Augmentation of Lipogenesis by 15-Deoxy-Δ12,14-Prostaglandin J2 in Hamster Sebaceous Glands: Identification of Cytochrome P-450-mediated 15-Deoxy-Δ12,14-Prostaglandin J2 Production
2005; Elsevier BV; Volume: 125; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2005.23866.x
ISSN1523-1747
AutoresChikakazu Iwata, Noriko Akimoto, Takashi Sato, Yuki Morokuma, Akira Itô,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoProstaglandins (PGs) play important roles in the regulation of cutaneous cell functions under physiological and pathological conditions. In this study, we examined the involvement of PGs in sebocyte lipogenesis using non-steroidal anti-inflammatory drugs in vivo and in vitro. Hamster auricle sebocytes spontaneously differentiated to accumulate intracellular triacylglycerol (TG), under which the relative levels of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) to PGF2α and PGE2 increased. 15d-PGJ2 was found to augment the formation of lipid droplets, which was because of an increase of TG synthesis by diacylglycerol acyltransferase (DGAT). Furthermore, sebocytes constitutively produced cyclooxygenase 2 (COX-2), but not COX-1, in vivo and in vitro. When sebocytes were treated with COX inhibitors such as indomethacin, diclofenac, or NS-398, the production of PGF2α and PGE2 decreased. The production of 15d-PGJ2, however, was increased in these inhibitor-treated sebocytes. In addition, indomethacin, diclofenac, and NS-398 augmented the synthesis of TG along with the increase in DGAT activity. Similarly, topical administration of indomethacin to hamster auricles caused the development of sebaceous glands with the augmentation of sebum deposition in vivo. Furthermore, indomethacin and NS-398-augmented 15d-PGJ2 production and TG synthesis were suppressed by a non-selective cytochrome P-450 (CYP) inhibitor, SKF-525A. A ligand activator of peroxisome proliferation activating receptor γ (PPARγ), troglitazone-induced synthesis of TG, however, was not altered even in the presence of SKF-525A. These results suggest that 15d-PGJ2 is a crucial stimulator of sebocyte lipogenesis by augmenting DGAT-mediated synthesis of TG. In addition to the COX-2-dependent pathway of PG synthesis, our findings suggest a sebocyte-specific pathway of 15d-PGJ2 production by CYP, the activity of which may be evoked by inhibiting COX-2. Prostaglandins (PGs) play important roles in the regulation of cutaneous cell functions under physiological and pathological conditions. In this study, we examined the involvement of PGs in sebocyte lipogenesis using non-steroidal anti-inflammatory drugs in vivo and in vitro. Hamster auricle sebocytes spontaneously differentiated to accumulate intracellular triacylglycerol (TG), under which the relative levels of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) to PGF2α and PGE2 increased. 15d-PGJ2 was found to augment the formation of lipid droplets, which was because of an increase of TG synthesis by diacylglycerol acyltransferase (DGAT). Furthermore, sebocytes constitutively produced cyclooxygenase 2 (COX-2), but not COX-1, in vivo and in vitro. When sebocytes were treated with COX inhibitors such as indomethacin, diclofenac, or NS-398, the production of PGF2α and PGE2 decreased. The production of 15d-PGJ2, however, was increased in these inhibitor-treated sebocytes. In addition, indomethacin, diclofenac, and NS-398 augmented the synthesis of TG along with the increase in DGAT activity. Similarly, topical administration of indomethacin to hamster auricles caused the development of sebaceous glands with the augmentation of sebum deposition in vivo. Furthermore, indomethacin and NS-398-augmented 15d-PGJ2 production and TG synthesis were suppressed by a non-selective cytochrome P-450 (CYP) inhibitor, SKF-525A. A ligand activator of peroxisome proliferation activating receptor γ (PPARγ), troglitazone-induced synthesis of TG, however, was not altered even in the presence of SKF-525A. These results suggest that 15d-PGJ2 is a crucial stimulator of sebocyte lipogenesis by augmenting DGAT-mediated synthesis of TG. In addition to the COX-2-dependent pathway of PG synthesis, our findings suggest a sebocyte-specific pathway of 15d-PGJ2 production by CYP, the activity of which may be evoked by inhibiting COX-2. 15-deoxy-Δ12,14-PGJ2 cyclooxygenase cytochrome P450 diacylglycerol acyltransferase epidermal growth factor prostaglandin peroxisome proliferation-activating receptor triacylglycerol By secreting sebum, sebaceous glands participate in the formation of a thin lipid layer on the skin surface as a physiological barrier (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (306) Google Scholar; Fluhr et al., 2003Fluhr J.W. Mao-Qiang M. Brown B.E. et al.Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice.J Invest Dermatol. 2003; 120: 728-737Crossref PubMed Scopus (184) Google Scholar). Sebaceous gland cells (sebocytes) differentiate to accumulate abundant cytoplasmic lipids, and, using a holocrine mechanism, lead to the secretion of lipids as sebum (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 (50) Google Scholar; Akamatsu et al., 1992Akamatsu H. Zouboulis ChC Orfanos C.E. Control of human sebocyte proliferation in vitro by testosterone and 5α-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., 1994Zouboulis 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 (41) 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 (124) Google Scholar). An excess secretion of sebum causes sebaceous-gland disorders such as acne vulgaris and seborrhea, 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 (93) 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 (189) Google Scholar). Recently, in vitro cultures of sebocytes have been established from sebaceous glands of humans (Xia et al., 1989Xia L.Q. Zouboulis ChC Detmar M. Mayer-da-Silva A. Stadler R. Orfanos C.E. Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model.J Invest Dermatol. 1989; 3: 315-321Crossref Google Scholar; Fujie et al., 1996Fujie T. Shikiji T. Uchida N. Urano Y. Nagae H. Arase S. Culture of cells derived from the human sebaceous gland under serum-free conditions without a biological feeder layer or specific matrices.Arch Dermatol Res. 1996; 288: 703-708Crossref PubMed Scopus (43) Google Scholar; Zouboulis et al., 1999Zouboulis ChC Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (277) Google Scholar; Thiboutot et al., 2003Thiboutot D. Jabara S. McAllister J.M. Sivarajah A. Gilliland K. Cong Z. Clawson G. Human skin is a steroidogenic tissue: Steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).J Invest Dermatol. 2003; 120: 905-914Crossref PubMed Scopus (239) Google Scholar) and rats (Laurent et al., 1992Laurent S.J. Mednieks M.I. Rosenfield R.L. Growth of sebaceous cells in monolayer culture.In Vitro Cell Dev Biol 28A. 1992: 83-89Crossref Scopus (22) Google Scholar).Plewig and Luderschmidt, 1977Plewig G. Luderschmidt C. Hamster ear model for sebaceous glands.J Invest Dermatol. 1977; 68: 171-176Crossref PubMed Scopus (75) Google Scholar reported that hamster sebaceous glands are similar to human glands with regard to size, response to androgens, and turnover time.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 described a culture method for hamster sebocytes from the auricles, and demonstrated that the proliferation of hamster sebocytes in response to androgens is similar to that of human sebocytes. Therefore, like human sebocytes, the cultured hamster sebocytes are a useful tool for studying the functions of sebaceous glands in vitro. These in vitro cell culture models have stimulated the elucidation of cellular properties of sebocytes, and studies on the pathological and physiological regulation of sebum synthesis by hormones (Xia et al., 1989Xia L.Q. Zouboulis ChC Detmar M. Mayer-da-Silva A. Stadler R. Orfanos C.E. Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model.J Invest Dermatol. 1989; 3: 315-321Crossref 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 (124) Google Scholar; Zouboulis et al., 1999Zouboulis ChC Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (277) Google Scholar; Thiboutot et al., 2003Thiboutot D. Jabara S. McAllister J.M. Sivarajah A. Gilliland K. Cong Z. Clawson G. Human skin is a steroidogenic tissue: Steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).J Invest Dermatol. 2003; 120: 905-914Crossref PubMed Scopus (239) Google Scholar). Sebum secretion and synthesis have been reported to be augmented by androgens such as testosterone and 5α-dihydrotestosterone (Akamatsu et al., 1992Akamatsu H. Zouboulis ChC Orfanos C.E. Control of human sebocyte proliferation in vitro by testosterone and 5α-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., 1994Zouboulis 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 (41) 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 (124) Google Scholar; Sato et al., 2001aSato T. Imai N. Akimoto N. Sakiguchi T. Kitamura K. Ito A. Epidermal growth factor and 1α,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebaceous gland cells in vitro.J Invest Dermatol. 2001; 117: 965-970Crossref PubMed Scopus (43) Google Scholar), and by insulin (Deplewski and Rosenfield, 1999Deplewski D. Rosenfield R.L. Growth hormone and insulin-like growth factors have different effects on sebaceous cell growth and differentiation.Endocrinology. 1999; 140: 4089-4094Crossref PubMed Scopus (113) Google Scholar) in humans, rats, and hamsters. In contrast, estrogen (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) and all-trans and 13-cis retinoic acids (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 (29) 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 (120) Google Scholar; Zouboulis et al., 1999Zouboulis ChC Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (277) Google Scholar; Sato et al., 2001aSato T. Imai N. Akimoto N. Sakiguchi T. Kitamura K. Ito A. Epidermal growth factor and 1α,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebaceous gland cells in vitro.J Invest Dermatol. 2001; 117: 965-970Crossref PubMed Scopus (43) Google Scholar) have been reported to suppress the formation of intracellular lipid droplets in vivo and in vitro. We reported that epidermal growth factor (EGF), basic fibroblast growth factor, transforming growth factor-α, keratinocyte growth factor, and 1α,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebocytes (Sato et al., 2001aSato T. Imai N. Akimoto N. Sakiguchi T. Kitamura K. Ito A. Epidermal growth factor and 1α,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebaceous gland cells in vitro.J Invest Dermatol. 2001; 117: 965-970Crossref PubMed Scopus (43) Google Scholar; Akimoto et al., 2002Akimoto N. Sato T. Sakiguchi T. Kitamura K. Kohno Y. Ito A. Cell proliferation and lipid formation in hamster sebaceous gland cells.Dermatology. 2002; 204: 118-123Crossref PubMed Scopus (37) Google Scholar). Prostaglandins (PGs) play important roles in the regulation of cellular functions under physiological and pathological conditions. Cyclooxygenase (COX)/prostaglandin endoperoxide H synthase, two types of which, COX-1 and COX-2, have been characterized in mammalians, is a rate-limiting enzyme complex for the production of PGs (Hruza and Pentland, 1993Hruza L.L. Pentland A.P. Mechanisms of UV-induced inflammation.J Invest Dermatol. 1993; 100: 35S-41SCrossref PubMed Scopus (294) Google Scholar; Narumiya et al., 1999Narumiya S. Sugimoto Y. Ushikubi F. Prostanoid receptors: Structures, properties, and functions.Physiol Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar; Tiano et al., 2002Tiano H.F. Loftin C.D. Akunda J. et al.Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis.Cancer Res. 2002; 62: 3395-3401PubMed Google Scholar). It has been reported that PGE2 participates in epidermal repair such as wound healing by its regulation of proliferation and differentiation of keratinocytes (Pentland and Needleman, 1986Pentland A.P. Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis.J Clin Invest. 1986; 77: 246-251Crossref PubMed Scopus (169) Google Scholar; Unemori et al., 1994Unemori E.N. Ehsani N. Wang M. Lee S. McGuire J. Amento E.P. Interleukin-1 and transforming growth factor-α: Synergistic stimulation of metalloproteinases, PGE2, and proliferation in human fibroblasts.Exp Cell Res. 1994; 210: 166-171Crossref PubMed Scopus (73) Google Scholar; Sato et al., 1997Sato T. Kirimura Y. Mori Y. The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts.J Invest Dermatol. 1997; 109: 334-339Crossref PubMed Scopus (55) Google Scholar). Sebocytes are classified as epithelial cells, and morphologically and functionally differentiate to form intracellular lipid droplets (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 (124) Google Scholar; Deplewski and Rosenfield, 1999Deplewski D. Rosenfield R.L. Growth hormone and insulin-like growth factors have different effects on sebaceous cell growth and differentiation.Endocrinology. 1999; 140: 4089-4094Crossref PubMed Scopus (113) Google Scholar). Therefore, it seems that, like keratinocytes, PGs may participate in the regulation of biological functions of sebaceous glands. It remains unclear, however, whether PGs are associated with the development of sebaceous glands and sebocyte lipogenesis. In this study, we investigated the possible involvement of PGs in the formation of intracellular lipid droplets in hamster sebaceous glands in vivo and in vitro. Our findings show that 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) plays a crucial role in the augmentation of lipid-droplet formation by increasing triacylglycerol (TG) synthesis in hamster sebocytes, and that the synthesis of 15d-PGJ2 is mediated by COX-2 and cytochrome P-450 (CYP)-dependent pathways. We first examined the relationship between PG levels and lipogenesis during the differentiation of hamster sebocytes. As intracellular lipid droplets accumulate in sebocytes mostly consist of TG (Sato et al., 2001aSato T. Imai N. Akimoto N. Sakiguchi T. Kitamura K. Ito A. Epidermal growth factor and 1α,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebaceous gland cells in vitro.J Invest Dermatol. 2001; 117: 965-970Crossref PubMed Scopus (43) Google Scholar), we evaluated sebocytic differentiation by measuring intracellular levels of TG. As shown in Figure 1a, levels of TG (closed squares) were found to increase in cell culture time dependently. The level of PGF2α (open squares) was somewhat higher than that of PGE2 (open triangles) and 15d-PGJ2 (open circles) on day 3 in culture, but was found to decrease time dependently for up to 12 d (to <10 pg per μg DNA). The level of PGE2 was considerably lower (<10 pg per μg DNA). 15d-PGJ2 levels were augmented for 6 d and this augmented level (20–30 pg per μg DNA) was sustained for up to 12 d, indicating that 15d-PGJ2 is predominant in differentiated hamster sebocytes. It was found that hamster sebocytes constitutively produce COX-2 with the same molecular weight as the human COX-2 protein and its production slightly decreased for up to 12 d (Figure 1b, lower panel). Furthermore, COX-2 was found to be expressed in sebaceous glands (see SG, Figure 1b, lower panel). There was no detectable COX-1 in either sebaceous glands or cultured sebocytes (see Figure 1b, upper panel). To clarify whether 15d-PGJ2 might induce the differentiation of sebocytes, we investigated the effect of exogenous 15d-PGJ2 on the formation of lipid droplets in hamster sebocytes. When 15d-PGJ2 (1–10 μM) was added in the sebocyte culture, the formation of intracellular lipid droplets (Figure 2b vs a) and TG levels (Figure 2c) increased in a concentration-dependent manner. As shown in Table I, 15d-PGJ2-induced augmentation of TG production was found to be associated with an increase in the activity of diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme of TG synthesis (p<0.001).Table IIncrease of DGAT activity by 15d-PGJ2 and COX inhibitors in hamster sebocytesTreatmentsμMDGAT activity (pmol per min per μg DNA)None—564.4±14.615d-PGJ210961.7±18.1ap<0.001, between untreated and COX inhibitors or 15d-PGJ2-treated cells.Indomethacin101142.0±36.9ap<0.001, between untreated and COX inhibitors or 15d-PGJ2-treated cells.Diclofenac101120.3±39.5ap<0.001, between untreated and COX inhibitors or 15d-PGJ2-treated cells.NS-39820869.7±63.2ap<0.001, between untreated and COX inhibitors or 15d-PGJ2-treated cells.DGAT, diacylglycerol acyltransferase; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; COX, cyclooxygenase.a p<0.001, between untreated and COX inhibitors or 15d-PGJ2-treated cells. Open table in a new tab DGAT, diacylglycerol acyltransferase; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; COX, cyclooxygenase. To clarify the possible involvement of 15d-PGJ2 in increasing sebocyte lipogenesis, we investigated the effects of COX inhibitors on the production of TG and the formation of intracellular lipid droplets in cultured hamster sebocytes. As shown in Figure 3, levels of PGE2 (panel A) and PGF2α (panel B) were significantly diminished (p<0.001) by non-selective COX inhibitors, indomethacin (10 μM) and diclofenac (10 μM), and by a COX-2-specific inhibitor, NS-398 (20 μM). The level of 15d-PGJ2 in the medium was found to increase in sebocytes treated with indomethacin (p<0.01), diclofenac (p<0.01), and NS-398 (p<0.001) (Figure 3c). In contrast, there was no change in the level of PGD2 between untreated and indomethacin (10 μM)-treated sebocytes (0.49±0.06 vs 0.54±0.09 pg per μg DNA, respectively). Neither COX-1 nor COX-2 production was altered in the COX inhibitor-treated cells (see Fig S1). Moreover, in primarily cultured hamster skin fibroblasts, these three inhibitors were found to inhibit the production of PGE2, PGF2α, and 15d-PGJ2 (data not shown). Download .jpg (.02 MB) Help with files Figure S1Expression of COX-1 and COX-2 in indomethacin- and NS-398-treated hamster sebocytes. Because of our finding that 15d-PGJ2 production was increased in sebocytes even when COX inhibitors were present, we examined whether the inhibitors could augment lipogenesis in sebaceous glands. As shown in Figure 4, the production of TG was found to increase concentration dependently in the presence of indomethacin (1–10 μM), diclofenac (0.1–10 μM), and NS-398 (5–20 μM). The formation of intracellular lipid droplets also increased (data not shown). There were no significant changes in the levels of free fatty acids and cholesterol compared with those of TG in the lipids (data not shown). The selective augmentation of TG production by indomethacin, diclofenac, and NS-398 was found to be associated with an increase in DGAT activity in hamster sebocytes (Table I). When auricles from 5-wk-old male golden hamsters were topically treated with 2% (wt/vol) indomethacin for 14 d, the development of sebaceous glands (arrow heads) with the deposition of sebum (asterisks) was observed (Figure 5b vs a).Figure 5Development of sebaceous glands by topically administering indomethacin in vivo. Histochemical staining of hamster auricles shows that topical treatment of indomethacin for 2 wk (B) leads to the development of sebaceous glands (arrow heads), and augments the deposition of sebum (asterisks) compared with vehicle-treated auricles (A). Three independent experiments were reproducible, and typical data are shown. Scale bar, 300 μm.View Large Image Figure ViewerDownload (PPT) Based on our finding that indomethacin, diclofenac, and NS-398 cause the selective augmentation of 15d-PGJ2 production in hamster sebocytes, a COX-independent pathway may be involved in the increase of 15d-PGJ2 production. As it has been reported that arachidonic acid is metabolized not only by COX but also by CYP (Laniado-Schwartzman et al., 1988Laniado-Schwartzman M. Davis K.L. McGiff J.C. Levere R.D. Abraham N.G. Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver.J Biol Chem. 1988; 263: 2536-2542Abstract Full Text PDF PubMed Google Scholar; Capdevila et al., 2000Capdevila J.H. Falck J.R. Harris R.C. Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase.J Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar), we investigated the effect of a CYP inhibitor on the production of 15d-PGJ2 and on the synthesis of intracellular TG in COX inhibitor-treated sebocytes. As shown in Figure 6a, a non-selective CYP inhibitor, SKF-525A (10 and 20 μM), suppressed indomethacin and NS-398-induced production of 15d-PGJ2. These COX inhibitor-augmented levels of intracellular TG were diminished by SKF-525A (Figure 6b). SKF-525A did not influence the basal levels of 15d-PGJ2 and TG in hamster sebocytes (Figure 6). Similar results were obtained when another non-selective CYP inhibitor, methoxsalen, was used (data not shown). As it has been reported that 15d-PGJ2 as well as PGJ2 is an endogenous activator of peroxisome proliferation-activating receptor γ (PPARγ) (Kliewer et al., 1995Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation.Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1863) Google Scholar; Yu et al., 1995Yu K. Bayona W. Kallen C.B. et al.Differential activation of peroxisome proliferator-activated receptors by eicosanoids.J Biol Chem. 1995; 270: 23975-23983Crossref PubMed Scopus (633) Google Scholar; Shibata et al., 2002Shibata T. Kondo M. Osawa T. Shibata N. Kobayashi M. Uchida K. 15-deoxy-Δ12,14-prostaglandin. J2 A prostaglandin D2 metabolite generated during inflammatory processes.J Biol Chem. 2002; 277: 10459-10466Crossref PubMed Scopus (356) Google Scholar; Scher and Pillinger, 2005Scher J.U. Pillinger M.H. 15d-PGJ2: The anti-inflammatory prostaglandin?.Clin Immunol. 2005; 114: 100-109Crossref PubMed Scopus (263) Google Scholar), and that the activation of PPARγ1 in sebocytes causes an increase in lipogenesis (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 (126) Google Scholar), we examined the effect of SKF-525A on the synthesis of TG in hamster sebocytes treated with a ligand activator of PPARγ, troglitazone. As shown in Table II, troglitazone-augmented TG synthesis was not influenced by SKF-525A (5–20 μM).Table IINo effect of CYP inhibitor on troglitazone-augmented TG synthesis in hamster sebocytesTreatmentsμMLevels of TG (μg per μg DNA)None—0.74±0.24Troglitazone102.37±0.17ap<0.001, between untreated and troglitazone-treated cells.Troglitazone+SKF-525A52.13±0.22102.51±0.30202.13±0.14CYP, cytochrome P-450; TG, triacylglycerol.a p<0.001, between untreated and troglitazone-treated cells. Open table in a new tab CYP, cytochrome P-450; TG, triacylglycerol. The proliferation and differentiation of keratinocytes have been reported to be regulated by PGE2, which may be associated with the development of the epidermis and epidermal repair under wound healing (Pentland and Needleman, 1986Pentland A.P. Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis.J Clin Invest. 1986; 77: 246-251Crossref PubMed Scopus (169) Google Scholar; Unemori et al., 1994Unemori E.N. Ehsani N. Wang M. Lee S. McGuire J. Amento E.P. Interleukin-1 and transforming growth factor-α: Synergistic stimulation of metalloproteinases, PGE2, and proliferation in human fibroblasts.Exp Cell Res. 1994; 210: 166-171Crossref PubMed Scopus (73) Google Scholar; Sato et al., 1997Sato T. Kirimura Y. Mori Y. The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts.J Invest Dermatol. 1997; 109: 334-339Crossref PubMed Scopus (55) Google Scholar). Sebocytes are classified as epithelial cells, and cause terminal differentiation with the intracellular accumulation of lipids (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 (124) Google Scholar; Deplewski and Rosenfield, 1999Deplewski D. Rosenfield R.L. Growth hormone and insulin-like growth factors have different effects on sebaceous cell growth and differentiation.Endocrinology. 1999; 140: 4089-4094Crossref PubMed Scopus (113) Google Scholar), it seems that PG might be involved in the regulation of sebaceous development and functions. In this study, the basal level of 15d-PGJ2 was found to increase, whereas that of PGF2α decreased and that of PGE2 did not change during spontaneous differentiation of hamster sebocytes in vitro. We demonstrated that the formation of intracellular lipid droplets was augmented by exogenously administering 15d-PGJ2, and was associated with an increase in DGAT-mediated TG biosynthesis in hamster sebocytes.Neufang et al., 2001Neufang G. Furstenberger G. Heidt M. Marks F. Muller-Decker K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.Proc Natl Acad Sci USA. 2001; 98: 7629-7634Crossref PubMed Scopus (184) Google Scholar reported that the development of sebaceous glands with sebum deposition is augmented in transgenic mice overexpressing COX-2 in the epidermis, in which the levels of PGF2α and PGE2 are elevated. But, they did not show data for 15d-PGJ2 in their transgenic mice. As a COX-mediated arachidonic acid metabolite, PGH2, is a common precursor for 15d-PGJ2, PGF2α, and PGE2 (Narumiya et al., 1999Narumiya S. Sugimoto Y. Ushikubi F. Prostanoid receptors: Structures, properties, and functions.Physiol Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar; Tiano et al., 2002Tiano H.F. Loftin C.D. Akunda J. et al.Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis.Cancer Res. 2002; 62: 3395-3401PubMed Google Scholar; Helliwell et al., 2004Helliwell R.J. Adams L.F. Mitchell M.D. Prostaglandin synthases: Recent developments and a novel hypothesis.Prostaglandins Leukot Essent Fatty Acids. 2004; 70: 101-113Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar; Scher and Pillinger, 2
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