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Endocrinology 146(6):2593–2601
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-1478
Isolation and Identification of Histone H3 Protein
Enriched in Microvesicles Secreted from
Cultured Sebocytes
Ayako Nagai, Takashi Sato, Noriko Akimoto, Akira Ito, and Michihiro Sumida
Department of Molecular and Cellular Biology (A.N., M.S.), Division of Biochemistry and Molecular Genetics, Ehime
University School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295, Japan; and Department of Biochemistry and
Molecular Biology (T.S., N.A., A.I.), School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo
192-0392, Japan
Secretion of microvesicles, defined as sebosomes, containing
lipid particles were discovered for the first time in cultured
sebocytes. After reaching confluency, hamster-cloned sebocytes released bubble-like microvesicles with a diameter
range of 0.5–5.0 ␮m. They had a complex structure containing
multiple Oil Red O-stainable particles. The lipid components
of the microvesicles were large amounts of squalene both of
hamster-cloned and rat primary cultured sebocytes. The microvesicles contained a concentrated 17-kDa cationic protein,
which was soluble in sulfate buffer including Nonidet P-40 at
pH 1.5. As the protein bound tightly to heparin-Sepharose and
eluted with 1.5 M NaCl, it was further purified from a SDSPAGE gel. Peptide sequencing identified the protein to be
histone H3. Polyclonal antibodies against the purified protein
detected the antigen in the microvesicles both in the hamster-
T
HE SEBACEOUS GLAND plays a protective role on the
skin surface by secreting sebum lipids that include
squalene, wax ester, and cholesterol esters (1). The development of the gland is regulated by various endocrine factors.
The proliferation of the sebocytes is stimulated by epidermal
growth factor (EGF), and their differentiation is induced by
steroid hormones such as testosterone and dihydroandrosterone (2). When their differentiation was induced by thioglitazone, an insulin sensitizer, its responsive adipogenic
transcription factor, peroxisome proliferator-activated receptor-␥ (3, 4), was expressed in sebocytes and deposited lipid
droplets (5). To study the characteristics of sebocytes including the regulation of the differentiation and lipid metabolism
of the cells, we attempted to establish a sebocyte clone from
a hamster auricle (6). The cloned cells proliferated and differentiated under control of EGF, TGF-␣, basic fibroblast
growth factor, keratinocyte growth factor, 1,25-dihydroxyvitamin D3, and androgen (7, 8). Recently, Zouboulis et al. (9)
also established the SZ-95 cell, a simian virus 40 large T
antigen immortalized human sebocyte, and they showed that
First Published Online March 3, 2005
Abbreviations: CBB, Coomassie Brilliant Blue; EGF, epidermal
growth factor; EGFP, enhanced green fluorescent protein; FITC,
fluorescein isothiocyanate; HRP, horseradish peroxidase; MV, microvesicles; PI, propidium iodide.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
cloned and rat primary cultured sebocytes. The antibodies
demonstrated a distribution of the protein within the nucleus, cytoplasm, and precursor microvesicles. When a gene
construct encoding histone H3-enhanced green fluorescent
protein was transfected to the sebocytes, fluorescence of the
fusion proteins was detected within both the nucleus and
the precursor microvesicles of the cytoplasm. The distribution of heparan sulfate was evident in the microvesicles,
and it suggested the possibility that the histone H3 protein
was recruited and then condensed to the secreted microvesicles by the molecules. In addition, the 14-3-3 protein,
which was detected in the microvesicles, also may help incorporate the histone H3 protein in the microvesicles because it can bind to both histone and lipid particles. (Endocrinology 146: 2593–2601, 2005)
the cells preserved the original properties of the gland (i.e.
undergoing differentiation in the presence of testosterone
and insulin and synthesized testosterone in response to
MSH) (10). Based on these observations, Zouboulis et al. (9)
proposed to define sebocytes as local endocrine cells.
The important function of the sebocyte is to protect the
skin surface from hazardous chemicals, UV, and microbes
by generating lipids layers with sebum lipid components,
including squalene and wax esters (11). It has been suggested that vitamin E also is secreted in the sebaceous
lipids (12) with protective roles against oxidative injury.
Animal sebum contains palmitoleic acid, which is exclusively secreted from the sebaceous gland with an antibacterial function (13). In the skin sebum, defensin, a cationic
protein, was identified in infected human skin with this
antibacterial function (14). It should be noted that histone
H2A and H2B, other cationic proteins, were found in fish
and frog skin with effective antibacterial activities (15, 16).
It was reported that the histone family in various tissues
exerted potential antibacterial functions, such as histone
H1 in fish skin (17) and H2B and H4 in placenta (18, 19)
and colon (20).
Here, we found that both primary cultured and cloned
sebocytes secreted membrane vesicles, defined as sebosomes, containing enriched histone H3 protein and
squalene-condensed lipid particles. These vesicles of the
sebocytes suggested the novel cell function that is associated with the secretion of antibacterial proteins and ste-
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Endocrinology, June 2005, 146(6):2593–2601
rol regulation, and they may play a crucial role in protecting the skin surface.
Materials and Methods
Cell culture
The cell line of hamster sebocytes has been established from sebaceous glands of auricles of 5-wk-old male golden hamsters as previously
described (6, 7). The sebocytes were cultured in DMEM/Ham’s F12
medium (1:1) (Invitrogen, Carlsbad, CA) supplemented with 6% (vol/
vol) heat-denatured fetal calf serum (Asahi Techno Glass, Tokyo, Japan),
2% (vol/vol) human serum (ICN Biochemicals, Costa Mesa, CA), 0.68
mm l-glutamine (Invitrogen), antibiotic antimycotic solution (Sigma
Chemical Co., St. Louis, MO), and recombinant human EGF (10 ng/ml)
(Progen Biotechnik GmbH, Heidelberg, Germany) as a standard culture
medium. The subconfluent cloned sebocytes were trypsinized and
seeded on a 10-cm-diameter culture dish (Corning Co., Corning, NY) for
the maintenance of the cells and their secreted microvesicles (MV) after
reaching confluency were harvested by centrifugation for 10 min at
6000 ⫻ g.
Animals and primary culture of rat sebocytes
Male Crj:Wistar rats, weighing 200 –300 g, were fed with a standard
laboratory diet (Oriental Yeast, Tokyo, Japan) and water ad libitum,
according to the animal-handling manual by the Laboratory of Animal
Center at Ehime University, School of Medicine. For the preparation of
primary cultured sebocytes, an isolated preputial gland from an anesthetized rat was digested by 300 U/ml collagenase (Sigma) and 2.4 U/ml
dispase (Roche, Mannheim, Germany) in a standard culture medium
and then gently dispersed by pipetting and finally placed in a 3.5-cm
culture dish (Corning) as previously reported (21). The outgrowth cells
of the adhered sebaceous glands were maintained in the standard culture medium, and the secreted MV in the medium from the cells were
harvested by centrifugation for 10 min at 6000 ⫻ g.
Oil Red O staining
Hamster-cloned sebocytes and rat preputial outgrowth cells were
fixed in 10% formalin and stained with 0.2% Oil Red O prepared in
isopropanol/water (4:3) as described previously (22). The released MV
were also stained by Oil Red O similar to the cells as described above.
Analysis of lipid components in MV
Analysis of lipid components of the MV was performed according to
the method described previously (7). Briefly, the total lipids of the MV
were extracted with chloroform/methanol (2:1, vol/vol) (23) and subjected to an automatic thin-layer chromatography Iatroscan (Iatron Laboratories, Tokyo, Japan) (24, 25). An initial development was performed
in hexane/benzene (35:35, vol/vol). After drying at room temperature
for 2 min, a second development was performed in hexane/diethyl
ether/formic acid (50:20:0.7, vol/vol) (26 –28). Standard lipids were tripalmitin (triglyceride), palmitic acid (free fatty acid), 1-monoglyceride,
squalene, cholesterol, cholesterol palmitate (cholesterol ester), phosphatidyl choline (phospholipid), and palmityl palmitate (wax ester).
Density of the isolated MV analyzed with sucrose
density gradient
The secreted MV from cultured sebocytes were harvested by centrifugation, resuspended to 2.0 ml of 20 mm Tris/HCl buffer (pH 8.0),
and applied to a discontinuous sucrose density gradient (29, 30), which
was formed above the 60% sucrose bed by adding 2.0 ml each of 50, 35,
25, and 5% sucrose solutions containing 20 mm Tris/HCl buffer (pH 8.0).
The tube was centrifuged at 10,000 ⫻ g for 1 h in an ultracentrifuge
(model SCP70H; Hitachi Co., Tokyo, Japan) at 4 C. After centrifugation,
MV samples were collected and stained with Oil Red O to study the lipid
particles in them.
Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
Isolation and purification of the MV proteins
The secreted MV from cultured sebocytes were harvested and
washed twice with chilled PBS. According to the method by Zhong et
al. (31), the MV proteins were extracted as follows: MV were exposed to
lysis buffer [10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 1.5 mm
phenylmethylsulfonyl fluoride, 5 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin] and incubated with sulfuric acid (final concentration, 0.2 m) for
60 min on ice. After centrifugation at 12,000 ⫻ g for 30 min, the supernatant was transferred to a fresh tube, diluted four times with distilled
water, and applied onto a heparin-Sepharose column (Amersham Biosciences, Uppsala, Sweden) preequilibrated with 10 mm NaCl. After
washing the column with 0.15 m NaCl, the bound proteins were eluted
by a stepwise gradient of 0.5, 1, 1.5, and 2 m NaCl, and proteins in each
fraction were precipitated on ice for 30 min by adding trichloroacetic
acid to a final concentration of 20% (18). These precipitated proteins were
centrifuged at 15,000 ⫻ g for 10 min at 4 C. The pellets were washed twice
with chilled acetone and stored at ⫺20 C until use.
Peptide sequence of the purified MV proteins
The purified MV proteins were separated by SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. Visualization of transferred protein bands was achieved by staining the membranes with Coomassie Brilliant Blue (CBB) and destaining with 20%
methanol and 10% acetic acid in water. The primary structures of the
proteins were determined by automated Edman degradation using a
peptide sequencer (Shimazu PSQ-2; Shimazu, Kyoto, Japan) (32).
Antibodies
Polyclonal antibodies were raised in a New Zealand White rabbit by
immunization with the purified 17-kDa MV protein as the antigen.
Anti-14-3-3 antibody was from Santa Cruz Biotechnology, Inc., Santa
Cruz, CA. Anti-heparan sulfate antibody (clone 10E4) was purchased
from Seikagaku Kogyo, Tokyo, Japan. Horseradish peroxidase (HRP)conjugated antirabbit IgG antibody was obtained from Zymed Laboratories, Inc., San Francisco, CA. HRP-conjugated antimouse IgM antibody, fluorescein isothiocyanate (FITC)-conjugated antimouse IgM
antibody, and FITC- or Cy3-conjugated antirabbit IgG antibody were
from Jackson Immuno Research Laboratories, Inc., West Grove, PA.
Western blotting
MV proteins (10 ␮g/well) were extracted and applied to perform
SDS-PAGE with 15% acrylamide gel in the Laemmli buffer system
(Laemmli sample buffer; Bio-Rad, Richmond, CA) (33). The proteins in
the gel were transblotted onto polyvinylidene difluoride membranes
(Bio-Rad) that were blocked with 2% BSA (fraction V; Nacalai Tesque,
Inc., Kyoto, Japan) solubilized in PBS containing 0.1% Tween 20 for 1 h.
The membrane was incubated with anti-17-kDa protein antiserum (1:500
dilution), anti-14-3-3 antibody (1:500 dilution), or anti-heparan sulfate
antibody (1:200 dilution) for 1 h. The membrane was rinsed five times
with 0.1% Tween 20 and incubated with a 1:500 diluted antirabbit IgG
HRP-labeled antibody or antimouse IgM HRP-labeled antibody (1:1000
dilution) for 1 h. After the membrane was rinsed five times with 0.1%
Tween 20, it was incubated with diaminobenzidine, and the signals of
the 17-kDa protein, 14-3-3 protein, or heparan sulfate were detected with
a densitometer (34). Each signal was analyzed with digitized images
using the NIH Image program. At least three experiments were done for
each experiment.
Immunocytochemical detection of the 17-kDa protein, 14-3-3
protein, and heparan sulfate
The cultured sebocytes were maintained in standard culture medium
with 10 ng/ml EGF. For an indirect immunofluorescence study, the cells
were washed with PBS and fixed with 100% methanol for 1 min and
incubated in 1 ml of 0.25% Triton X-100 in PBS on ice for 5 min. To block
nonspecific binding of antibodies, the cells were incubated with 2% BSA,
except the cells that were prepared for staining with anti-heparan sulfate
antibody, and sequentially the cells were incubated in 10% donkey
serum (Chemicon, Temecula, CA) in PBS for 1 h and 10% human serum
Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
derived from a healthy volunteer in PBS for 1 h. The cells were incubated
in the anti-17-kDa protein antiserum, anti-14-3-3 antibody, anti-heparan
sulfate antibody, or control IgG prepared from nonimmunized rabbit
serum diluted 1:200 in PBS containing 1% BSA for 2 h at room temperature. After the incubation with the first antibodies, the cells were rinsed
once with PBS containing 0.1% Tween 20 and twice with PBS and
additionally incubated with the FITC- or Cy3-conjugated donkey antirabbit IgG antibody or FITC-conjugated antimouse IgM antibody, which
was diluted 1:100 in PBS containing 1% BSA for 1 h. The cells were
incubated with 4 ␮g/ml propidium iodide (PI) (Sigma) in PBS at room
temperature for 5 min. These treated cells were observed under fluorescence microscopy using an inverted microscope (Nikon Eclipse TE
300, Tokyo, Japan) equipped with filter systems [excitation filter 460 –500
and barrier filter 510 –560 for enhanced green fluorescent protein (EGFP)
and FITC; excitation filter 510 –560 and barrier filter 590 for PI). Doublestaining analysis of FITC and Cy3 was performed with laser confocal
microscopy (Nikon C1 confocal microscopy) equipped with filter systems (excitation filter 488 and barrier filter 515/30 for FITC; excitation
filter 543 and barrier filter 605/75 for Cy3). Pictures of images were taken
by a CCD camera (Cool SNAP; Roper, Atlanta, GA) and processed with
the computer program Adobe Photoshop (Adobe Systems Inc., Mountain View, CA) (35, 36). The experiments were repeated at least four
times.
Construction of expression plasmids and gene transfection
Total RNA was isolated from the proliferating and differentiated
cultured sebocytes or rat preputial sebaceous gland by the acid phenol/
guanidine thiocyanate procedure (37) and used as templates for RT
reaction using Ready-To-Go You-Prime First-Strand beads (Amersham)
to synthesize the cDNA. The primer sets for the PCR of the open reading
frame of histone H3 cDNA were 5⬘-ATATCTCGAGCACCATGGCCCGTACGAAGCAGACCG-3⬘ and 5⬘-ATATAAGCTTAGCGCGCTCCCCACGGAT-3⬘ according to mouse histone H3 (GenBank accession no.
X16148). The primer sets for the PCR open reading frame of 14-3-3␥
cDNA were 5⬘-ATATCTCGAGCACCATGGTGGACCGCGAGCAA-3⬘
and 5⬘-ATATAAGCTTGTTGTTGCCTTCACCGCC-3⬘ according to 143-3␥ (GenBank accession no. AF058799). The PCR condition with Taq
polymerase (Applied Biosystems, Foster City, CA) was as follows: 94 C
for 5 min and 40 cycles of 94 C for 30 sec, 55 C for 45 sec, and 72 C for
60 sec using GeneAmp PCR System 9700 Thermal cycler (Applied
Biosystems). The amplified cDNAs of histone H3 protein and 14-3-3␥
were cloned into mammalian expression vector pEGFP-N1 (BD Clontech, Palo Alto, CA) to express EGFP-fusion proteins. Nucleotide sequencing was performed using a Big-Dye Terminator Kit (Applied
Biosystems) with an automated capillary electrophoresis DNA sequ-
FIG. 1. Secreted MV from cultured sebocytes. Secreted MV (C, D, and E) from and
intracellular precursor MV (A and B) of
cultured sebocytes were isolated and
stained with Oil Red O. A, C, and D, Hamster-cloned sebocytes; B and E, rat primary
cultured sebocytes. Scale bars, 10 ␮m (A
and B) and 5 ␮m (C–E).
Endocrinology, June 2005, 146(6):2593–2601
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encer (ABI PRISM 310 Genetic Analyzer; Applied Biosystems). The
pEGFP plasmid constructs were transfected into the cultured sebocytes
using DoFect Transfection Reagent (DOJINDO, Kumamoto, Japan).
Immunofluorescence images of the pEGFP, pEGFP-histone H3, and
pEGFP-14-3-3-transfected sebocytes were observed with a fluorescent
microscope and photographed with a digital imaging camera as described above (38, 39).
Results
Secretion of MV
Generation and secretion of MV with measured diameter
of 0.5–5 ␮m were demonstrated in cultured hamster sebocytes after reaching confluency, and multiple Oil Red Ostainable lipid particles were contained in the MV (Fig. 1, A,
C, and D). Density of the MV was determined with discontinuous sucrose density gradient centrifugation to be 1.15–
1.18 g/ml. Rat primary cultured outgrowth sebocytes also
generated and secreted large MV with bubble-like morphology containing multiple lipid particles (Fig. 1, B and E). When
0.1 mm oleic acid was added to the culture medium, the size
of the secreted MV from the cloned sebocytes was increased,
and their density was decreased to 1.09 –1.14 g/ml, accompanied by the increased lipid particle contents (Fig. 1D). The
generation and secretion of the MV were lowered by adding
an excess amount of EGF (50 ng/ml) to the cells, and the cells
started proliferation when they were trypsinized and seeded
at subconfluent cell density (data not shown).
Components of lipid particles in MV
We extracted lipids from the secreted MV and analyzed
their components with Iatroscan and found that high concentrations of squalene (33% of total MV lipids) and phospholipid (59.7%) were detected with free cholesterol (4.1%),
fatty acid (0.97%), triglyceride (0.58%), and trace amount of
wax ester (Fig. 2A). When 0.1 mm oleic acid was added to the
culture medium, the contents of squalene (49%), fatty acid
(12.2%), and triglyceride (6.0%) of the MV were increased
and phospholipid (30.1%) was decreased (Fig. 2B). The
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Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
FIG. 2. Analysis of lipid components of
the secreted MV. Lipids were extracted
from secreted MV from cultured sebocytes and analyzed by Iatroscan. A and
B, Secreted MV from hamster-cloned
sebocytes that were cultured in standard medium (A) or in medium supplemented with 0.1 mM oleic acid (B) for
24 h; C, secreted MV from rat primary
cultured sebocytes cultured in standard medium.
squalene content of the secreted MV from the rat primary
cultured sebocytes was high (61.6%), but the contents of
cholesterol (1.0%), fatty acid (2.8%), triglyceride (1.5%),
monoacylglycerol (1.6%), and wax ester (0.05%) were much
lower (Fig. 2C).
cleus nor the MV precursor (Fig. 4D). The histone H3
protein was identified both in the nucleus and in the MV
precursor in the cytoplasm in rat primary cultured sebocytes that grew from the tissue fragment (Fig. 4E).
Components of MV proteins
MV proteins of hamster-cloned sebocytes were analyzed
with SDS-PAGE, and a distinctive CBB staining pattern including several enriched proteins of low molecular masses
were demonstrated (Fig. 3A). As shown in Fig. 3, a 17-kDa
protein of the MV was concentrated and the content in the
MV was at least 3.5 times higher than the protein among the
total cell proteins (Fig. 3B). The 17-kDa protein was solubilized with 0.25% Nonidet P-40 and did not bind to the
anion exchanger, such as diethylaminoethyl-Sepharose and
diethyl[2-hydroxypropyl] aminoethyl (QAE)-Sepharose (data
not shown), but bound to heparin-Sepharose and was soluble
in sulfate buffer at pH 1.5. We tried to isolate the protein with
a heparin-Sepharose column that was eluted with 1.5 m NaCl
(Fig. 3C, lane 2). The eluted fraction was applied to SDSPAGE, and the 17-kDa proteins were extracted from the gel
(Fig. 3C, lane 3). The N-terminal peptide sequence of the
purified protein was determined to be MARTKQTARKS using Edman degradation, and the protein was identified to be
histone H3 protein using the BLAST search program (http://
www.expasy.org/tools/blast/).
Localization of histone H3 protein in the sebocytes
Polyclonal antibodies against the purified histone H3
protein were raised by injecting the purified histone H3
protein from the secreted MV. Western blot analysis with
the antibodies identified the 17-kDa protein in the MV
(Fig. 4A). Localization of histone H3 protein in the hamster-cloned cells was studied with the antibodies, and it
was shown only in the nucleus in the proliferating cells
(Fig. 4B) but was shown both in the nucleus and in the MV
precursor in the cytoplasm of the MV-generating cells (Fig.
4C). Nonimmunized control IgG stained neither the nu-
FIG. 3. Analysis of protein components of the secreted MV. A and B,
Proteins of the secreted MV from hamster-cloned sebocytes (A, lane
1, and B1) or of the whole cells (A, lane 2, and B2) were separated by
SDS-PAGE. A, CBB staining of the separated proteins; B, densitometric analysis of the CBB staining proteins; A, lane M, molecular
mass marker. C, Lane M, molecular mass marker; lane 1, proteins of
the secreted MV from hamster-cloned sebocytes; lane 2, MV proteins
that were eluted with 1.5 M NaCl from heparin-Sepharose; lane 3,
purified 17-kDa protein extracted from SDS-PAGE gel.
Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
Endocrinology, June 2005, 146(6):2593–2601
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FIG. 4. Localization of the 17-kDa protein in cultured sebocytes. A, Western blot analysis of MV protein; lane M, molecular mass marker; lane 1, CBB
staining of the MV proteins; lane 2, Western blot of MV proteins using the polyclonal antibodies against the purified histone H3 protein. B–D,
Immunohistochemistry of hamster sebocytes with the ammonium sulfate-purified polyclonal antibodies against the purified histone H3 protein; B,
proliferating cloned sebocytes; C, sebocytes containing MV precursors; D, sebocytes containing MV precursors stained with nonimmunized control
IgG; E, immunofluorescent staining of rat primary cultured sebocytes with the antibodies against the purified histone H3 protein. Panel 1 in B–E,
FITC fluorescence, panel 2, PI staining; panel 3, phase-contrast images. Scale bar, 10 ␮m. F, Immunofluorescent staining of the secreted MV with
FITC-conjugated antibodies against heparan sulfate (F1) and with Cy3-conjugated antibodies against purified histone H3 protein (F2) and the merged
images of F1 and F2 (F3). Scale bar, 1.0 ␮m.
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Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
To examine the colocalization of heparan sulfate and histone H3 protein in the secreted MV, double staining of the
heparan sulfate with FITC (Fig. 4F1) and histone H3 with Cy3
(Fig. 4F2) was carried out. Their fluorescences were later
observed with laser confocal microscopy. As shown in these
figures, the MV stained with both Cy3 and FITC, indicating
that all of the MV contained both histone H3 and heparan
sulfate. The majority of the merged image of FITC overlapped with Cy3 and turned into a yellowish color indicating
the colocalization of heparan sulfate and histone H3 in the
secreted MV (Fig. 4F3).
To confirm the localization of histone H3 proteins in the
cells and in the secreted MV, we constructed a histone H3EGFP plasmid and transfected the hamster-cloned sebocytes
with it. In the proliferating cells, the fluorescence of the
histone H3-EGFP fusion protein was detected only in the
nucleus (Fig. 5A) but both in the nucleus and in the MV
precursor in the cytoplasm after reaching confluency (Fig.
5B). Fluorescence of the histone H3-EGFP also was detected
in the secreted MV in cultured medium from the cells (Fig.
5C). In control cells in which EGFP was expressed, its fluorescence was detected in cytoplasm but not in the nucleus
or in the precursor (Fig. 5D) and secreted MV (data not
shown).
MV-associated proteins
To study sorting mechanisms how histone H3 concentrated in the secreted MV, we observed the localization of
heparan sulfate of the cloned sebocytes, because histone H3
protein bound tightly to heparan sulfate and eluted only in
high-salt solution (Fig. 3). Antibodies against heparan sulfate
bound to 30- and 17-kDa proteins in the secreted MV (Fig. 6A,
lane 2). Immunohistochemical studies with the antibodies
demonstrated the distribution of heparan sulfate on the precursor MV in addition to the plasma membrane (Fig. 6B).
These results indicated that heparan sulfate recruited histone
H3 to the secreted MV.
In our observation, Western blot analysis with anti-14-3-3
antibodies exhibited the 14-3-3 protein incorporated in the
secreted MV (Fig. 7A). Immunohistochemical studies
showed the distribution of the 14-3-3 protein both in the
cytoplasm and on the precursor MV in the confluent sebocytes (Fig. 7B). In addition, the localization of the 14-3-3EGFP fusion protein was detected in the secreted MV (Fig.
7D) and in the precursor MV in the cytoplasm (Fig. 7C). These
results suggest that the 14-3-3 protein contribute to the recruitment of the histone H3 protein into the secreted MV.
Discussion
We established hamster-cloned sebocytes from the auricular sebaceous glands (6). The cells proliferated in response
to androgens, and the lipogenesis was augmented by testosterone and 5␣-dihydrotestosterone but suppressed by
EGF, 1,25-dihydroxyvitamin D3, and all-trans retinoic acid
(7). The mitogenic and antilipogenic activities of the cells also
were evidenced by growth factors, including TGF-␣ and
basic fibroblast growth factor (8). In addition to these characteristics of the sebocytes, we found that the cells secrete
unique MV extensively after reaching confluency (Fig. 1).
FIG. 5. Localization of histone H3-EGFP in hamster-cloned sebocytes
and their secreted MV. Shown are images of fluorescent microscopy
(A–D) and of phase-contrast microscopy (a– d) of stably transfected
hamster sebocytes expressing histone H3-EGFP. A and a, Proliferating sebocytes; B and b, transfected sebocytes containing the MV
precursors; C and c, secreted MVs from the cells; D and d, control
sebocytes expressing only EGFP. Scale bar, 10 ␮m.
When the MV are compared with other exosomes secreted
from various tissue cells such as dendritic cells (40), intestinal
mucosal cells (41), B cells (42), tumor cells (43), reticulocytes
(44), and morphogenic cells (45), sebaceous MV have much
larger vesicle sizes and contain exclusively multiple lipid
particles (Fig. 1, C and D). It is likely that squalene in the
sebum originated in the secreted MV because the squalene
content extracted from the cells was very low in amount (7).
Components of MV proteins also were exclusive, including
highly concentrated 17-kDa protein (Fig. 3). In addition to
cloned sebocytes (Fig. 1, A, C, and D), primary cultured
sebocytes prepared from rat preputial tissues also generated
and secreted the MV that included the lipid particles (Fig. 1,
B and E). Although they were more expanded than cloned
cell-derived MV, the secretion of MV seemed to be the common characteristic of the confluent sebocytes.
Nagai et al. • Histone H3 in Sebosomes Secreted from Sebocyte
FIG. 6. Distribution of heparan sulfate in hamster sebocytes and
their secreted MV. A, Western blot analysis of the MV proteins with
anti-heparan sulfate antibodies; lane 1, CBB staining of the MV
proteins; lane 2, Western blot of MV proteins using anti-heparan
sulfate antibodies in secreted MV from the secobytes. B, Immunofluorescent staining (B1) and phase-contrast microscopy (B2) of the
sebocytes containing the MV with anti-heparan sulfate antibodies.
Scale bar, 10 ␮m.
Profiles of the lipid components of the secreted MV from
the sebocytes were analyzed, and the profiles showed that a
large amount of squalene, one of the main sebum components (11), was identified with detectable levels of cholesterol
and triglyceride (Fig. 2A). Because the amount of squalene,
triglyceride, and fatty acid in the secreted MV was largely
increased when oleic acid was added to the culture medium
(Fig. 2B), lipid contents and numbers of lipid particles in MV
were suggested to be controlled by the nutritional lipid conditions of the cells.
Analysis of the protein components of the secreted MV
with SDS-PAGE demonstrated that a 17-kDa protein was
enriched in the MV (Fig. 3). We characterized that the protein
was cationic and had high affinity to heparan sulfate and
FIG. 7. Distribution of 14-3-3 protein in hamster sebocytes and their secreted MV. A, Western blot analysis of secreted MV proteins with
anti-14-3-3 protein antibodies. B, Immunofluorescent staining of the precursor MV in hamster sebocytes with anti-14-3-3 protein antibodies (B1), PI staining of the precursor MV (B2),
and image of phase-contrast microscopy of the
precursor MV (B3). C and D, Localization of
14-3-3-EGFP protein in hamster sebocytes (C1
and C2) and in secreted MV from the sebocytes
(D1 and D2) and images of fluorescent microscopy (C1 and D1) and of phase-contrast microscopy (C2 and D2). Scale bar, 10 ␮m.
Endocrinology, June 2005, 146(6):2593–2601
2599
determined it to be histone H3 protein by N-terminal peptide
sequencing. We raised polyclonal antibodies against the purified protein and showed its localization in the precursor
MV both in the cytoplasm of the confluent cells and in the
nucleus in the MV-generating cells (Fig. 4), although the
protein was detected only in the nucleus in the proliferating
cells (Fig. 4). To confirm the sorting of the histone H3 protein
into the secreted MV, we expressed histone H3-EGFP fusion
protein in the cloned sebocytes and visualized its localization
both in the nucleus and on the MV precursor in the cytoplasm
of the MV-generating cells (Fig. 5). These results coincided
well with those that were revealed by immunohistochemical
studies (Fig. 4).
It can be predicted that the secreted histone H3 protein has
an antimicrobial function because similar cationic proteins,
such as histone H2A and H2B proteins, were demonstrated
to exert an antibacterial function in the cytoplasm of syncytiotrophoblasts and amnion cells (18). Histone H1 protein
was found in the cytoplasm of villus epithelial cells in human
gastrointestinal tract and was shown to protect the colonic
lumen against microorganism penetration (46). Furthermore,
the antimicrobial function of the histone family has been
reported in the skin mucosa of fish (15, 17), in frog skin (16),
in human placenta (18), and in human colon mucosa (20).
Although histone H3 proteins have been reported neither in
animal nor in human sebum components, its antibacterial
function on the skin surface will be expected when the protein in the MV is secreted through hair and sebaceous follicles. In our preliminary antimicrobial experiments of the
solubilized MV and purified histone H3 with radial diffusion
assay, both of them depressed proliferation of Escherichia coli,
DH-5␣ (data not shown).
2600
Endocrinology, June 2005, 146(6):2593–2601
To study the sorting mechanism of the histone H3 protein
into the secreted MV, we visualized the distribution of heparan sulfate in the MV-generating cells, because the heparan
sulfate proteoglycans were reported to distribute dominantly on the plasma membrane and membrane vesicles,
argosomes, which secrete from morphogenic tissues (45, 47).
In fact, antibodies against heparan sulfate bound to the 17kDa protein in the secreted MV (Fig. 6A) and stained the
precursor MV in the cultured sebocytes (Fig. 6B). Because
isolated histone H3 protein from the MV bind with high
affinity to heparan sulfate (Fig. 3), the protein located in the
cytoplasm might be recruited to the secreted MV by heparan
sulfate. In our observation, the secreted MV contained 14-3-3
protein, which was reported to be an exosomal protein in the
dendritic cells (40). Because the 14-3-3 protein is associated
with histone (48) and lipid droplets in Chinese hamster ovary
K2 cells (49), the protein may contribute to the sorting of
histone H3 protein to the secreted MV that contained multiple lipid particles, although a precise mechanism still needs
to be clarified.
We examined whether the MV secretion from the sebocytes are related to the apoptosis of the cells and concluded
that the cause of the secretion probably was not related to the
apoptosis for the following reasons: 1) most of the MVgenerating cells started proliferation after they were reseeded and cultured at lower cell density in the presence of
EGF (data not shown) and 2) the secreted MV was not stained
with PI (Fig. 4) or with annexin V-EGFP (data not shown).
The secretion of the MV from the primary cultured sebocytes
also might not be related to apoptosis because the cells secreted continuously the membrane vesicles for several weeks
(Fig. 1B) and survived in the presence of supplemented EGF
(data not shown).
Based on these results, we believe that the newly discovered secreted MV from the cultured sebocytes have a protective function on the skin surface by supplying a squalene
coating and an antimicrobial protein. A detailed analysis of
the molecular mechanisms of the MV secretion is necessary
to activate their secretion efficiently and persistently from the
sebocytes to maintain the skin’s homeostasis.
Acknowledgments
We are grateful to Dr. Takeshi Takaku and Mr. Masachika Syudo in
the Division of Medical Bioscience, Integrated Center for Sciences, at
Ehime University for their excellent technical support. We also are very
indebted to Dr. Minoru Hamada in Translational Research Center at
Kurume University for his critical suggestions. We thank Mr. Kaipo
Ikemoto for revising the manuscript. We are thankful to Applied Biosystems Japan Ltd. (Tokyo, Japan) for peptide sequencing and their
analysis.
Received November 15, 2004. Accepted February 22, 2005.
Address all correspondence and requests for reprints to: Michihiro
Sumida Ph.D.,1Department of Molecular and Cellular Biology, Division
of Biochemistry and, Molecular Genetics, Ehime University School of
Medicine, Shitsukawa, Toon-city, Ehime 791-0295, Japan, E-mail:
[email protected]
This work was supported by the Softwater Research Center of Miura
Co., Ltd., Ehime, Japan.
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