ORIGINAL ARTICLE

PPAR and LXR Activators Regulate ABCA12 Expression in Human Keratinocytes Yan J. Jiang1, Biao Lu1, Peggy Kim1, Gyorgy Paragh2, Gerd Schmitz2, Peter M. Elias3 and Kenneth R. Feingold1

ATP-binding cassette (ABC) transporter, family 12 (ABCA12), a member of the ABC superfamily, facilitates the delivery of lipids to lamellar bodies (LB) in keratinocytes, which is critical for permeability barrier function. Recently, of ABCA12 were found to underlie Harlequin and , two devastating disorders. Previously we and others have demonstrated that peroxisome proliferators-activated receptors (PPARs) and liver X receptor (LXR) activation improved epidermal permeability barrier homeostasis by stimulating keratinocyte differentiation, lipid synthesis, and increasing LB formation/secretion. Here we report that both PPAR-g and -b/d activators markedly stimulate ABCA12 mRNA expression in cultured human keratinocyte (CHK) in a dose- and time-dependent manner. Increased ABCA12 mRNA levels are accompanied by an increase in ABCA12 , suggesting biological importance of this upregulation. LXR activators also increase ABCA12 mRNA levels in CHK, but to a lesser extent. In contrast, activators of PPAR-a, RAR, RXR, or vitamin D receptor did not alter ABCA12 expression. Two major ABCA12 alternative transcripts and their corresponding are also expressed and upregulated by PPAR or LXR activator in both undifferentiated and differentiated CHK. Together, our data demonstrate that PPAR and LXR activators increase ABCA12 expression, providing an additional mechanism by which PPAR and LXR activators promote epidermal permeability barrier homeostasis. Journal of Investigative Dermatology (2008) 128, 104–109; doi:10.1038/sj.jid.5700944; published online 5 July 2007

INTRODUCTION and one pseudogene (ABCA11), and all mediate lipid ATP-binding cassettes (ABC) are a large group of proteins that transport (Schmitz et al., 2000). Most importantly, mutations mediate the transport of a variety of different substrates across in ABCA proteins have been implicated in several autosomal cellular membranes. These proteins contain two transmem- recessive disorders of lipid metabolism, including ABCA1 in brane domains and two ATP-binding domains or nucleotide- (Brooks-Wilson et al., 1999), ABCA4 in binding folds. Forty-eight ABC have been identified to (Allikmets et al., 1997), and ABCA3 in fatal date, which have been further divided into seven subfamilies, surfactant deficiency in newborns (Shulenin et al., 2004). based on and organization of the ABCA12 is a member of the ABCA subfamily (Akiyama, nucleotide-binding folds (Allikmets et al., 1996; Dean 2006). The functional importance of ABCA12 was revealed et al., 2001; Borst and Elferink, 2002; Akiyama et al., when a missense was first reported to cause type 2 2005). The ABCA subfamily comprises 12 full transporters lamellar ichthyosis (Lefevre et al., 2003). Very recently, severe mutations in ABCA12 were further identified to cause Harlequin ichthyosis, a severe keratinizing disorder, which is 1 Metabolism Section, Department of Veterans Affairs Medical Center, fatal in most affected newborns (Lefevre et al., 2003; Kelsell Northern California Institute for Research and Education, University of California at San Francisco, San Francisco, California, USA; 2Institute of et al., 2005). Evidence from morphological and biochemical Clinical Chemistry, University of Regensburg, Franz-Josef-Strauss-Allee 11, studies indicate that ABCA12 is important for lipid (gluco- Regensburg, Germany and 3Dermatology Service, Veterans Affairs Medical sylceramide) loading into lamellar body (LB) and its absence Center, Northern California Institute for Research and Education, University of results in a severe abnormality in permeability barrier California at San Francisco, San Francisco, California, USA formation and extraordinary, intrauterine thickening of the Correspondence: Dr Yan J. Jiang, Metabolism Section (111F), Department of Veterans Affairs Medical Center, Northern California Institute for Research stratum corneum, ultimately resulting in high rates of prenatal and Education, University of California at San Francisco, 4150 Clement Street, mortality (Akiyama et al., 2005). Strikingly, genetic correction San Francisco, California 94121, USA. E-mail:[email protected] of ABCA12 deficiency in patients’ keratinocytes by gene Abbreviations: ABCA12, ATP-binding cassette (ABC) transporter, family 12; transfer normalized loading of glucosylceramides into LB CHK, cultured human keratinocyte; Cig, ciglitazone; GW, GW610742; LB, (Akiyama et al., 2005). Glucosylceramides are major lamellar body; LXR, liver X receptor; PPAR, peroxisome proliferators- activated receptor; TO, TO901317; VD3,1a, 25-dihydroxyvitamin D3 precursors of ceramides, which comprises 50% of the lipid Received 18 January 2007; revised 2 April 2007; accepted 30 April 2007; that forms the barrier (Uchida et al., 2000). Moreover, as published online 5 July 2007 protein loading into LB is dependent on prior or concurrent

104 Journal of Investigative Dermatology (2008), Volume 128 & 2007 The Society for Investigative Dermatology YJ Jiang et al. Upregulation of ABCA12 by PPAR and LXR in Keratinocytes

lipid loading (Rassner et al., 1999), the failure to deliver lipids a *** 1,400 *** 1,203 may result in a deficiency in proteases, accounting in part for 1,176 the severe keratinizing disorder. Together, these studies 1,200 1,000 ** strongly indicate a critical role for ABCA12 in epidermal 642 physiology, specifically, the formation of LB and permeability 800 ** barrier homeostasis. Hence, understanding how this protein 600 514 328** is regulated could be great importance. Currently, however, 400 173 the factors that regulate ABCA12 expression in keratinocytes 200 100 103 140 94 130 65 remain unknown, although ABCA12 expression in cultured hABCA12 mRNA (% of control) 0 ) ) ) ) ) ) ) ) ) ) ) human keratinocyte (CHK) increases with keratinocyte M M M M M M M M M M M differentiation (Akiyama et al., 2005). Control  Gl (10 TO (10 WY (20 GW (8  22R (10 Cig (7.5 Tro (7.5 ATRA (1 VD3 (0.1 Previously, we and others have demonstrated that peroxi- CLO (400 9-cisRA (1 some proliferators-activated receptor (PPAR)-a,-b/d,-g,as LXR PPAR PPAR PPAR RAR RXR VDR well as liver X receptor (LXR)-a and -b, are expressed in 0.03 mM Ca2+, 24 hours incubation murine and human keratinocytes (Hanley et al., b 1,200 *** 2000a, b; Westergaard et al., 2001; Komuves et al., 2002). 1,041 Activation of PPARs and LXRs have numerous differentiation- 1,000 related effects on keratinocytes, including (1) stimulating 800 corneocyte envelope precursor expression (Hanley et al., 1998, 1999, 2000b); (2) increasing the expression of 600 *** 437 Sult2B1b, which catalyzes the synthesis of cholesterol sulfate 400 ** (Jiang et al., 2005); (3) stimulating epidermal lipid synthesis 275 200 (Man et al., 2006); (4) acceleration of LB secretion (Man 81 100 99 hABCA12 mRNA (% of control) et al., 2006); and (5) improving epidermal permeability 0 ) ) ) ) barrier homeostasis (Hanley et al., 1998, 1999, 2000a, b; 2+ M M M M Ca M Control Komuves et al., 2000, 2002; Jiang et al., 2005; Man et al.,  TO (10  Cig (5 GW (8  2006). Very recently, we demonstrated that ABCA1, a WY (20 0.03 m 2+ member of ABCA family that transports cholesterol and 1.2 mM Ca , 24 hours incubation phospholipids out of cells (Schmitz and Langmann, 2005), is Figure 1. Activation of PPAR-c, PPAR-b/d, and LXR increase ABCA12 mRNA not only expressed in CHK and murine epidermis, but also expression in CHK. (a) Cells were incubated with either vehicle control, or markedly stimulated by LXR, PPAR-a, PPAR-b/d activation activators of LXR (10 mM 22R or 10 mM TO); PPAR-g (7.5 mM Cig, or 7.5 mM (Jiang et al., 2006). Based on these findings, we hypothesized troglitazone (Tro), or 10 mM GI); PPAR-b/d GW (8 mM); PPARa (20 mM WY14643 or 400 mM CLO); RAR (1 mM all-trans retinoic acid); RXR (1 mM 9-cis-retinoic that activation of PPARs and LXR may also stimulate ABCA12 acid); vitamin D receptor (1a, 25-dihydroxyvitamin D3 0.1 mM) in low calcium expression in keratinocytes. medium for 24 hours (h). (b) Alternatively, cells were incubated in either 2 þ 2 þ 2 þ 0.03 mM Ca medium, or 1.2 mM Ca medium alone, or 1.2 mM Ca RESULTS AND DISCUSSION medium with 10 mM TO, or 5 mM Cig, or 8 mM GW, or 20 mM WY14643 for Initially, we examined the effect of the activation of LXR, 24 hours. Real-time PCR was performed to measure mRNA levels of ABCA12 PPARs, RAR, RXR, and vitamin D receptor (VDR) on ABCA12 and cyclophilin (internal control), as described in ‘‘Materials and Methods’’. mRNA levels. The optimized dose for each activator was Data are expressed as percentage of control (100%) and presented as mean7SEM (n ¼ 3 in each group). Similar results were obtained when the determined, as described previously (Jiang et al., 2005). As experiment was repeated with a different batch of cells. **Po0.01; shown in Figure 1a, ABCA12 mRNA levels increased ***Po0.001. markedly following treatment with PPAR-g activators (cigli- tazone (Cig): 11.8-fold; troglitazone: 12-fold; GI 251929X: 6.4-fold) and PPAR-b/d activator (GW610742 (GW): 5.1- induced by high calcium (1.2 mM). As shown in Figure 1b, fold), after 24 hours incubation. Furthermore, the upregula- although high calcium itself did not induce significant tion of ABCA12 by PPAR-g (Cig) occurred rapidly, was amount of ABCA12 mRNA, ligand activation of either sustained over an extend period of time (Figure 2a), and PPAR-g or -b/d increased ABCA12 mRNA levels by 11- and occurred in a dose-dependent manner (Figure 2b). Similar 4.5-fold, respectively, even in high calcium. Similarly, LXR time- and dose-dependent upregulation of ABCA12 mRNA activation by TO increased ABCA12 mRNA level by 2.3-fold was induced by PPAR-b/d (GW) activation (Figure S1). In (Po0.01). Consistent with the observations in undifferen- addition, although LXR activators modestly increased tiated CHK (above), PPAR-a activation had no effect on ABCA12 mRNA level (TO901317 (TO): 3.3-fold), activators ABCA12 mRNA (Figure 1b). Furthermore, ligand treatment of PPAR-a, RAR, RXR, or vitamin D receptor had no effect (5 mm Cig or 8 mm GW) of CHK for 4 days in high calcium (Figure 1a). As previous studies showed that keratinocyte medium increased, ABCA12 mRNA 2.2- and 1.8-fold, differentiation induced by calcium stimulate ABCA12 mRNA respectively, compared with high calcium alone (data not expression after 4 days incubation (Akiyama et al., 2005), we shown). Thus, under our experimental condition, certain next assessed whether PPAR and LXR activators would further PPAR and LXR activators stimulate ABCA12 mRNA expres- increase ABCA12 mRNA levels in differentiated CHK, sion, independent of keratinocyte differentiation.

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a cDNA 1,000 hABCA12, 2q34–q35, ~206 kb ** 12 3789456 53 Exon 800 694 ABCA12-L * ABCA12-S 600 * 454 TISS1 TISS2 361 Protein 400 328 aa ABCA12-L * (2595 aa) 136 10 aa 200 100 ABCA12-S (2277 aa) hABCA12 mRNA (% of control) 0 b 800 0 hour 6 hours 16 hours 24 hours 48 hours ABCA12-L ** 700 ABCA12-S 0.03 mM Ca2+, 5 M Cig

2+ 600 **

3,000 Ca 500 b *** M 2,479 400 2,500 *** 300 *** ** ** 2,000 200 hABCA12 transcripts (% of 0.03 m *** 100 1,500 1,176 0 ) ) ) ) 2+ 2+ M M M M 1,000 Ca Ca M M *** Cig (5 GW (8  22R (10 WY (20 *** 380 1.2 m 0.03 m 500 242 1.2 mM Ca2+, 24 hours incubation

hABCA12 mRNA (% of control) 100 0 Figure 3. Alternate transcripts of ABCA12 are expressed in CHK and 0 M 2.5 M 5.0 M 7.5 M 10 M upregulated by PPAR-c, PPAR-b/d, and LXR activators. (a) Genomic structure

0.03 mM Ca2+, 24 hours incubation of human ABCA12 locus and ABCA12 isoforms. Two major hABCA12 cDNAs, ABCA12-L and -S, are transcribed by alternative usage of two Figure 2. Activation of PPAR-c increases ABCA12 mRNA level in a time- and transcription initiation start sites, TISS1 and TISS2. Exon 1–8 are found only in dose-dependent manner. (a) Cells were incubated with 5 mM Cig for various ABCA12-L transcript. Two major human ABCA12 protein isoforms are 2 þ periods of time (0, 6, 16, 24, and 48 h) in 0.03 mM Ca medium. synthesized accordingly. ABCA12-L and -S proteins share a large common (b) Alternatively, cells were incubated with Cig at various concentrations (0, C-terminus, however, with a unique 328 amino acids (aa) or 10 aa at 2.5, 5.0, 7.5, 10 mM) in the same medium for 24 hours. ABCA12 and N-terminus, respectively. (b) Cells were incubated in either 0.03 mM or 2 þ 2 þ cyclophilin mRNA levels were measured as described. Data are expressed as 1.2 mM Ca medium alone, or 1.2 mM Ca medium with 10 mM TO, or 7 percentage of vehicle control (100%) and presented as mean SEM (n ¼ 3in 5 mM Cig, or 8 mM GW, or 20 mM WY14643 for 24 hours. Human ABCA12-L, each group). Similar results were obtained when the experiment was repeated -S, and cyclophilin mRNA levels were determined by real-time PCR. Under

with a different batch of cells. *Po0.05; **Po0.01; ***Po0.001. our experimental conditions, the absolute Ct values (mean from triplicates) for the basal levels of these two transcripts, hABCA12-L and -S are 25.4 and 30.9, respectively. Data are expressed as percentage of control (100%) and Previously, we reported that three alternate variants of presented as mean7SEM (n ¼ 6 in each group). **Po0.01; ***Po0.001. ABCA1 are expressed in CHK, which contribute equally to the increase in ABCA1 mRNA in response to LXR activation (Jiang et al., 2006). As in the case of ABCA1 (Singaraja et al., 2001, 2005), two major splicing transcripts of ABCA12 have 2.5-fold, respectively (Figure 3b). Again, activation of PPAR-a recently been described (Lefevre et al., 2003), which were had no effect on either transcript (Figure 3b). When assigned as hABCA12-L and hABCA12-S in this study (Figure undifferentiated keratinocytes were used, similar results were 3a). ABCA12-L (NM_173076) gene consists of 53 exons with obtained (data not shown). a coding sequence of 9112 bp, which is translated into a Most importantly, polyclonal antibodies against human protein of 2595 amino acids with a calculated molecular ABCA12 recognized both 293.3 kDa (upper) and 257 kDa weight of 293.4 kDa. ABCA12-S (NM_015657) gene consists (lower bands) under basal condition, corresponding to gene of 8097 bp, with 45 exons coding for a protein of 2277 amino products of ABCA12-L and -S, respectively (Figure 4a). As acids with a calculated molecular weight of 257 kDa (Figure expected, activation of PPAR-g by Cig increased ABCA12 3a). To determine which of these transcripts are expressed in protein expression (upper and lower band, 220 and 317%, CHK, we designed specific primer pairs (Table 1) for both respectively) in differentiated CHK (Figure 4). Furthermore, hABCA12-L and -S, and used quantitative real-time PCR to activation of PPAR-b/d by GW also caused a 175–182% measure their mRNA levels. As shown in Figure 3b, both increase in ABCA12 protein in these cells (Figure S2). Finally, transcripts are expressed at moderate levels in Ca2 þ - both Cig and GW also stimulated ABCA12 protein in differentiated CHK; following 24 hours incubation, PPARg undifferentiated CHK (data not shown). Thus, PPAR-g and activator Cig increased both transcripts by 4.4- and 5.8-fold, -b/d activators upregulate both transcripts of ABCA12 and respectively. Similarly, PPAR-b/d (GW) or LXR activator their corresponding isoforms. The function of these isoforms, (22(R)-OH-cholesterol) increased both transcripts by 2- and however, remains unknown.

106 Journal of Investigative Dermatology (2008), Volume 128 YJ Jiang et al. Upregulation of ABCA12 by PPAR and LXR in Keratinocytes

Table 1. The human primer sequence for real-time PCR Gene Primer sequence Amplicon (bp) A Accession no.

ABCA12 (F) 50-ACAGGAATGGCCTTCATCAC-30 317 AY033486 (R) 50-AACATGGTGCCCTGAGAAAC-30 ABCA12-L (F) 50-TGCAACTAACGAAGGTTTCAGA-30 116 NM_173076 (R) 50-CATCCTCATTCCACACATGC-30

ABCA12-S (F) 50-ACCTGGGATGGTTTTCACTG-30 144 NM_015657 (R) 50-TGCTTGGAGATAAGGTCTGTCC-30

Cyclophilin (F) 50-TCTCCTTTGAGCTGTTTGCAG-30 326 Y00052 (R) 50-CACCACATGCTTGCCATC-30 ABCA12, ATP-binding cassette transporter, family 12.

48 hours (high Ca2+) and secretion of LBs, which is associated with alterations in the distribution pattern of glucosylceramide (Akiyama et al., 293 kDa 2005). It is hypothesized that ABCA12 mediates the transport ABCA12 257 kDa of sphingolipids (i.e., glucosylceramide) into LB, which is critical for a competent barrier formation. Control Cig (5 M) Previously, we have shown that activation of LXR, PPARa, b 400 PPARb/d, and PPARg improves permeability barrier home- 317 ostasis (Hanley et al., 1999, 2000a, b; Komuves et al., 2000, 300 2002). This could be attributed to their effects on both the 220 protein (corneocytes) ‘‘brick’’ and the lipid ‘‘mortar’’ components of the stratum corneum. Corneocytes provide a 200 scaffold for the organization of the lipid matrix into highly 100 100 organized lammelar membranes. Activation of PPARs and (% of control) 100 ABCA12 protein LXR has been shown to stimulate keratinocyte differentiation and the formation of the cornified envelope (Hanley et al., 0 1998, 1999, 2000a, b; Komuves et al., 1999; Westergaard Control  Control M Cig (5 M) Cig (5 ) et al., 2001; Mao-Qiang et al., 2004; Schmuth et al., 2004). Upper bands Lower bands Activation of PPARs and LXR has also been shown to Figure 4. PPAR-c activation increases ABCA1 protein expression. (a) Cells stimulate LB secretion, epidermal lipid synthesis, and lipid 2 þ were incubated with vehicle or 5 mM Cig in 1.2 mM Ca medium for processing in the stratum corneum (Man et al., 2006), as well 48 hours. The whole cell extract was prepared and subjected to Western blot as stimulate SULT2B1b expression, the key enzyme in the analyses to determine ABCA12 protein as described. A representative blot is synthesis of cholesterol sulfate (Jiang et al., 2005). Together, shown. (b) The densitometry values from a typical Western blot are expressed these effects on both the bricks and mortar portions of the as percentage of control (100%), plotted, and presented as mean values (n ¼ 2 stratum corneum could account for the improvement in in each group). Similar results were obtained when the experiment was permeability barrier homeostasis induced by PPAR and LXR repeated with a different batch of cells. activator treatment. In this study, we further demonstrate that PPAR and LXR activators increase ABCA12 expression, which could facilitate lipid movement and LB formation, thereby The epidermal permeability barrier, which is a prerequisite providing another mechanism by which PPAR and LXR for terrestrial life, resides in the outermost layers of the activators promote epidermal permeability barrier home- mammalian epidermis and is comprised of lamellar mem- ostasis. branes enriched in non-polar lipids that are derived from the secretion of LB by stratum granulosum cells (Elias and MATERIALS AND METHODS Menon, 1991). Abnormal LB formation or secretion leads to Materials the failure of the formation of a functional barrier; a severe 22(R)-OH-cholesterol, clofibric acid (CLO), WY14643, all-trans- case occurs in the disease Harlequin ichthyosis. Ultra- retinoic acid, and 9-cis-retinoic acid were purchased from Sigma structurally, in cultured Harlequin ichthyosis keratinocytes, (St Louis, MO). Cig, troglitazone, and TO were purchased from ABCA12 deficiency leads to an abnormality in the formation Cayman Chemical Co. (Ann Arbor, MI). Synthetic PPAR-d activator

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GW and PPAR-g activator GI 251929X were generous gifts from Dr Statistical analysis 7 Tim Willson (GlaxoSmithKline). 1a, 25-Dihydroxyvitamin D3 was All data are expressed as mean SEM. Comparison between two purchased from BIOMOL International (Plymouth Meeting, PA). groups is undertaken using two-tail and unpaired t-test. Differences Molecular grade chemicals such as TRI Reagent were obtained from in values are considered significant if Po0.05. either Sigma or Fisher Scientific (Fairlawn, NJ). The iScriptTMcDNA Synthesis Kit for first-strand cDNA synthesis was purchased from Institutional approval BIO-RAD Laboratories (Hercules, CA). All reagents and supplies for The use of human keratinocytes and all experimental procedures were Real-time PCR were purchased from Applied Biosystems (Foster approved by the appropriate committees including the Committees of City, CA). Primary polyclonal antibody against human ABCA12 was Human Research at the University of California, San Francisco and obtained from the University of Regensburg, Germany. All other the San Francisco Veterans Affairs Medical Center. reagents for Western blot, including NuPAGEs Novex Pre-cast gradient gels (3–8% Tris-Acetate), buffers, protein standards, and CONFLICT OF INTEREST detection kits, were purchased from Invitrogen (Carlsbadm, CA). The authors state no conflict of interest.

Keratinocyte culture ACKNOWLEDGMENTS This study was supported by National Institutes of Health grants: AR050629, The second passage of human foreskin keratinocytes were seeded AR39448, and the Research Service, Department of Veterans Administration and maintained as described (Jiang et al., 2006). In a typical at San Francisco. We thank Ms Sally Pennypacker for her excellent assistance experiment, cells were treated with reagent at optimized concentra- on cell culture. tion at pre-confluence (60–70%) in either low or high calcium conditions for 24 hours, and were harvested at 80–100% confluence. SUPPLEMENTARY MATERIAL Alternatively, cells were incubated in the presence or absence of Figure S1. Activation of PPAR-b/d increases ABCA12 mRNA levels in a ligands for 4 days in high calcium medium. Control keratinocytes time- and dose-dependent manner. were treated with vehicle (0.05% ethanol or DMSO). Figure S2. PPAR-b/d activation also increases ABCA1 protein expression.

Real-time PCR REFERENCES Real-time PCR was carried out as described previously (Jiang et al., Akiyama M (2006) Pathomechanisms of Harlequin ichthyosis and ABCA 2006). Briefly, following RNA isolation, cDNA was synthesized to transporters in human diseases. Arch Dermatol 142:914–8 measure the relative mRNA levels of hABCA12 (full length), Akiyama M, Sugiyama-Nakagiri Y, Sakai K, McMillan JR, Goto M, Arita K alternative transcripts (hABCA12-L and -S). The primer sequences et al. (2005) Mutations in lipid transporter ABCA12 in Harlequin ichthyosis and functional recovery by corrective gene transfer. J Clin for PCR are listed in Table 1. A mixture of (20 ml) individual PCR Invest 115:1777–84 reaction contains 30 ng cDNA, 450–600nM forward or reverse Allikmets R, Gerrard B, Hutchinson A, Dean M (1996) Characterization of the primers and 10 mlof2 SYBR Green quantitative PCR Master Mix human ABC superfamily: isolation and mapping of 21 new genes using (BIO-RAD). The PCR reaction was performed at 501C for 2 minutes, the expressed sequence tags database. Hum Mol Genet 5:1649–55 951C for 10 minutes, and then 40 cycles of amplification of melting Allikmets R, Shroyer NF, Singh N, Seddon JM, Lewis RA, Bernstein PS et al. at 951C for 30 seconds, annealing at 601C for 30 seconds, and (1997) Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science 277:1805–7 extension at 721C for 45 seconds, respectively. The PCR reaction was performed in duplicate, with 3–4 samples in each group (n ¼ 3–4). Borst P, Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71:537–92 Experiments were repeated at least once using a different batch of Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M et al. cells to ensure reproducibility. Gel electrophoresis and melting (1999) Mutations in ABC1 in Tangier disease and familial high-density curve analyses were performed to confirm accurate PCR product lipoprotein deficiency. Nat Genet 22:336–45 sizes and absence of nonspecific bands. The expression levels of Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette each gene were normalized against cyclophilin (an invariant (ABC) transporter superfamily. Genome Res 11:1156–66

transcript) using the comparative Ct method, and expressed as Elias PM, Menon GK (1991) Structural and lipid biochemical correlates of the percentage of control, with the control as 100%. epidermal permeability barrier. Adv Lipid Res 24:1–26 Hanley K, Jiang Y, He SS, Friedman M, Elias PM, Bikle DD et al. (1998) Western blot analysis Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARalpha. J Invest Dermatol 110:368–75 Western blots were carried out according to the manufacturer’s Hanley K, Komuves LG, Bass NM, He SS, Jiang Y, Crumrine D et al. (1999) protocol. Briefly, the whole cell extract was prepared from CHK and Fetal epidermal differentiation and barrier development In vivo is 80–100 mg proteins were fractionated on pre-cast gels (3–8%) and accelerated by nuclear hormone receptor activators. J Invest Dermatol transferred to polyvinylidine difluoride membranes, overnight at 113:788–95 41C. The proteins on the membrane were subsequently probed with Hanley K, Komuves LG, Ng DC, Schoonjans K, He SS, Lau P et al. (2000a) polyclonal primary antibodies against human ABCA12 (1:1000), and Farnesol stimulates differentiation in epidermal keratinocytes via then visualized by horseradish peroxidase-conjugated anti-rabbit PPARalpha. J Biol Chem 275:11484–91 secondary antibody (1:7500) using the Enhanced Chemilumines- Hanley K, Ng DC, He SS, Lau P, Min K, Elias PM et al. (2000b) Oxysterols induce differentiation in human keratinocytes and increase Ap-1- cence (ECL) Western Blotting Detection System Kit. Membranes dependent involucrin transcription. J Invest Dermatol 114:545–53 were then exposed to CL-XPosure film. An identical blot was probed Jiang YJ, Kim P, Elias PM, Feingold KR (2005) LXR and PPAR activators with anti-GAPDH antibody to verify the equal loading of protein stimulate cholesterol sulfotransferase type 2 isoform 1b in human (data not shown). keratinocytes. J Lipid Res 46:2657–66

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