Induction of SREBP-1c mRNA by Differentiation and LXR Ligand in Human Keratinocytes

ORIGINAL ARTICLE

Induction of SREBP-1c mRNA by Differentiation and LXR Ligand in Human Keratinocytes Ai Yokoyama1,2, Makoto Makishima1, Mihwa Choi1, Yoshitake Cho1, Shigeru Nishida1, Yuichi Hashimoto3 and Tadashi Terui2 The epidermis is an active site of lipid metabolism, and the synthesis of fatty acids and cholesterol is required for cutaneous homeostasis. Liver X receptor-a (LXRa) and LXRb are nuclear receptors that are activated by oxysterols and regulate cholesterol and fatty acid metabolism. LXRs, predominantly LXRb, have been shown to be involved in keratinocyte differentiation and epidermal permeability barrier function. Although LXR regulates hepatic lipogenesis by inducing sterol-regulatory element-binding protein-1c (SREBP-1c), SREBP-1c induction by LXR in the epidermis has not been studied. In this study, we report that SREBP-1c mRNA increased during differentiation of human keratinocyte HaCaT cells and that LXR agonist effectively induced expression of LXR target genes, including SREBP-1c and ATP-binding cassette transporter A1, in differentiated HaCaT cells. Differentiation-associated and LXR-enhanced expression of SREBP-1c was also observed in malignant human keratinocyte A431 cells and primary human keratinocytes. A synthetic LXR antagonist inhibited confluencydependent expression of SREBP-1c. Thus, SREBP-1c expression increases during keratinocyte differentiation, and LXR activation enhances its expression. Journal of Investigative Dermatology (2009) 129, 1395–1401; doi:10.1038/jid.2009.15; published online 26 February 2009

INTRODUCTION The epidermis functions as a permeability barrier to prevent the loss of water and electrolytes and the entry of toxic compounds. The permeability barrier comprises extracellular lipid-enriched membranes in the stratum corneum that contain fatty acids, cholesterol, and ceramides (Feingold, 2007). Their precursor lipids, such as phospholipids, cholesterol sulfate, glucosylceramides, and sphingomyelins, are delivered to the extracellular space by the secretion of lamellar bodies and metabolized at the boundary between the stratum granulosum and the stratum corneum by enzymes that are cosecreted in lamellar bodies (Uchida and Holleran, 2008). The lipids contained in the lamellar bodies are derived from epidermal lipid synthesis and extracutaneous sources such as diet (Feingold, 2007). Although the lamellar bodies 1

Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan; 2Division of Cutaneous Science, Department of Dermatology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan and 3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan Correspondence: Professor Makoto Makishima, Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan. E-mail: [email protected] Abbreviations: ABCA1, ATP-binding cassette transporter A1; FAS, fatty acid synthase; LXR, liver X receptor; NHEK, normal human epidermal keratinocyte; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol-regulatory element-binding protein

Received 8 July 2008; revised 1 December 2008; accepted 20 December 2008; published online 26 February 2009

& 2009 The Society for Investigative Dermatology

are not present in the undifferentiated basal layer of the epidermis, they appear in the upper stratum spinosum layer and are accumulated along with keratinocyte differentiation. Sterol-regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of enzymes involved in cholesterol and fatty acid synthesis (Goldstein et al., 2006). SREBP-1a and 1c are the protein products of a single gene with different promoters and alternatively spliced transcripts. SREBP-2, a gene product distinct from SREBP-1, is primarily involved in stimulating cholesterol synthesis, whereas SREBP-1c induces fatty acid synthesis. SREBP-2 was reported to regulate cholesterol and fatty acid synthesis as the predominant form in keratinocytes (Harris et al., 1998). The increase in lipid synthesis induced by keratinocyte differentiation appears to be due to activation of SREBPs. The nuclear receptors liver X receptor-a (LXRa) and LXRb play a role in lipid metabolism (Makishima, 2005). LXRa and LXRb are expressed in murine and human keratinocytes (Hanley et al., 2000; Komuves et al., 2002). LXR activators stimulate keratinocyte differentiation and the formation of the cornified envelope in an LXRb-dependent manner. LXR activation also stimulates epidermal lipid synthesis, lamellar body secretion, and lipid processing in the stratum corneum (Man et al., 2005). Thus, LXRs regulate a variety of skin functions, including lipid metabolism and permeability barrier function. SREBP-1c is a direct target gene of LXRs, and administration of synthetic LXR agonists in mice increases liver and plasma triglycerides and phospholipids through SREBP-1c-dependent induction of hepatic lipogenic www.jidonline.org 1395

A Yokoyama et al. SREBP-1c Induction in Keratinocytes

genes (Schultz et al., 2000; Liang et al., 2002). These findings suggest that the LXR-SREBP-1c cascade plays a role in lipid metabolism in keratinocytes. In this study, we examined the effects of LXR ligand administration on the expression of genes involved in lipid metabolism in keratinocytes.

As the spontaneously immortalized keratinocyte cell line HaCaT retains differentiation potential in vivo and in vitro, we utilized these cells to examine the effects of an LXR ligand on induction of genes involved in lipid metabolism (Ryle et al., 1989). When HaCaT cells were plated at a concentration of 1  105 cells per well on a six-well plate (9.6 cm2 per well), the cells proliferated until they became confluent on day 7 (Figure 1a and b). A time-dependent increase in the expression of involucrin, a keratinocyte differentiation marker, was observed. Involucrin induction may be due to density-dependent differentiation of HaCaT cells (Ryle et al., 1989). T0901317 (N-(2,2,2-trifluoroethyl)N-[4–(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl] benzenesulfonamide) is a potent and selective agonist for both LXRa and LXRb (Schultz et al., 2000). We treated cells with T0901317 (Figure 1c) and examined the expression of LXR target genes. Interestingly, the expression of SREBP-1c, an LXR target that plays a role in fatty acid synthesis, was enhanced in cells treated with T0901317 with increasing cellular confluency (Figure 1d). SREBP-1c expression was also increased in confluent cells in the absence of ligand. T0901317 treatment induced the expression of another LXR target gene, ATP-binding cassette transporter A1 (ABCA1), more effectively in confluent cells than in non-confluent cells. Expression of SREBP-1c and ABCA1 was not further increased at day 9. This may be due to decreased cell viability (Figure 1b). Expression of fatty acid synthase (FAS), a target of both SREBP-1 and LXRs (Joseph et al., 2002), was slightly increased in confluent cells without LXR ligand, and T0901317 treatment was not effective on FAS induction. The mRNA levels of LXRa and LXRb were slightly increased in confluent cells and were unaffected by T0901317 treatment. Thus, the induction of SREBP-1c and ABCA1 mRNA expression by LXR ligand was enhanced by the duration of cell culture, and enhanced expression may be associated with confluency-dependent differentiation of HaCaT cells. We examined protein expression of SREBP-1 and LXRs by immunoblotting. As SREBP-1 proteins undergo rapid degradation (Wang et al., 1994), we could not detect SREBP-1 proteins without treating the cells with a protease inhibitor (data not shown). Then, we treated the cells with an inhibitor of neutral cysteine proteases and examined SREBP-1 protein expression in nuclear extracts. SREBP-1c protein levels are increased from day 1 to day 3 in HaCaT cells, and T0901317 treatment increased SREBP-1 proteins in both precursor and mature forms at day 3 and day 7 (Figure 1e). There was no increase in SREBP-1 expression in T0901317-treated cells from day 3 to day 7. LXRb protein was observed in HaCaT cells, but LXRa was not detected. 1396 Journal of Investigative Dermatology (2009), Volume 129

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Figure 1. Confluency-dependent induction of LXR target genes in HaCaT cells. (a) HaCaT cells proliferate until reaching confluency at day 7. Bar ¼ 50 mm. (b) Cell number reaches maximum at day 7. (c) Experimental procedure for T0901317 treatment in cells of different confluency. HaCaT cells were treated with vehicle control or T0901317 (1 mM) at day 2, 4, 6, or 8; cultured for 24 hours; and harvested for examination of mRNA (d) and protein expression (e). (d) mRNA expression of SREBP-1c, ABCA1, FAS, involucrin, LXRa, and LXRb. *Po0.05; **Po0.01. (e) Nuclear expression of SREBP-1, LXRb, and lamin B. Western blotting was repeated twice with similar results.

Density-dependent differentiation and enhanced expression of lipogenic genes

To examine the correlation between density-dependent differentiation and enhanced expression of LXR target genes, we plated HaCaT cells at a low density (1  105 cells per well) and at a high density (4  105 cells per well) in six-well plates, cultured them for 1 day, and then treated them with

A Yokoyama et al. SREBP-1c Induction in Keratinocytes

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Figure 2. Density-dependent expression of genes involved in lipid metabolism in HaCaT cells. (a) Experimental procedure for T0901317 treatment in HaCaT cells cultured at low density or high density. HaCaT cells were plated at 1  105 cells per well or at 4  105 cells per well in a 9.6 cm2 per well plate and treated with vehicle control or 1 mM T0901317 for 24 hours. (b) mRNA expression of keratinocyte differentiation markers (involucrin and transglutaminase 1), genes involved in lipid metabolism (SREBP-1c, SCD-1, ABCA1, FAS, acetyl CoA carboxylase (ACC), SREBP-2, HMG-CoA synthase, HMG-CoA reductase), and LXRs. *Po0.05; **Po0.01.

T0901317 (Figure 2a). Increased expression of the keratinocyte differentiation markers involucrin and transglutaminase 1 was observed in cells cultured at a high density (Figure 2b). Without T0901317 treatment, expression of SREBP-1c and another lipogenic gene, stearyl CoA desaturase-1 (SCD-1), which is a SREBP-1c and LXR target (Chu et al., 2006), was increased in high-density culture. T0901317-induced expression of these genes was enhanced in high-density cells. Expression of ABCA1 and FAS did not change between highdensity culture and low-density culture in the absence of LXR ligand, and T0901317 increased gene expression in a density-independent manner. The expression of acetyl CoA carboxylase was slightly induced by T0901317 and there was no density-dependent enhancement. The expression of a cholesterol metabolism regulator (SREBP-2) and cholesterol synthetic enzymes (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase) was not affected by cell density or T0901317 treatment. LXRa and LXRb expression did not differ in high-density culture and

low-density culture. Therefore, increased expression of SREBP-1c and SCD-1 and enhanced induction of these genes by LXR activation were associated with density-dependent differentiation. Differentiation-dependent but cell contact-independent induction of SREBP-1c

Confluent cells exhibit increased intercellular adhesion, and the cell–cell adhesion interaction induces various cellular responses (Mu¨ller et al., 2008). To discriminate between effects of cellular differentiation and differentiation-independent effects in confluency-dependent induction of SREBP-1c, we utilized a suspension culture of HaCaT cells. Keratinocytes cultured in suspension, in which the cell–matrix attachment is disrupted, undergo terminal differentiation (Watt et al., 1988). We cultured HaCaT cells in a plate coated with poly(2-hydroxyethyl methacrylate) to inhibit cell–matrix attachment and examined the expression of involucrin and SREBP-1c. In suspension culture, the www.jidonline.org 1397

A Yokoyama et al. SREBP-1c Induction in Keratinocytes

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Figure 3. Differentiation of HaCaT cells in suspension culture increases expression of SREBP-1c. (a) mRNA expression of involucrin and SREBP-1c in a regular plate (non-coated) and in a poly(2-hydroxyethyl methacrylate)coated plate for suspension (coated) for 1 (D1), 2 (D2), or 3 days (D3). (b) Effect of T0901317 on SREBP-1c expression in HaCaT cells in suspension culture. HaCaT cells were cultured in suspension for 3 days and treated with vehicle control or 1 mM T0901317 for 24 hours. *Po0.05; **Po0.01.

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Expression of SREBP-1c in malignant keratinocytes and primary keratinocytes

To examine whether confluency-dependent induction of SREBP-1c is observed in malignant keratinocytes and primary keratinocytes, we cultured epidermoid carcinoma A431 cells and normal human epidermal keratinocytes (NHEK) in the absence or presence of T0901317. A431 cells grew until they became confluent at day 11 (Figure 4a). Expression of involucrin was increased at day 5 and was unaffected by continuous culture (Figure 4b). Expression of SREBP-1c, FAS, and ABCA1 increased slightly from day 5. T0901317 treatment minimally decreased the involucrin expression in A431 cells, and it effectively induced SREBP-1c, FAS, and ABCA1 in involucrin-expressing cells. Thus, the expression of SREBP-1c, FAS, and ABCA1 and their induction by LXR are associated with differentiation of A431 cells. Microscopic observations showed that primary keratinocyte NHEK stopped proliferation before they became confluent (data not shown), and NHEK 1398 Journal of Investigative Dermatology (2009), Volume 129

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differentiation marker involucrin was strongly induced and the expression of SREBP-1c was also enhanced (Figure 3a). T0901317 treatment further increased SREBP-1c expression in HaCaT cells cultured in a suspension (Figure 3b). Thus, the induction of SREBP-1c mRNA expression is associated with cellular differentiation.

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Figure 4. Differentiation-associated expression of LXR target genes in epidermoid carcinoma A431 cells. (a) Number of A431 cells. A431 carcinoma cells proliferated until day 11. (b) mRNA expression of involucrin, SREBP-1c, FAS, and ABCA1 in A431 cells. Cells were cultured, treated with vehicle control or 1 mM T0901317 for 24 hours, and harvested at the indicated day as shown in Figure 1c. *Po0.05; **Po0.01.

increased involucrin expression at day 5 (Figure 5a). A timedependent increase in the SREBP-1c expression was also observed in NHEK. As in HaCaT cells, high-density culture of NHEK increased the expression of involucrin and SREBP-1c but not of ABCA1 (Figure 5b). T0901317 increased SREBP-1c expression in both low-density culture and high-density culture. T0901317 increased ABCA1 expression in a densityindependent manner. Although differentiation mechanisms may differ in NHEK, HaCaT, and A431 cells, differentiated cells enhance SREBP-1c mRNA expression through LXR activation.

A Yokoyama et al. SREBP-1c Induction in Keratinocytes

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Figure 6. Effect of LXR antagonist on expression of SREBP-1c in HaCaT cells. (a) Experimental procedure for treatment with LXR agonist and antagonist in HaCaT cells. Cells were cultured for 6 days and treated with vehicle control or 10 mM 5CPPSS-50 in the absence or presence of 0.3 mM T0901317 for 24 hours. (b) Effect of 5CPPSS-50 on confluency-dependent expression of ABCA1 and SREBP-1c. *Po0.05; **Po0.01.

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shown). We examined the effect of 5CPPSS-50 on SREBP-1c expression in HaCaT cells. Confluent HaCaT cells were treated with 5CPPSS-50 (10 mM) with or without T0901317 (0.3 mM) (Figure 6a). Treatment with 5CPPSS-50 suppressed confluency-dependent expression of ABCA1 and SREBP-1c (Figure 6b), suggesting that their mRNA expression is mediated by endogenous ligand-dependent LXR activation. T0901317 addition increased the ABCA1 and SREBP-1 expression suppressed by 5CPPSS-50. These data suggest that a ligand-dependent mechanism is involved in the increased expression of SREBP-1c in differentiated keratinocytes.

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Figure 5. Expression of involucrin and SREBP-1c in primary NHEK. (a) Time course of mRNA expression of involucrin and SREBP-1c. **Po0.01 compared with expression at day 3. (b) Effect of T0901317 on expression of involucrin and SREBP-1c in NHEK. Cells were plated at low density or high density and treated with vehicle control or 1 mM T0901317 for 24 hours, as shown in Figure 2a. *Po0.05; **Po0.01.

Suppression of SREBP-1c expression by an LXR antagonist

5CPPSS-50 (5-chloro-N-20 -n-pentylphenyl-1,3-dithiophthalimide) is an LXR antagonist with IC50 values of 10 and 13 mM for LXRa and LXRb, respectively (Noguchi-Yachide et al., 2007). 5CPPSS-50 (10 mM) did not inhibit ligand-dependent activation of peroxisome proliferator-activated receptor-a (PPARa) or PPARg in transfection experiments (data not

DISCUSSION In this study, we investigated the expression of LXR target genes in keratinocytes and found exogenous ligand-dependent and independent induction of LXR target genes in differentiated keratinocytes. SREBP-1c plays an important role in hepatic lipogenesis (Goldstein et al., 2006). LXR agonists stimulate hepatic lipogenesis through SREBP-1c-dependent induction of lipogenic genes (Schultz et al., 2000; Liang et al., 2002). In the epidermis, LXR agonists also stimulate fatty acid synthesis, and topical treatment with LXR agonists enhances permeability barrier function (Komuves et al., 2002; Feingold, 2007). These findings suggest that the LXR-SREBP-1c cascade plays a role in lipogenic gene induction in the epidermis. However, SREBP-1 was reported to be absent from the epidermis, and SREBP-2 has been proposed to induce both fatty acid and cholesterol synthesis (Harris et al., 1998). As LXR induces expression of SREBP-1c but not of SREBP-1a or SREBP-2 (Repa et al., 2000; Schultz et al., 2000), LXR-dependent stimulation of lipogenesis in the epidermis would be expected to be mediated by SREBP1c. We detected SREBP-1c mRNA and protein in HaCaT cells, and T0901317 increased SREBP-1c expression. Harrison et al. (2007) reported that SREBP-1 is expressed in keratinocytes as well as in sebocytes. Thus, SREBP-1c is induced by LXR activation and is suggested to play a role in lipogenic gene regulation in keratinocytes. www.jidonline.org 1399

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Liver X receptors have been shown to play an important role in cutaneous homeostasis (Schmuth et al., 2008). Although both LXRa and LXRb are expressed in cultured human keratinocytes and throughout all layers of the human epidermis (Hanley et al., 2000; Russell et al., 2007), LXRb is the predominant isoform transcribed in mouse epidermis (Komuves et al., 2002). LXR activators stimulate keratinocyte differentiation in an LXRb-dependent manner (Komuves et al., 2002). We detected LXRb and not LXRa protein in HaCaT cells, although real-time quantitative reverse transcription-PCR analysis showed mRNA expression for both isoforms, a finding that supports a predominant role for LXRb in keratinocytes. Oxysterols are in vivo ligands for LXR in the liver (Chen et al., 2007). Although mechanisms of oxysterol metabolism in the epidermis have not been elucidated, increased oxysterol production may activate LXR and induce SREBP-1c expression in differentiated keratinocytes. LXRs are common inducers of SREBP-1 and ABCA1 gene expression (Makishima, 2005), and administration of an LXR antagonist decreased their expression in confluent cells. These cells are suggested to contain increased endogenous LXR ligands, such as oxysterols, although the possibility that 5CPPSS-50 has LXR-independent effects cannot be ruled out. We need to develop more potent antagonists to elucidate the role of endogenous liganddependent activation of LXRs in keratinocytes. LXRa and LXRb exhibit ligand-independent activation by interacting with other proteins, such as retinoid X receptor and coactivators (Wiebel et al., 1999; Son et al., 2008). Expression of coactivator proteins changes during keratinocyte differentiation (Oda et al., 2003). Although LXRs are necessary factors for SREBP-1c expression, several mechanisms, including insulin signaling, regulate SREBP-1c expression (Tobin et al., 2002). LXR ligand-independent mechanisms may also be involved in SREBP-1c mRNA expression. PPARs are expressed in the epidermis (Schmuth et al., 2008), and activation of PPARs, especially PPARd, stimulates keratinocyte differentiation (Westergaard et al., 2001). However, PPARa activation suppresses LXR-dependent induction of the SREBP-1c gene in hepatocytes (Ide et al., 2003). PPARs may not be involved in mRNA expression of SREBP-1c in keratinocytes. Nuclear SREBP-1c protein expression was increased from day 1 to day 3 in HaCaT cells, and T0901317 treatment increased SREBP-1 proteins in both precursor and mature forms. However, a differentiation-associated increase in SREBP-1 proteins was not observed in T0901317-treated cells. SREBP-1 protein levels are regulated by multiple mechanisms, such as synthesis, proteolytic processing, translocation, and degradation (Goldstein et al., 2006). PPARd inhibits the proteolytic processing of SREBP-1 through the induction of insulin-induced gene-1 in hepatocytes (Qin et al., 2008). Differentiation-associated expression of SREBP-1 proteins may be regulated by mRNA expression and additional mechanisms. Further studies are required to elucidate differentiation-associated induction of SREBP-1c. Taken together, these studies show that basal mRNA expression of SREBP-1c increases during keratinocyte differentiation, and the LXR agonist further enhances its expression. 1400 Journal of Investigative Dermatology (2009), Volume 129

LXR ligands should prove to be useful agents in stimulating epidermal lipogenic activity.

MATERIALS AND METHODS Chemical compounds T0901317 was purchased from Cayman Chemical (Ann Arbor, MI), and 5CPPSS-50 was synthesized in our laboratory (Noguchi-Yachide et al., 2007).

Cell culture The human immortalized keratinocyte cell line HaCaT (Boukamp et al., 1988) and epidermoid carcinoma cell line A431 (Giard et al., 1973) were cultured in DMEM containing 10% fetal bovine serum, 100 U ml1 penicillin, and 100 mg ml1 streptomycin at 371C in a humidified atmosphere containing 5% CO2. NHEK (Kurabo Industries, Osaka, Japan) were cultured in serum-free medium (HumediaKG2; Kurabo Industries) containing 10 mg ml1 insulin, 0.1 ng ml1 human recombinant epidermal growth factor, 0.5 mg ml1 hydrocortisone, 0.4% bovine pituitary extract, 100 U ml1 penicillin, and 100 mg ml1 streptomycin according to the manufacturer’s recommendations (Komatsu et al., 2003). For suspension culture, cells were seeded on dishes that were pre-coated with poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich, St Louis, MO) (Watt et al., 1988). Cells were counted in a Z1S Coulter Counter (Beckman Coulter, Fullerton, CA).

Real-time quantitative reverse transcription-PCR analysis Total RNAs from samples were prepared with RNAiso (Takara Bio, Otsu, Japan), and cDNAs were synthesized using the ImProm-II reverse transcription system (Promega, Madison, WI). Real-time quantitative reverse transcription-PCR was performed on the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq (Takara Bio) as described previously (Kalaany et al., 2005; Ishizawa et al., 2008). Primer sequences are listed in Table S1. The RNA values were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA.

Immunoblotting Cells were treated with 25 mg ml1 calpain inhibitor (ALLN; EMD Chemicals, San Diego, CA) for 2 hours before harvest, and nuclear extracts were prepared as described previously (Wang et al., 1994). The proteins were separated by SDS-PAGE and were transferred to a nitrocellulose membrane; probed with anti-SREBP-1 antibody (BD Biosciences, San Jose, CA), anti-LXRb antibody (Perseus Proteomics, Tokyo, Japan), or anti-lamin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA); and visualized with an alkaline phosphatase conjugate substrate system (Inaba et al., 2007). The anti-SREBP-1 antibody recognizes the N-terminal basic helix-loop-helix domain of human SREBP-1 and is not cross-reactive with SREBP-2 (Sakai et al., 1997).

Statistics All values are shown as means±1 SD of triplicate assays. The unpaired two-group Student t-test was performed to assess significant differences. CONFLICT OF INTEREST The authors state no conflict of interest.

A Yokoyama et al. SREBP-1c Induction in Keratinocytes

ACKNOWLEDGMENTS We thank the members of the Makishima, Terui, and Hashimoto laboratories for technical assistance and helpful comments. This study was supported in part by grants from the Ministry of Health, Labor, and Welfare, Japan (Research on Measures for Intractable Diseases, 2008); the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and Nihon University School of Medicine.

SUPPLEMENTARY MATERIAL Table S1. Primer sequences for real-time quantitative reverse transcriptionPCR.

REFERENCES Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761–71 Chen W, Chen G, Head DL, Mangelsdorf DJ, Russell DW (2007) Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice. Cell Metab 5:73–9 Chu K, Miyazaki M, Man WC, Ntambi JM (2006) Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation. Mol Cell Biol 26:6786–98 Feingold KR (2007) The role of epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid Res 48:2531–46 Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H et al. (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51:1417–23 Goldstein JL, DeBose-Boyd RA, Brown MS (2006) Protein sensors for membrane sterols. Cell 124:35–46 Hanley K, Ng DC, He SS, Lau P, Min K, Elias PM et al. (2000) Oxysterols induce differentiation in human keratinocytes and increase Ap-1dependent involucrin transcription. J Invest Dermatol 114:545–53 Harris IR, Farrell AM, Holleran WM, Jackson S, Grunfeld C, Elias PM et al. (1998) Parallel regulation of sterol regulatory element binding protein-2 and the enzymes of cholesterol and fatty acid synthesis but not ceramide synthesis in cultured human keratinocytes and murine epidermis. J Lipid Res 39:412–22 Harrison WJ, Bull JJ, Seltmann H, Zouboulis CC, Philpott MP (2007) Expression of lipogenic factors galectin-12, resistin, SREBP-1, and SCD in human sebaceous glands and cultured sebocytes. J Invest Dermatol 127:1309–17 Ide T, Shimano H, Yoshikawa T, Yahagi N, Amemiya-Kudo M, Matsuzaka T et al. (2003) Cross-talk between peroxisome proliferator-activated receptor (PPAR) a and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. II. LXRs suppress lipid degradation gene promoters through inhibition of PPAR signaling. Mol Endocrinol 17:1255–67 Inaba Y, Yamamoto K, Yoshimoto N, Matsunawa M, Uno S, Yamada S et al. (2007) Vitamin D3 derivatives with adamantane or lactone ring side chains are cell type-selective vitamin D receptor modulators. Mol Pharmacol 71:1298–311 Ishizawa M, Matsunawa M, Adachi R, Uno S, Ikeda K, Masuno H et al. (2008) Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia. J Lipid Res 49:763–72

Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, Brown MS (2002) Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory elementbinding protein-1c. J Biol Chem 277:9520–8 Makishima M (2005) Nuclear receptors as targets for drug development: regulation of cholesterol and bile acid metabolism by nuclear receptors. J Pharmacol Sci 97:177–83 Man M-Q, Choi E-H, Schmuth M, Crumrine D, Uchida Y, Elias PM et al. (2005) Basis for improved permeability barrier homeostasis induced by PPAR and LXR activators: liposensors stimulate lipid synthesis, lamellar body secretion, and post-secretory lipid processing. J Invest Dermatol 126:386–92 Mu¨ller EJ, Williamson L, Kolly C, Suter MM (2008) Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation? J Invest Dermatol 128:501–16 Noguchi-Yachide T, Miyachi H, Aoyama H, Aoyama A, Makishima M, Hashimoto Y (2007) Structural development of liver X receptor (LXR) antagonists derived from thalidomide-related glucosidase inhibitors. Chem Pharm Bull 55:1750–4 Oda Y, Sihlbom C, Chalkley RJ, Huang L, Rachez C, Chang C-PB et al. (2003) Two distinct coactivators, DRIP/Mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation. Mol Endocrinol 17:2329–39 Qin X, Xie X, Fan Y, Tian J, Guan Y, Wang X et al. (2008) Peroxisome proliferator-activated receptor-d induces insulin-induced gene-1 and suppresses hepatic lipogenesis in obese diabetic mice. Hepatology 48:432–41 Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I et al. (2000) Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRa and LXRb. Genes Dev 14:2819–30 Russell LE, Harrison WJ, Bahta AW, Zouboulis CC, Burrin JM, Philpott MP (2007) Characterization of liver X receptor expression and function in human skin and the pilosebaceous unit. Exp Dermatol 16:844–52 Ryle CM, Breitkreutz D, Stark HJ, Leigh IM, Steinert PM, Roop D et al. (1989) Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones. Differentiation 40:42–54 Sakai J, Nohturfft A, Cheng D, Ho YK, Brown MS, Goldstein JL (1997) Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavageactivating protein. J Biol Chem 272:20213–21 Schmuth M, Jiang YJ, Dubrac S, Elias PM, Feingold KR (2008) Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology. J Lipid Res 49:499–509 Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L et al. (2000) Role of LXRs in control of lipogenesis. Genes Dev 14:2831–8 Son YL, Park OG, Kim GS, Lee JW, Lee YC (2008) RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal co-integrator-2. Biochem J 410:319–30 Tobin KAR, Ulven SM, Schuster GU, Steineger HH, Andresen SM, Gustafsson J-A et al. (2002) Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis. J Biol Chem 277:10691–7 Uchida Y, Holleran WM (2008) Omega-O-acylceramide, a lipid essential for mammalian survival. J Dermatol Sci 51:77–87

Joseph SB, Laffitte BA, Patel PH, Watson MA, Matsukuma KE, Walczak R et al. (2002) Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors. J Biol Chem 277:11019–25

Wang X, Sato R, Brown MS, Hua X, Goldstein JL (1994) SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77:53–62

Kalaany NY, Gauthier KC, Zavacki AM, Mammen PP, Kitazume T, Peterson JA et al. (2005) LXRs regulate the balance between fat storage and oxidation. Cell Metab 1:231–44

Watt FM, Jordan PW, O0 Neill CH (1988) Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc Natl Acad Sci USA 85:5576–80

Komatsu N, Takata M, Otsuki N, Toyama T, Ohka R, Takehara K et al. (2003) Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages. J Invest Dermatol 121:542–9

Westergaard M, Henningsen J, Svendsen ML, Johansen C, Jensen UB, Schroder HD et al. (2001) Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid. J Invest Dermatol 116:702–12

Komuves LG, Schmuth M, Fowler AJ, Elias PM, Hanley K, Man M-Q et al. (2002) Oxysterol stimulation of epidermal differentiation is mediated by liver X receptor-b in murine epidermis. J Invest Dermatol 118:25–34

Wiebel FF, Steffensen KR, Treuter E, Feltkamp D, Gustafsson J-A (1999) LigandIndependent coregulator recruitment by the triply activatable OR1/retinoid X receptor-a nuclear receptor heterodimer. Mol Endocrinol 13:1105–18

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