essary 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
condi-tions 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
pre-pared specific antibodies against mouse 17b-HSD11 to
detect endogenous protein: 17b-HSD11 tagged with
GFP 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 that
17b-HSD11 is localized both on the endoplasmic
retic-ulum (ER) and on LDs, depending on physiologic
conditions
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 intes-tine 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 syn-thetic 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 pro-teins were separated by SDS⁄ PAGE for western blot-ting analysis Both antibodies specifically recognized the same protein on the SDS⁄ PAGE gels having a molecular mass of 34 kDa (Fig 1A), and a representa-tive result obtained using postnuclear fractions of the intestinal mucosa and liver and antibodies to recombi-nant 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 17b-HSD11 along the gut, proteins from portions of stom-ach, 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 duo-denum and jejunum, with a similar expression pattern
to that of L-FABP (Fig 2B) Absence of these pro-teins 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 duode-num than in the jejuduode-num, whereas 17b-HSD11 levels
Trang 3were almost the same in both sites, with a drop at the
boundary between the two sites, showing a biphasic
expression pattern (Fig 2C)
17b-HSD11 colocalized with an ER marker protein
in transfected Chinese hamster ovary (CHO) cells
We next investigated the intracellular location of 17b-HSD11 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 17b-HSD11, when it was detected by the indirect immuno-fluorescence method using a specific antibody against the protein These microscopic observations suggested that 17b-HSD11 is associated with the ER in the trans-fected CHO cells under normal cell culture conditions
4 5
6
9 10
1
2 3
7
8
stomach
jejunum
colon
ileum duodenum
caecum A
B 1 2 3 4 5 6 7 8 9 10
1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’
17 β-HSD11
17 β-HSD11
L-FABP
L-FABP C
Fig 2 Expression of 17b-HSD11 protein in the gastrointestinal tract (A) The gastrointestinal tract of mice [29] fed a diet containing Wy-14 643 for 5 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.
75
37 50
25
(kDa)
75
M
Peptide Recombinant Wy14,643
Western blot
50
25
Intestine Liver
+ + + +
Wy14,643
17 β-HSD11
CBB-stain
B
A
Fig 1 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
(Recombi-nant) 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
blot-ting 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
frac-tions 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.
Trang 4This 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 3
(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
demon-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 plas-mid, and were then cultured in a medium supple-mented 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 under-went a marked change to largely localized signals around the LDs upon their formation in the
transfect-ed 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 17b-HSD11-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
HSD-Myc
Merged(Calnexin + HSD)
HSD-Myc
Merged (PI + HSD)
Fig 3 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
594-labeled secondary antibodies (red) Areas of colocalization appear as
yellow in the merged images (merged) The bar represents 10 lm.
Trang 5the 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
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 5 days induced the expression of the
pro-tein in both tissues to such an extent that it was
easily detected by enzyme-linked
immunohistochemis-try In the small intestine, the cytoplasm or the
retic-ular structure of microvillous epithelia was heavily
stained with the antibodies only after treatment of
mice with a PPARa agonist, and only a few
micro-vesicular structures were detected that were
sur-rounded by 17b-HSD11 (Fig 6A) These patterns are
consistent 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 Substantial
induction of 17b-HSD11 was also confirmed in the
liver, and the parenchymal cells were heavily stained However, 17b-HSD11 was concentrated on vesicular structures (Fig 6B), suggesting that most of the 17b-HSD11 molecules are associated with LDs in the liver
Oil Red O
HSD-GFP
Calnexin PI
Merged (PI + HSD) Merged (Calnexin + HSD)
B
A
Fig 4 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 l M 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
LD-containing 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
localiza-tion, using Arexa 594-labeled secondary antibodies (red) Areas of
colocalization appear as yellow in the merged images (merged).
The bar represents 10 lm.
Trang 617b-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
associa-tion 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
subcel-lular fractionation of the liver homogenate followed by
western blot analysis, because the method of
subcellu-lar fractionation has been established to be optimal for
the 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 fractionated
by the differential centrifugation method, and the
pro-teins 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
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 con-trast, the distribution of an ER protein, calnexin, did not 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 a quantitative viewpoint, however, the recovery of 17b-HSD11 (10–20%) in the top fraction after centrifuga-tion 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
Before LD formation After LD formation
Halo-tag Ligand
DiAcFAM
Anti-17 ββ-HSD11
merged
Fig 5 Redistribution of 17b-HSD11 from the ER to LDs 17b-HSD11 with Halo-tag-expressing vector was transiently
transfect-ed into CHO cells and incubattransfect-ed with Halo-tag ligand for 15 min after protein expres-sion 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 l M oleic acid to induce formation of LDs The chased 17b-HSD11 was analyzed for its redistribution in the cells with LDs Bars represent 10 lm.
Trang 7and tissue types should also be considered In
princi-ple, the microscopic observations on the distribution
of 17b-HSD11 under various conditions were
essen-tially 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
induc-tion is more effective than in the liver parenchymal
cells 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 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 vec-tors [15] Although it has not been finally demon-strated that the 17b-HSD11 gene is directly regulated
by PPARa and its ligand in the intestine, because of methodologic difficulties, involvement of a tissue-spe-cific 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
50 50
Wy14,643 Control
Fig 6 Immunohistochemical staining of 17b-HSD11 in mouse tissues The intestine (A) and liver (B) tissue sections from mice fed a con-trol diet (concon-trol) or one containing Wy-14 643 (Wy14 643) for 5 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 2,4-diaminobutyric acid The brown reaction product produced by the 2,4-2,4-diaminobutyric acid reaction denotes positively stained cells The tis-sue sections were counterstained using hematoxylin.
Trang 817b-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 4 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
subcellu-lar 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
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 distri-bution by western blot and immunocytochemistry showed that most of the 17b-HSD11 was localized in the LD-enriched fraction, whereas another protein iden-tified 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 phospho-lipid 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 corre-spond 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-termi-nal 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 character-ized, 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 sup-port the basic structure and function of the LDs, as
Wy14,643
Control
PNS Mito Micro Cyto Top PNS Mito Micro Cyto Top
17 β-HSD11
Calnexin
L-FABP
CBB-stain
(100) 49 11 32 8 (100) 58 9 25 8 (%)
protein recovery
Fig 7 Subcellular fractionation studies on the localization of
17b-HSD11 in mouse liver The livers of mice fed a control diet (–) or
one containing Wy-14 643 for 5 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.
Trang 9supported 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 pathway
during LD regression was also reported [25] ACSL3
was 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 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
sug-gested its involvement in the metabolic response to
fast-ing [23] Tissue-specific association of these accessory
proteins with the LDs may play a key role in whole
body energy homeostasis, including dietary lipid
metab-olism, especially in the case of 17b-HSD11, which is
uniquely induced in the small intestine and liver
Fur-ther 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 6 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]ace-tic 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]
Peroxi-dase-conjugated secondary antibody and an enhanced
chemiluminescent kit (Super Signal West Pico, Pierce,
Rich-mond, IL, USA) were used Rabbit anti-mouse calnexin
serum was purchased from Stressgen (Victoria, Canada)
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 frag-ment 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 manu-facturer’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 3 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 min at
Trang 10room temperature After being washed with NaCl⁄ Pi, cells
were permeabilized with 0.1% Triton X-100 in NaCl⁄ Pifor
30 min at room temperature The cells, preincubated with
5% BSA in NaCl⁄ Pi for 30 min at room temperature to
block nonspecific binding, were then incubated with the
antibodies for 30 min 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
iso-thiocyanate (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
cover-slides were labeled with 1 lm Halo-tag diAcFAM ligand
for 15 min 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 1 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
fur-ther 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
micro-scopic observation
Immunohistochemistry
Tissue samples from mice fed a control diet or one
contain-ing 0.05% Wy-14 643 were fixed in a formaldehyde-based
fixing solution (Genostaff, Inc., Tokyo, Japan) overnight at
4C, embedded in paraffin, and cut into 6-lm-thick
sec-tions Deparaffinized and rehydrated slides were subjected
to microwave antigen retrieval by boiling for 10 min in
10 mm citric acid buffer (pH 6.0), and then treating with
3% H2O2 in methanol for 15 min at room temperature
Slides were washed in Tris-buffered saline (NaCl⁄ Tris), and
then blocked with Dako Protein Block (Dako X0909) for
10 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 at
4C 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 using
hematoxylin
Subcellular fractionation Subcellular fractionation was performed as described previ-ously [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,
1 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 5 min at 800 g (Kubota 6500 with AG-508R rotor; Kubota, Tokyo, Japan) The supernatant was recentrifuged for 20 min at
25 400 g (Kubota 6500 with AG-508R rotor) The pellet was suspended in five volumes of ice-cold homogenization buffer (‘Mito’ fraction) The supernatant was recentrifuged for 60 min at 100 000 g (CB80WX centrifuge with RPS40 rotor; Hitachi Koki, Tokyo, Japan) to obtain the superna-tant (‘Cyto’ fraction) The top white fraction was separated
as the ‘Top’ fraction The pellet was suspended in five vol-umes of ice-cold homogenization buffer (‘Micro’ fraction) Acknowledgements
We thank Dr K Higashi and the Motojima laboratory members for helpful discussions This work was sup-ported in part by the Meiyaku Open Research Project and grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science
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