Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3 Suppress Lipogenesis in Hamster Sebaceous Gland Cells In Vitro
2001; Elsevier BV; Volume: 117; Issue: 4 Linguagem: Inglês
10.1046/j.0022-202x.2001.01516.x
ISSN1523-1747
AutoresTakashi Sato, Noriyasu Imai, Noriko Akimoto, Akira Itô, T. Sakiguchi, Kenji Kitamura,
Tópico(s)melanin and skin pigmentation
ResumoWe have previously reported the establishment of a culture system of hamster auricular sebocytes. Although their morphologic and biochemical properties are very similar to those of human sebocytes, the regulation of lipogenesis is not clear. Therefore, we investigated the effect of epidermal growth factor, all-trans retinoic acid, 1α,25-dihydroxyvitamin D3, and androgens such as testosterone and 5α-dihydrotestosterone on lipogenesis in cultured hamster sebocytes. Intracellular lipid droplets detected with Oil-Red-O staining were observed in 5 d cultures and increased in a time-dependent manner; 40.7% ± 1.11% of 2 wk cultured cells tested lipid-positive by flow cytometric analysis. When the hamster sebocytes were cultured in the presence of epidermal growth factor, all-trans retinoic acid, or 1α,25-dihydroxyvitamin D3, the intracellular lipid droplets were diminished by all-trans retinoic acid and epidermal growth factor, and slightly by 1α,25-dihydroxyvitamin D3. The intracellular lipid droplets consisted mainly of triglycerides (71.8%) and, as minor components, cholesterol (18.0%), wax esters (3.6%), and free fatty acids (6.6%). Epidermal growth factor and all-trans retinoic acid decreased the intracellular accumulation of triglycerides (92.6% and 96.1% inhibition, respectively) and free fatty acids (54.3% and 62.6% inhibition, respectively) in the sebocytes. In addition, 1α,25-dihydroxyvitamin D3 decreased the triglyceride level (34.3% inhibition), but augmented the accumulation of wax esters (30% increase). There was no difference in the level of cholesterol as a result of these treatments, however. In contrast, 5α-dihydrotestosterone augmented the formation of intracellular lipid droplets along with an increase in the accumulation of triglycerides in hamster sebocytes. Our findings that regulation of lipogenesis by all-trans retinoic acid and androgen in hamster sebocytes is identical to regulation in humans suggest that hamster sebocytes are useful for the elucidation of sebaceous function at the cellular level. Furthermore, this is the first evidence that epidermal growth factor and 1α,25-dihydroxyvitamin D3 may act as suppressors in the regulation of lipogenesis in hamster sebocytes in vitro. We have previously reported the establishment of a culture system of hamster auricular sebocytes. Although their morphologic and biochemical properties are very similar to those of human sebocytes, the regulation of lipogenesis is not clear. Therefore, we investigated the effect of epidermal growth factor, all-trans retinoic acid, 1α,25-dihydroxyvitamin D3, and androgens such as testosterone and 5α-dihydrotestosterone on lipogenesis in cultured hamster sebocytes. Intracellular lipid droplets detected with Oil-Red-O staining were observed in 5 d cultures and increased in a time-dependent manner; 40.7% ± 1.11% of 2 wk cultured cells tested lipid-positive by flow cytometric analysis. When the hamster sebocytes were cultured in the presence of epidermal growth factor, all-trans retinoic acid, or 1α,25-dihydroxyvitamin D3, the intracellular lipid droplets were diminished by all-trans retinoic acid and epidermal growth factor, and slightly by 1α,25-dihydroxyvitamin D3. The intracellular lipid droplets consisted mainly of triglycerides (71.8%) and, as minor components, cholesterol (18.0%), wax esters (3.6%), and free fatty acids (6.6%). Epidermal growth factor and all-trans retinoic acid decreased the intracellular accumulation of triglycerides (92.6% and 96.1% inhibition, respectively) and free fatty acids (54.3% and 62.6% inhibition, respectively) in the sebocytes. In addition, 1α,25-dihydroxyvitamin D3 decreased the triglyceride level (34.3% inhibition), but augmented the accumulation of wax esters (30% increase). There was no difference in the level of cholesterol as a result of these treatments, however. In contrast, 5α-dihydrotestosterone augmented the formation of intracellular lipid droplets along with an increase in the accumulation of triglycerides in hamster sebocytes. Our findings that regulation of lipogenesis by all-trans retinoic acid and androgen in hamster sebocytes is identical to regulation in humans suggest that hamster sebocytes are useful for the elucidation of sebaceous function at the cellular level. Furthermore, this is the first evidence that epidermal growth factor and 1α,25-dihydroxyvitamin D3 may act as suppressors in the regulation of lipogenesis in hamster sebocytes in vitro. 1α, 25-dihydroxyvitamin D3 all-trans retinoic acid Ca2+ and Mg2+ free phosphate-buffered saline forward scatter side scatter triglyceride Sebaceous glands are important skin appendages and sebum excretion is considered to be associated with the functional maintenance of the cutaneous surface as a biologic barrier (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (291) Google Scholar). The development of sebaceous glands is dependent on androgens and sebocytic differentiation sequentially occurs with accumulating abundant cytoplasmic lipids (Sawaya et al., 1988Sawaya M.E. Honig L.S. Hsia S.L. Increased androgen binding capacity in sebaceous glands in scalp of male-pattern baldness.J Invest Dermatol. 1988; 92: 91-95Crossref Scopus (48) Google Scholar;Akamatsu et al., 1992Akamatsu H. Zouboulis ChC Orfanos C.E. Control of human sebocyte proliferation in vitro by testosterone and 5-alpha-dihydrotestosterone is dependent on the localization of the sebaceous glands.J Invest Dermatol. 1992; 99: 509-511Abstract Full Text PDF PubMed Google Scholar;Zouboulis et al., 1994aZouboulis ChC Akamatsu H. Stephanek K. Orfanos C.E. Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.Skin Pharmacol. 1994; 7: 33-40Crossref PubMed Scopus (37) Google Scholar;Rosenfield et al., 1998Rosenfield R.L. Eplewski D. Kentsis A. Ciletti N. Mechanisms of androgen induction of sebocyte differentiation.Dermatology. 1998; 196: 43-46Crossref PubMed Scopus (120) Google Scholar). An excess increase in the endogenous level of androgens causes sebaceous-gland disorders such as acne and seborrhoea, which are the most common skin diseases (Harris et al., 1983Harris H.H. Downing D.T. Stewart M.E. Strauss J.S. Sustainable rates of sebum secretion in acne patients and matched normal control subjects.J Am Acad Dermatol. 1983; 8: 200-203Abstract Full Text PDF PubMed Scopus (88) Google Scholar;Piérard et al., 1987Piérard G.E. Pierard-Franchimont C. Le T. Seborrhoea in acne-prone and acne-free patients.Dermatologica. 1987; 175: 5-9PubMed Google Scholar;Zouboulis et al., 1998Zouboulis ChC Xia L. Akamatsu H. et al.The human sebocyte culture model provides new insights into development and management of seborrhoea and acne.Dermatology. 1998; 196: 21-31Crossref PubMed Scopus (183) Google Scholar). Proliferation and lipogenesis in sebocytes of human and rodent sebaceous glands are known to be augmented by testosterone and 5α-dihydrotestosterone (5α-DHT) in vivo and in vitro (Sawaya et al., 1988Sawaya M.E. Honig L.S. Hsia S.L. Increased androgen binding capacity in sebaceous glands in scalp of male-pattern baldness.J Invest Dermatol. 1988; 92: 91-95Crossref Scopus (48) Google Scholar;Akamatsu et al., 1992Akamatsu H. Zouboulis ChC Orfanos C.E. Control of human sebocyte proliferation in vitro by testosterone and 5-alpha-dihydrotestosterone is dependent on the localization of the sebaceous glands.J Invest Dermatol. 1992; 99: 509-511Abstract Full Text PDF PubMed Google Scholar;Zouboulis et al., 1994aZouboulis ChC Akamatsu H. Stephanek K. Orfanos C.E. Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.Skin Pharmacol. 1994; 7: 33-40Crossref PubMed Scopus (37) Google Scholar;Rosenfield et al., 1998Rosenfield R.L. Eplewski D. Kentsis A. Ciletti N. Mechanisms of androgen induction of sebocyte differentiation.Dermatology. 1998; 196: 43-46Crossref PubMed Scopus (120) Google Scholar). In addition to androgen, other hormones and growth factors have been shown to participate in the development of sebaceous glands. Epidermal growth factor (EGF) stimulates sebaceous glands in the pinna of hamster to proliferate in vivo (Matias and Orentreich, 1983Matias J.R. Orentreich N. Stimulation of hamster sebaceous glands by epidermal growth factor.J Invest Dermatol. 1983; 80: 516-519Crossref PubMed Scopus (17) Google Scholar). In contrast, estrogens reduce lipogenesis in human and rat sebaceous glands (Strauss et al., 1962Strauss J.S. Kligman A.M. Pochi P.E. Effects of androgens and estrogens on human sebaceous glands.J Invest Dermatol. 1962; 39: 139-155Abstract Full Text PDF PubMed Scopus (129) Google Scholar;Ebling and Skinner, 1983Ebling F.J. Skinner J. The local effects of topically applied estradiol, cyproterone acetate, and ethanol on sebaceous secretion in intact male rats.J Invest Dermatol. 1983; 81: 448-451Crossref PubMed Scopus (16) Google Scholar). All-trans and 13-cis retinoic acids also prevent lipid formation in human and rodent sebaceous gland cells in vivo and in vitro (Strauss et al., 1980Strauss J.S. Stranieri A.M. Farrell L.N. Downing D.T. The effect of marked inhibition of sebum production with 13-cis-retinoic acid on skin surface lipid composition.J Invest Dermatol. 1980; 74: 66-67Crossref PubMed Scopus (90) Google Scholar;Jones et al., 1983Jones D.H. King K. Miller A.J. Cunliffe W.J. A dose–response study of 13-cis-retinoic acid in acne vulgaris.Br J Dermatol. 1983; 108: 333-345Crossref PubMed Scopus (98) Google Scholar;Zouboulis et al., 1991Zouboulis ChC Korge B. Akamatsu H. Xia L. Schiller S. Gollnick H. Orfanos C.E. Effects of 13-cis-retinoic acid, all-trans-retinoic acid and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro.J Invest Dermatol. 1991; 96: 792-797Abstract Full Text PDF PubMed Google Scholar;Hommel et al., 1996Hommel L. Geiger J.M. Harms M. Saurat J.H. Sebum excretion rate in subjects treated with oral all-trans-retinoic acid.Dermatology. 1996; 193: 127-130Crossref PubMed Scopus (27) Google Scholar;Orfanos and Zouboulis, 1998Orfanos C.E. Zouboulis ChC Oral retinoids in the treatment of seborrhoea and acne.Dermatology. 1998; 196: 140-147Crossref PubMed Scopus (113) Google Scholar). Recently,Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar have established a preparation of hamster sebocytes from the auricle and demonstrated that the proliferation of hamster sebocytes in response to androgens is similar to that of human sebocytes. In addition,Plewig and Luderschmidt, 1977Plewig G. Luderschmidt C. Hamster ear model for sebaceous glands.J Invest Dermatol. 1977; 68: 171-176Crossref PubMed Scopus (72) Google Scholar reported that the hamster sebaceous gland is similar to the human gland with regard to its size, response to androgens, and turnover time. Therefore, this culture system is considered to be a useful tool for studying functions of sebaceous glands in vitro. The regulation of lipogenesis in hamster sebocytes remains unclear, however. In this study, we investigated the effect of growth factor and hormones on lipid formation and demonstrated that testosterone and 5α-DHT augmented lipogenesis, but EGF, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], and all-trans retinoic acid (atRA) suppressed it in hamster sebocytes in vitro. Hamster sebocytes have been established from sebaceous glands of auricles of 5-wk-old male golden hamsters (Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar) and cultured in culture dishes of 60 mm diameter (1 × 103 cells per cm2) (Becton Dickinson, Tokyo, Japan) in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium (1:1) (Life Technologies, Grand Island, MD) supplemented with 6% heat-denatured fetal bovine serum (FBS) (BioWhittaker, Walkersville, MD), 2% human serum (ICN Biochemicals, Costa Mesa, CA), and 0.68 mM L-glutamine (Life Technologies) in the presence or absence of recombinant human EGF (Progen Biotechnik, Heidelberg, Germany), 1,25(OH)2D3, atRA, testosterone or 5α-DHT (Sigma, St. Louis, MO) for up to 14 d. Cultured hamster sebocytes were washed once with Ca2+- and Mg2+-free phosphate-buffered saline [PBS(–)] and fixed with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan) diluted with PBS(–) for 1 h at room temperature. The cells were stained first with Mayer's hematoxylin solution (Wako) at room temperature for 5 min and then with 0.3% Oil-Red-O (Sigma) at 37°C for 15 min. In addition, the number of cells in six randomly chosen areas per dish was counted under a light microscope at 20× magnification. Hamster sebocytes were maintained for 2 wk in DMEM/Ham's F12 medium (1:1) supplemented with 6% heat-denatured FBS, 2% human serum, and 0.68 mM L-glutamine, and then were stained with Nile red (Sigma) to detect lipid-positive cells (final concentration of 100 ng per ml) in PBS(–) for 20 min at room temperature in the dark. The dye solution was removed and then the cells were washed twice with PBS(–). The cells treated with Nile red were harvested with 0.25% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) in PBS(–) and then resuspended in the culture medium. Cell size and cellular fluorescence were measured for 10,000 cells using a FACScan flow cytometer (Becton Dickinson). Excitation laser light was 488 nm and the fluorescent intensity at 530 ± 15 nm was detected. The cell distribution was expressed as forward and side scatters (FSC and SSC, respectively). The cells were washed twice with PBS(–) and then harvested with 0.25% trypsin and 0.02% EDTA in PBS(–). The cells were sonicated and then mixed with chloroform:methanol (2:1) for 5 min at room temperature. The mixture was centrifuged at 2000 rpm for 5 min at room temperature following addition of 0.88% KCl, and then the methanol (the upper phase) was removed. Lipids in chloroform (the lower phase) were collected and then subjected to an automatic thin-layer chromatography Iatroscan (Iatron Laboratories, Tokyo, Japan) (Shantha, 1992Shantha N.C. Thin-layer chromatography-flame ionization detection Iatroscan system.J Chromatogr. 1992; 624: 21Crossref PubMed Scopus (63) Google Scholar;Pisch et al., 1997Pisch S. Bornscheuer U.T. Meyer H.H. Schmid R.D. Properties of unusual phospholipids IV. Chemienzymatic synthesis of phospholipids bearing acetylenic fatty acids.Tetrahedron. 1997; 53: 14627-14634Crossref Scopus (13) Google Scholar). Tripalmitin (triglyceride; TG), palmitic acid (free fatty acid), cholesterol, cholesterol palmitate (cholesterol ester) (Doosan Serdary Research Laboratories, Englewood Cliffs, NJ), and palmityl palmitate (wax ester) (Nu-Chek-Prep, Elysian, MN) were used as lipid standards in the chromatography. The amounts of lipid components were calculated by an internal control concomitantly performed using authentic cholesterol acetate (2 µg) (Doosan). The cell lysate (100 µl) prepared above was coated in triplicate on wells in 24-well multiplates and then incubated with 3,5-diaminobenzoic acid dihydrochloride (Sigma) (400 mg per ml) at 60°C for 45 min according to the method ofJohnson-Wint and Hollis, 1982Johnson-Wint B. Hollis S. A rapid in situ deoxyribonucleic acid assay for determining cell number in culture and tissue.Anal Biochem. 1982; 122: 338-344Crossref PubMed Scopus (100) Google Scholar. After the reaction, the fluorescent intensity was measured by excitation at 365 nm and emission at 530 nm following the addition of 1 M hydrochloride. The content of intracellular DNA was calculated using a standard curve concomitantly produced using authentic salmon sperm DNA (6.125–200 µg per ml). Data were analyzed by Student's t test; p <0.05 was considered to be statistically significant. When hamster sebocytes were cultured for 14 d, lipid droplets in the cytoplasm were obviously stained with Oil-Red-O Figure 1a. The cellular morphology was distorted and nuclei were difficult to observe because the cytoplasm was filled with lipid droplets. In addition, accumulation of intracellular lipid droplets was observed as early as at 5 d in culture and increased in a time-dependent manner Figure 1b. During cultivation, small lipid droplets were determined in the cytoplasm within 1 wk by Oil-Red-O staining and grew to large droplets fusing together (data not shown). Furthermore, a similar histochemical observation was obtained by utilizing the other dye reagent, Nile red (data not shown). On the other hand, we performed flow cytometric analysis to determine the percentage of lipid-accumulated cells. We first identified an area with lower FSC and SSC as Gate 1, in which there were more than 95% of lipid-negative sebocytes induced by EGF (see Figure 3) (Figure 2a, insert). Differentiated sebocytes with intracellular lipids seemed to be granular so that the cellular population was shifted to an area of larger SSC, termed Gate 2. When sebocytes were labeled with Nile red reagent (Greenspan et al., 1985Greenspan P. Mayer E.P. Fowler S.D. Nile red: a selective fluorescent stain for intracellular lipid droplets.J Cell Biol. 1985; 100: 965-973Crossref PubMed Scopus (1690) Google Scholar) and then subjected to flow cytometric analysis, most sebocytes in the 2-wk culture were distributed in both Gates 1 and 2 Figure 2a. The fluorescence intensity of cells in Gate 2 was about 40 times higher than that in Gate 1 Figure 2b. We also obtained similar results in the flow cytometric analysis when the cells were stained with Oil-Red-O (data not shown), indicating that the cells in Gate 2 were lipid-positive. Furthermore, we calculated that 40.7% ± 1.11% of the cultured sebocytes accumulated intracellular lipid droplets Figure 2. Thus, these results suggest that hamster sebocytes are able to spontaneously accumulate intracellular lipids under these culture conditions in vitro.Figure 2Characterization of lipid-positive hamster sebocytes. Hamster sebocytes at the second passage were cultured for 2 wk, and then stained with Nile red as described in Materials and Methods. The Nile-red-stained cells were subjected to flow cytometric analysis in SSC and FSC (A) and then the fluorescence intensity was determined for characterization of lipid-positive and lipid-negative cells (B). Inserted panel, EGF-treated sebocytes. Gates 1 and 2 correspond to the lipid-negative and lipid-positive sebocytes, respectively.View Large Image Figure ViewerDownload (PPT)Figure 3Intracellular lipid formation is inhibited by EGF, atRA, and 1,25(OH)2D3 in hamster sebocytes. Hamster sebocytes at the second passage were untreated (A) or treated with EGF (100 ng per ml) (B), atRA (1 µM) (C) and 1,25(OH)2D3 (100 nM) (D) every 3 d for 2 wk. After fixing, the cells were stained with Oil-Red-O and Mayer's hematoxylin as described in Materials and Methods.View Large Image Figure ViewerDownload (PPT) To clarify the regulation of lipogenesis in hamster sebocytes, we investigated the effect of EGF, 1,25(OH)2D3, and atRA in the accumulation of intracellular lipids. The level of intracellular lipids was much less in the EGF-treated sebocytes compared to control culture Figure 3a, b. In addition, lipid formation no longer occurred in the atRA-treated cells Figure 3c. Furthermore, 1,25(OH)2D3 decreased lipogenesis, and even in the Oil-Red-O-positive cells the amount of intracellular lipids became smaller than in the untreated cells Figure 3d. We next quantitatively analyzed the composition of intracellular lipids in hamster sebocytes by thin-layer chromatography using a flame ionization detector. As shown in Figure 4b, the intracellular lipids in hamster sebocytes were found to consist of TG, wax esters, free fatty acids, and cholesterol. The lipids consisted of TG (71.8%) as the major lipid component, and free fatty acids (6.6%), cholesterol (18.0%), and wax esters (3.6%) were detected as minor components (Figure 5, control). In addition, we confirmed the time-dependent increase in total lipid biosynthesis resulting mostly from the augmentation of the TG level in cultured hamster sebocytes (data not shown).Figure 5Alteration of lipid composition in EGF, atRA, and 1,25(OH)2D3-treated hamster sebocytes. Hamster sebocytes at the second passage were untreated or treated with EGF (100 ng per ml), 1,25(OH)2D3 (100 nM), and atRA (1 µM) as described in Figure 3. The composition of intracellular lipids extracted from the cells was analyzed by thin-layer chromatography as described in Materials and Methods. Data represent the mean ± SD of triplicate dishes. *, **, ***, Significantly different from each composition in control (p <0.05, 0.01, and 0.001, respectively). Total, sum of each composition; TGs, triglycerides; WAXs, wax esters; FFAs, free fatty acids; CHO, cholesterol.View Large Image Figure ViewerDownload (PPT) When the sebocytes were treated with EGF, atRA, and 1,25(OH)2D3, the intracellular accumulation of TGs was suppressed by EGF and atRA (92.6% and 96.1% inhibition, respectively). In addition, the level of free fatty acids was similarly decreased by EGF and atRA (54.3% and 62.6% inhibition, respectively). Furthermore, 1,25(OH)2D3 slightly decreased the TG level (34.3% inhibition) but slightly augmented the accumulation of wax esters (30% increase). In contrast, there was no difference in the level of cholesterol under these treatments Figure 5. Therefore, these results suggest that the suppression of lipogenesis by EGF, atRA, and 1,25(OH)2D3 is due to a decrease in the accumulation of TG in hamster sebocytes. 5α-DHT and testosterone have been shown to augment the biosynthesis of lipids in human and rat sebaceous glands in vivo and in vitro (Sawaya et al., 1988Sawaya M.E. Honig L.S. Hsia S.L. Increased androgen binding capacity in sebaceous glands in scalp of male-pattern baldness.J Invest Dermatol. 1988; 92: 91-95Crossref Scopus (48) Google Scholar;Akamatsu et al., 1992Akamatsu H. Zouboulis ChC Orfanos C.E. Control of human sebocyte proliferation in vitro by testosterone and 5-alpha-dihydrotestosterone is dependent on the localization of the sebaceous glands.J Invest Dermatol. 1992; 99: 509-511Abstract Full Text PDF PubMed Google Scholar;Zouboulis et al., 1994aZouboulis ChC Akamatsu H. Stephanek K. Orfanos C.E. Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.Skin Pharmacol. 1994; 7: 33-40Crossref PubMed Scopus (37) Google Scholar;Rosenfield et al., 1998Rosenfield R.L. Eplewski D. Kentsis A. Ciletti N. Mechanisms of androgen induction of sebocyte differentiation.Dermatology. 1998; 196: 43-46Crossref PubMed Scopus (120) Google Scholar).Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar also reported that the proliferation of hamster sebocytes is androgen-dependent. We demonstrated that the accumulation of intracellular lipids in cytoplasm was augmented in 5α-DHT-treated hamster sebocytes Figure 6, and that the lipid-positive cells in 5α-DHT treatment amounted to 52.1% ± 1.58% and increased 1.3-fold in comparison to untreated cells (p <0.001) (data not shown). Furthermore, as shown in Figure 7, 5α-DHT increased the amount of TG whereas the levels of wax esters, free fatty acids, and cholesterol were not influenced, indicating that the increased lipid formation was mostly due to the increase in TG level. Therefore, these results suggest that, as in human and rat sebaceous glands, androgens act as inducers for lipogenesis in hamster sebocytes in vitro.Figure 75α-DHT increases intracellular TG level in hamster sebocytes. Hamster sebocytes at the second passage were untreated or treated with 5α-DHT (10 nM) and then the extracted lipids were analyzed as described in Figure 5. Data represent the mean ± SD of triplicate dishes. *Significantly different from control (p <0.05). Total, sum of each composition; TGs, triglycerides; WAXs, wax esters; FFAs, free fatty acids; CHO, cholesterol.View Large Image Figure ViewerDownload (PPT) Sebocytes are epithelial cells. Accumulation of lipid droplets in their cytoplasm reflects terminal differentiation (Zouboulis et al., 1994bZouboulis ChC Krieter A. Gollnick H. Mischke D. Orfanos C.E. Progressive differentiation of human sebocytes in vitro is characterized by increased cell size and altered antigenic expression and is regulated by culture duration and retinoids.Exp Dermatol. 1994; 3: 151-160Crossref PubMed Scopus (53) Google Scholar). So far, many investigators have reported the function and regulation of sebum under physiologic and pathologic conditions (Harris et al., 1983Harris H.H. Downing D.T. Stewart M.E. Strauss J.S. Sustainable rates of sebum secretion in acne patients and matched normal control subjects.J Am Acad Dermatol. 1983; 8: 200-203Abstract Full Text PDF PubMed Scopus (88) Google Scholar;Piérard et al., 1987Piérard G.E. Pierard-Franchimont C. Le T. Seborrhoea in acne-prone and acne-free patients.Dermatologica. 1987; 175: 5-9PubMed Google Scholar;Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (291) Google Scholar;Zouboulis et al., 1998Zouboulis ChC Xia L. Akamatsu H. et al.The human sebocyte culture model provides new insights into development and management of seborrhoea and acne.Dermatology. 1998; 196: 21-31Crossref PubMed Scopus (183) Google Scholar). The regulatory mechanism of lipogenesis in sebaceous glands is not fully understood, however. Therefore, it is of interest to clarify physiologic candidate(s), which negatively regulate(s) lipid formation in sebaceous glands. In this study, we demonstrated that EGF, 1,25(OH)2D3, and atRA inhibited lipogenesis in cultured hamster sebocytes. It has been reported that organ culture systems of sebaceous glands are established in human and rodents (Strauss et al., 1962Strauss J.S. Kligman A.M. Pochi P.E. Effects of androgens and estrogens on human sebaceous glands.J Invest Dermatol. 1962; 39: 139-155Abstract Full Text PDF PubMed Scopus (129) Google Scholar;Strauss et al., 1980Strauss J.S. Stranieri A.M. Farrell L.N. Downing D.T. The effect of marked inhibition of sebum production with 13-cis-retinoic acid on skin surface lipid composition.J Invest Dermatol. 1980; 74: 66-67Crossref PubMed Scopus (90) Google Scholar;Ebling and Skinner, 1983Ebling F.J. Skinner J. The local effects of topically applied estradiol, cyproterone acetate, and ethanol on sebaceous secretion in intact male rats.J Invest Dermatol. 1983; 81: 448-451Crossref PubMed Scopus (16) Google Scholar;Jones et al., 1983Jones D.H. King K. Miller A.J. Cunliffe W.J. A dose–response study of 13-cis-retinoic acid in acne vulgaris.Br J Dermatol. 1983; 108: 333-345Crossref PubMed Scopus (98) Google Scholar;Sawaya et al., 1988Sawaya M.E. Honig L.S. Hsia S.L. Increased androgen binding capacity in sebaceous glands in scalp of male-pattern baldness.J Invest Dermatol. 1988; 92: 91-95Crossref Scopus (48) Google Scholar). Recently, in vitro culture systems of human and rat sebocytes have also been reported, and the accumulation of intracellular lipids is augmented by androgens such as testosterone and 5α-DHT and inhibited by retinoic acids (Jones et al., 1983Jones D.H. King K. Miller A.J. Cunliffe W.J. A dose–response study of 13-cis-retinoic acid in acne vulgaris.Br J Dermatol. 1983; 108: 333-345Crossref PubMed Scopus (98) Google Scholar;Zouboulis et al., 1994aZouboulis ChC Akamatsu H. Stephanek K. Orfanos C.E. Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.Skin Pharmacol. 1994; 7: 33-40Crossref PubMed Scopus (37) Google Scholar;Rosenfield et al., 1998Rosenfield R.L. Eplewski D. Kentsis A. Ciletti N. Mechanisms of androgen induction of sebocyte differentiation.Dermatology. 1998; 196: 43-46Crossref PubMed Scopus (120) Google Scholar). The hamster sebaceous gland is similar to the human gland with regard to its size, response to androgens, and turnover time in vivo (Plewig and Luderschmidt, 1977Plewig G. Luderschmidt C. Hamster ear model for sebaceous glands.J Invest Dermatol. 1977; 68: 171-176Crossref PubMed Scopus (72) Google Scholar).Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar have established a culture method of hamster sebocytes, and, like human sebocytes, the proliferation of hamster sebocytes is androgen-dependent. Furthermore, the accumulated intracellular lipids consist of TG, wax esters, cholesterol, and free fatty acids; these are identical to accumulated intracellular lipids in human sebocytes except for squalene and cholesterol esters (Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar). We demonstrated that the accumulation of intracellular lipids in hamster sebocytes was augmented by both testosterone (data not shown) and 5α-DHT and suppressed by atRA. Therefore, these results suggest that the cell proliferation and lipogenesis of hamster sebocytes in response to androgens and retinoic acids are similar to those of human sebocytes. We characterized lipid-positive hamster sebocytes as amounting to 40.7% ± 1.11% of the cultured cells, and 5α-DHT treatment further augmented this percentage (30% increase) (data not shown).Rosenfield et al., 1999Rosenfield R.L. Kentsis A. Deplewski D. Ciletti N. Rat preputial sebocyte differentiation involves peroxisome proliferator-activated receptors.J Invest Dermatol. 1999; 112: 226-232Crossref PubMed Scopus (119) Google Scholar also reported that in rat sebocytes only 5% of the cells were lipid-positive, but 5α-DHT treatment increased the percentage to approximately 15% in culture. Although the culture conditions and analytical procedures for evaluating lipid-positive cells are different for hamster and rat sebocytes, the differential potency in sebocytes is likely to be higher in hamster than in rat. Various endogenous factors such as growth factors and hormones are known to participate in maintaining skin homeostasis in vivo. EGF plays an important role in maintaining cutaneous events such as the proliferation and keratinization of the epidermis (Frati et al., 1977Frati L. D'Armiento M. Gulletta E. Verna R. Covelli I. The control of epidermis proliferation by epidermal growth factor (EGF). Relationship with cyclic nucleotides systems.Pharmacol Res Commun. 1977; 9: 815-822Crossref PubMed Scopus (12) Google Scholar;Green et al., 1983Green M.R. Basketter D.A. Couchman J.R. Rees D.A. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth.Dev Biol. 1983; 100: 506-512Crossref PubMed Scopus (91) Google Scholar;Bhora et al., 1995Bhora F.Y. Dunkin B.J. Batzri S. Aly H.M. Bass B.L. Sidawy A.N. Harmon J.W. Effect of growth factors on cell proliferation and epithelialization in human skin.J Surg Res. 1995; 59: 236-244Abstract Full Text PDF PubMed Scopus (136) Google Scholar) and the remodeling of extracellular matrix (McDonnell et al., 1990McDonnell S.E. Kerr L.D. Matrisian L.M. Epidermal growth factor stimulation of stromelysin mRNA in rat fibroblasts requires induction of proto-oncogenes c-fos and c-jun and activation of protein kinase C.Mol Cell Biol. 1990; 10: 4284-4293Crossref PubMed Scopus (163) Google Scholar;McCawley et al., 1998McCawley L.J. O'Brien P. Hudson L.G. Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)-mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP) -9.J Cell Physiol. 1998; 176: 255-265Crossref PubMed Scopus (179) Google Scholar). It has been reported that EGF receptors exist on sebaceous glands in rat (Green et al., 1983Green M.R. Basketter D.A. Couchman J.R. Rees D.A. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth.Dev Biol. 1983; 100: 506-512Crossref PubMed Scopus (91) Google Scholar). In addition,Matias and Orentreich, 1983Matias J.R. Orentreich N. Stimulation of hamster sebaceous glands by epidermal growth factor.J Invest Dermatol. 1983; 80: 516-519Crossref PubMed Scopus (17) Google Scholar reported that administration of EGF into the pinna of hamster increases the number of cells per sebaceous gland unit in vivo. We demonstrated that the cellular DNA content in EGF-treated hamster sebocytes was 2.6-fold higher than in untreated cells (data not shown). In contrast,Zouboulis et al., 1998Zouboulis ChC Xia L. Akamatsu H. et al.The human sebocyte culture model provides new insights into development and management of seborrhoea and acne.Dermatology. 1998; 196: 21-31Crossref PubMed Scopus (183) Google Scholar reported that EGF does not affect the proliferation of human sebocytes in vitro. Therefore, it is likely that the involvement of EGF in sebaceous proliferation may be dependent on cell species (Nikkari, 1974Nikkari T. Comparative chemistry of sebum.J Invest Dermatol. 1974; 62: 257-267Crossref PubMed Scopus (104) Google Scholar) and at least different between hamster and human sebocytes. On the other hand, we demonstrated that the accumulation of intracellular lipids was inhibited in EGF-treated hamster sebocytes. Similar observations have been reported in organ cultures of the human sebaceous gland, in which lipogenesis is increased with EGF depletion from the culture medium (Guy and Kealey, 1998Guy R. Kealey T. The organ-maintained human sebaceous gland.Dermatology. 1998; 196: 16-20Crossref PubMed Scopus (14) Google Scholar). These results therefore suggest that EGF acts not only as a mitogenic factor but also as an antilipogenic factor in hamster sebocytes. 1,25(OH)2D3 has been shown to play an important role in epidermal differentiation. 1,25(OH)2D3 inhibits the proliferation but augments the differentiation of human keratinocytes (Gibbs et al., 1996Gibbs S. Backendorf C. Ponec M. Regulation of keratinocyte proliferation and differentiation by all-trans-retinoic acid, 9-cis-retinoic acid and 1,25-dihydroxyvitamin D3.Arch Dermatol Res. 1996; 288: 729-738Crossref PubMed Scopus (47) Google Scholar;Sorensen et al., 1997Sorensen S. Solvsten H. Politi Y. Kragballe K. Effects of vitamin D3 on keratinocyte proliferation and differentiation in vitro: modulation by ligands for retinoic acid and retinoid X receptors.Skin Pharmacol. 1997; 10: 144-152Crossref PubMed Scopus (19) Google Scholar). In addition, the administration of 1,25(OH)2D3 to psoriasis patients is efficacious for repairing the organization of epidermis in vivo (van de Kerkhof, 1998van de Kerkhof P.C. An update on vitamin D3 analogues in the treatment of psoriasis.Skin Pharmacol Appl Skin Physiol. 1998; 11: 2-10Crossref PubMed Scopus (36) Google Scholar;Peters et al., 2000Peters B.P. Weissman F.G. Gill M.A. Pathophysiology and treatment of psoriasis.Am J Health Syst Pharm. 2000; 57: 645-659PubMed Google Scholar). We demonstrated that 1,25(OH)2D3 suppressed the accumulation of intracellular lipid droplets in hamster sebocytes, but the extent of suppression was less than that of EGF or atRA treatment. In addition, like EGF and atRA, 1,25(OH)2D3 decreased the level of TG but slightly augmented that of wax esters, whereas the cellular DNA content was not altered in 1,25(OH)2D3-treated hamster sebocytes. Thus, this is the first evidence that 1,25(OH)2D3 may be involved in the suppressive regulation of lipogenesis in hamster sebocytes. Brind et al., 1986Brind J.L. Alani E. Wheatley V.R. Orentreich N. Analysis of ear sebum of the Syrian hamster (Mesocricetus auratus) reveals pronounced sexual dimorphism.Comp Biochem Physiol B. 1986; 84: 403-407PubMed Google Scholar showed that sterol esters are major components of ear sebum from Syrian hamster in vivo. Concerning the discrepancy between that report and our findings, we suggest a possibility that the lipid composition in sebaceous glands in vivo may result from the modulation of lipogenesis by various growth factors and hormones. In this study, the level of cholesterol in EGF- or atRA-treated sebocytes was higher than that of TG. In addition, the level of wax ester was almost equal to that of TG under these treatments. Therefore, we speculate that the substantial composition of hamster sebum may be similar in vitro and in vivo. Further experiments will be required to address the hypothesis. In conclusion, we demonstrated that, as in human and rat sebocytes, lipogenesis in hamster sebocytes is augmented by androgens and inhibited by atRA. Taken together with previous reports (Ito et al., 1998Ito A. Sakiguchi T. Kitamura K. Akamatsu H. Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells.Dermatology. 1998; 197: 238-244Crossref PubMed Scopus (27) Google Scholar), this suggests that the hamster sebocyte culture system will be useful for the elucidation of sebaceous function at the cellular level. Furthermore, this is the first evidence that EGF and 1,25(OH)2D3 may act as suppressors to control sebum excretion in hamster sebaceous glands.
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