Isolation and Identification of Histone H3 Protein Enriched in Microvesicles Secreted from Cultured Sebocytes
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内分泌学杂志 2005年第6期
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
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: sumida@m.ehime-u.ac.jp
Abstract
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 μm. 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 SDS-PAGE 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-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.
Introduction
THE 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 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 sterol 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 x 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 x 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, 27, 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 x 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.
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 μg/ml aprotinin, and 10 μg/ml leupeptin] and incubated with sulfuric acid (final concentration, 0.2 M) for 60 min on ice. After centrifugation at 12,000 x 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 x 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 μg/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 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 μg/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). Double-staining 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 14-3-3 (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 sequencer (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 μm were demonstrated in cultured hamster sebocytes after reaching confluency, and multiple Oil Red O-stainable 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).
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 μm (A and B) and 5 μm (C–E).
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 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).
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.
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 SDS-PAGE, 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/).
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.
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 nucleus 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).
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 μm. 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 μm.
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 H3-EGFP 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).
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 μm.
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.
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 μm.
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 14-3-3-EGFP 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.
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 μm.
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). 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.
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 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).
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 17-kDa 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 MV-generating 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.
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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: sumida@m.ehime-u.ac.jp
Abstract
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 μm. 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 SDS-PAGE 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-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.
Introduction
THE 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 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 sterol 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 x 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 x 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, 27, 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 x 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.
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 μg/ml aprotinin, and 10 μg/ml leupeptin] and incubated with sulfuric acid (final concentration, 0.2 M) for 60 min on ice. After centrifugation at 12,000 x 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 x 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 μg/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 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 μg/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). Double-staining 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 14-3-3 (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 sequencer (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 μm were demonstrated in cultured hamster sebocytes after reaching confluency, and multiple Oil Red O-stainable 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).
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 μm (A and B) and 5 μm (C–E).
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 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).
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.
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 SDS-PAGE, 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/).
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.
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 nucleus 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).
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 μm. 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 μm.
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 H3-EGFP 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).
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 μm.
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.
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 μm.
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 14-3-3-EGFP 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.
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 μm.
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). 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.
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 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).
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 17-kDa 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 MV-generating 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.
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