Introduction

ATP-binding cassettes (ABC) are a large group of proteins that mediate the transport of a variety of different substrates across cellular membranes. These proteins contain two transmembrane domains and two ATP-binding domains or nucleotide-binding folds. Forty-eight ABC genes have been identified to date, which have been further divided into seven subfamilies, based on sequence homology and organization of the nucleotide-binding folds (Allikmets et al., 1996; Dean et al., 2001; Borst and Elferink, 2002; Akiyama et al., 2005). The ABCA subfamily comprises 12 full transporters and one pseudogene (ABCA11), and all mediate lipid transport (Schmitz et al., 2000). Most importantly, mutations in ABCA proteins have been implicated in several autosomal recessive disorders of lipid metabolism, including ABCA1 in Tangier disease (Brooks-Wilson et al., 1999), ABCA4 in Stargardt disease (Allikmets et al., 1997), and ABCA3 in fatal surfactant deficiency in newborns (Shulenin et al., 2004).

ABCA12 is a member of the ABCA subfamily (Akiyama, 2006). The functional importance of ABCA12 was revealed when a missense mutation was first reported to cause type 2 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 fatal in most affected newborns (Lefevre et al., 2003; Kelsell et al., 2005). Evidence from morphological and biochemical studies indicate that ABCA12 is important for lipid (glucosylceramide) loading into lamellar body (LB) and its absence results in a severe abnormality in permeability barrier formation and extraordinary, intrauterine thickening of the stratum corneum, ultimately resulting in high rates of prenatal mortality (Akiyama et al., 2005). Strikingly, genetic correction of ABCA12 deficiency in patients' keratinocytes by gene transfer normalized loading of glucosylceramides into LB (Akiyama et al., 2005). Glucosylceramides are major precursors of ceramides, which comprises 50% of the lipid that forms the barrier (Uchida et al., 2000). Moreover, as protein loading into LB is dependent on prior or concurrent lipid loading (Rassner et al., 1999), the failure to deliver lipids may result in a deficiency in proteases, accounting in part for the severe keratinizing disorder. Together, these studies strongly indicate a critical role for ABCA12 in epidermal physiology, specifically, the formation of LB and permeability barrier homeostasis. Hence, understanding how this protein is regulated could be great importance. Currently, however, the factors that regulate ABCA12 expression in keratinocytes remain unknown, although ABCA12 expression in cultured human keratinocyte (CHK) increases with keratinocyte differentiation (Akiyama et al., 2005).

Previously, we and others have demonstrated that peroxisome proliferators-activated receptor (PPAR)-, -/, -, as well as liver X receptor (LXR)- and -, are expressed in murine epidermis and human keratinocytes (Hanley et al., 2000a, 2000b; Westergaard et al., 2001; Komuves et al., 2002). Activation of PPARs and LXRs have numerous differentiation-related effects on keratinocytes, including (1) stimulating corneocyte envelope precursor expression (Hanley et al., 1998, 1999, 2000b); (2) increasing the expression of Sult2B1b, which catalyzes the synthesis of cholesterol sulfate (Jiang et al., 2005); (3) stimulating epidermal lipid synthesis (Man et al., 2006); (4) acceleration of LB secretion (Man et al., 2006); and (5) improving epidermal permeability barrier homeostasis (Hanley et al., 1998, 1999, 2000a, 2000b; Komuves et al., 2000, 2002; Jiang et al., 2005; Man et al., 2006). Very recently, we demonstrated that ABCA1, a member of ABCA family that transports cholesterol and phospholipids out of cells (Schmitz and Langmann, 2005), is not only expressed in CHK and murine epidermis, but also markedly stimulated by LXR, PPAR-, PPAR-/ activation (Jiang et al., 2006). Based on these findings, we hypothesized that activation of PPARs and LXR may also stimulate ABCA12 expression in keratinocytes.

Results and Discussion

Initially, we examined the effect of the activation of LXR, PPARs, RAR, RXR, and vitamin D receptor (VDR) on ABCA12 mRNA levels. The optimized dose for each activator was determined, as described previously (Jiang et al., 2005). As shown in Figure 1a, ABCA12 mRNA levels increased markedly following treatment with PPAR- activators (ciglitazone (Cig): 11.8-fold; troglitazone: 12-fold; GI 251929X: 6.4-fold) and PPAR-/ activator (GW610742 (GW): 5.1-fold), after 24 hours incubation. Furthermore, the upregulation of ABCA12 by PPAR- (Cig) occurred rapidly, was sustained over an extend period of time (Figure 2a), and occurred in a dose-dependent manner (Figure 2b). Similar time- and dose-dependent upregulation of ABCA12 mRNA was induced by PPAR-/ (GW) activation (Figure S1). In addition, although LXR activators modestly increased ABCA12 mRNA level (TO901317 (TO): 3.3-fold), activators of PPAR-, RAR, RXR, or vitamin D receptor had no effect (Figure 1a). As previous studies showed that keratinocyte differentiation induced by calcium stimulate ABCA12 mRNA expression after 4 days incubation (Akiyama et al., 2005), we next assessed whether PPAR and LXR activators would further increase ABCA12 mRNA levels in differentiated CHK, induced by high calcium (1.2 mM). As shown in Figure 1b, although high calcium itself did not induce significant amount of ABCA12 mRNA, ligand activation of either PPAR- or -/ increased ABCA12 mRNA levels by 11- and 4.5-fold, respectively, even in high calcium. Similarly, LXR activation by TO increased ABCA12 mRNA level by 2.3-fold (P<0.01). Consistent with the observations in undifferentiated CHK (above), PPAR- activation had no effect on ABCA12 mRNA (Figure 1b). Furthermore, ligand treatment (5 m Cig or 8 m GW) of CHK for 4 days in high calcium medium increased, ABCA12 mRNA 2.2- and 1.8-fold, respectively, compared with high calcium alone (data not shown). Thus, under our experimental condition, certain PPAR and LXR activators stimulate ABCA12 mRNA expression, independent of keratinocyte differentiation.

Figure 1.

Activation of PPAR-, PPAR-/, and LXR increase ABCA12 mRNA expression in CHK. (a) Cells were incubated with either vehicle control, or activators of LXR (10 M 22R or 10 M TO); PPAR- (7.5 M Cig, or 7.5 M troglitazone (Tro), or 10 M GI); PPAR-/ GW (8 M); PPAR (20 M WY14643 or 400 M CLO); RAR (1 M all-trans retinoic acid); RXR (1 M 9-cis-retinoic acid); vitamin D receptor (1, 25-dihydroxyvitamin D₃ 0.1 M) in low calcium medium for 24 hours (h). (b) Alternatively, cells were incubated in either 0.03 mM Ca²⁺ medium, or 1.2 mM Ca²⁺ medium alone, or 1.2 mM Ca²⁺ medium with 10 M TO, or 5 M Cig, or 8 M GW, or 20 M WY14643 for 24 hours. Real-time PCR was performed to measure mRNA levels of ABCA12 and cyclophilin (internal control), as described in "Materials and Methods". Data are expressed as percentage of control (100%) and presented as meanSEM (n=3 in each group). Similar results were obtained when the experiment was repeated with a different batch of cells. ^**P<0.01; ^***P<0.001.

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Figure 2.

Activation of PPAR- increases ABCA12 mRNA level in a time- and dose-dependent manner. (a) Cells were incubated with 5 M Cig for various periods of time (0, 6, 16, 24, and 48 h) in 0.03 mM Ca²⁺ medium. (b) Alternatively, cells were incubated with Cig at various concentrations (0, 2.5, 5.0, 7.5, 10 M) in the same medium for 24 hours. ABCA12 and cyclophilin mRNA levels were measured as described. Data are expressed as percentage of vehicle control (100%) and presented as meanSEM (n=3 in each group). Similar results were obtained when the experiment was repeated with a different batch of cells. ^*P<0.05; ^**P<0.01; ^***P<0.001.

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Previously, we reported that three alternate variants of 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 recently been described (Lefevre et al., 2003), which were assigned as hABCA12-L and hABCA12-S in this study (Figure 3a). ABCA12-L (NM_173076) gene consists of 53 exons with a coding sequence of 9112 bp, which is translated into a protein of 2595 amino acids with a calculated molecular weight of 293.4 kDa. ABCA12-S (NM_015657) gene consists of 8097 bp, with 45 exons coding for a protein of 2277 amino acids with a calculated molecular weight of 257 kDa (Figure 3a). To determine which of these transcripts are expressed in CHK, we designed specific primer pairs (Table 1) for both hABCA12-L and -S, and used quantitative real-time PCR to measure their mRNA levels. As shown in Figure 3b, both transcripts are expressed at moderate levels in Ca²⁺-differentiated CHK; following 24 hours incubation, PPAR activator Cig increased both transcripts by 4.4- and 5.8-fold, respectively. Similarly, PPAR-/ (GW) or LXR activator (22(R)-OH-cholesterol) increased both transcripts by 2- and 2.5-fold, respectively (Figure 3b). Again, activation of PPAR- had no effect on either transcript (Figure 3b). When undifferentiated keratinocytes were used, similar results were obtained (data not shown).

Figure 3.

Alternate transcripts of ABCA12 are expressed in CHK and upregulated by PPAR-, PPAR-/, and LXR activators. (a) Genomic structure of human ABCA12 locus and ABCA12 isoforms. Two major hABCA12 cDNAs, ABCA12-L and -S, are transcribed by alternative usage of two transcription initiation start sites, TISS1 and TISS2. Exon 1–8 are found only in ABCA12-L transcript. Two major human ABCA12 protein isoforms are synthesized accordingly. ABCA12-L and -S proteins share a large common C-terminus, however, with a unique 328 amino acids (aa) or 10 aa at N-terminus, respectively. (b) Cells were incubated in either 0.03 mM or 1.2 mM Ca²⁺ medium alone, or 1.2 mM Ca²⁺ medium with 10 M TO, or 5 M Cig, or 8 M GW, or 20 M WY14643 for 24 hours. Human ABCA12-L, -S, and cyclophilin mRNA levels were determined by real-time PCR. Under our experimental conditions, the absolute C_t 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 presented as meanSEM (n=6 in each group). ^**P<0.01; ^***P<0.001.

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Table 1 - The human primer sequence for real-time PCR.

Full table

Most importantly, polyclonal antibodies against human ABCA12 recognized both 293.3 kDa (upper) and 257 kDa (lower bands) under basal condition, corresponding to gene products of ABCA12-L and -S, respectively (Figure 4a). As expected, activation of PPAR- by Cig increased ABCA12 protein expression (upper and lower band, 220 and 317%, respectively) in differentiated CHK (Figure 4). Furthermore, activation of PPAR-/ by GW also caused a 175–182% increase in ABCA12 protein in these cells (Figure S2). Finally, both Cig and GW also stimulated ABCA12 protein in undifferentiated CHK (data not shown). Thus, PPAR- and -/ activators upregulate both transcripts of ABCA12 and their corresponding isoforms. The function of these isoforms, however, remains unknown.

Figure 4.

PPAR- activation increases ABCA1 protein expression. (a) Cells were incubated with vehicle or 5 M Cig in 1.2 mM Ca²⁺ medium for 48 hours. The whole cell extract was prepared and subjected to Western blot analyses to determine ABCA12 protein as described. A representative blot is shown. (b) The densitometry values from a typical Western blot are expressed as percentage of control (100%), plotted, and presented as mean values (n=2 in each group). Similar results were obtained when the experiment was repeated with a different batch of cells.

Full figure and legend (58K)

The epidermal permeability barrier, which is a prerequisite for terrestrial life, resides in the outermost layers of the mammalian epidermis and is comprised of lamellar membranes enriched in non-polar lipids that are derived from the secretion of LB by stratum granulosum cells (Elias and Menon, 1991). Abnormal LB formation or secretion leads to the failure of the formation of a functional barrier; a severe case occurs in the disease Harlequin ichthyosis. Ultra-structurally, in cultured Harlequin ichthyosis keratinocytes, ABCA12 deficiency leads to an abnormality in the formation and secretion of LBs, which is associated with alterations in the distribution pattern of glucosylceramide (Akiyama et al., 2005). It is hypothesized that ABCA12 mediates the transport of sphingolipids (i.e., glucosylceramide) into LB, which is critical for a competent barrier formation.

Previously, we have shown that activation of LXR, PPAR, PPAR/, and PPAR improves permeability barrier homeostasis (Hanley et al., 1999, 2000a, 2000b; Komuves et al., 2000, 2002). This could be attributed to their effects on both the protein (corneocytes) "brick" and the lipid "mortar" components of the stratum corneum. Corneocytes provide a scaffold for the organization of the lipid matrix into highly organized lammelar membranes. Activation of PPARs and LXR has been shown to stimulate keratinocyte differentiation and the formation of the cornified envelope (Hanley et al., 1998, 1999, 2000a, 2000b; Komuves et al., 1999; Westergaard et al., 2001; Mao-Qiang et al., 2004; Schmuth et al., 2004). Activation of PPARs and LXR has also been shown to stimulate LB secretion, epidermal lipid synthesis, and lipid processing in the stratum corneum (Man et al., 2006), as well as stimulate SULT2B1b expression, the key enzyme in the synthesis of cholesterol sulfate (Jiang et al., 2005). Together, these effects on both the bricks and mortar portions of the stratum corneum could account for the improvement in permeability barrier homeostasis induced by PPAR and LXR 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 providing another mechanism by which PPAR and LXR activators promote epidermal permeability barrier homeostasis.

Materials and Methods

Materials

22(R)-OH-cholesterol, clofibric acid (CLO), WY14643, all-trans-retinoic acid, and 9-cis-retinoic acid were purchased from Sigma (St Louis, MO). Cig, troglitazone, and TO were purchased from Cayman Chemical Co. (Ann Arbor, MI). Synthetic PPAR- activator GW and PPAR- activator GI 251929X were generous gifts from Dr Tim Willson (GlaxoSmithKline). 1, 25-Dihydroxyvitamin D₃ was purchased from BIOMOL International (Plymouth Meeting, PA). Molecular grade chemicals such as TRI Reagent were obtained from either Sigma or Fisher Scientific (Fairlawn, NJ). The iScript^TMcDNA Synthesis Kit for first-strand cDNA synthesis was purchased from BIO-RAD Laboratories (Hercules, CA). All reagents and supplies for Real-time PCR were purchased from Applied Biosystems (Foster City, CA). Primary polyclonal antibody against human ABCA12 was obtained from the University of Regensburg, Germany. All other reagents for Western blot, including NuPAGE^® Novex Pre-cast gradient gels (3–8% Tris-Acetate), buffers, protein standards, and detection kits, were purchased from Invitrogen (Carlsbadm, CA).

Keratinocyte culture

The second passage of human foreskin keratinocytes were seeded and maintained as described (Jiang et al., 2006). In a typical experiment, cells were treated with reagent at optimized concentration at pre-confluence (60–70%) in either low or high calcium conditions for 24 hours, and were harvested at 80–100% confluence. Alternatively, cells were incubated in the presence or absence of ligands for 4 days in high calcium medium. Control keratinocytes were treated with vehicle (0.05% ethanol or DMSO).

Real-time PCR

Real-time PCR was carried out as described previously (Jiang et al., 2006). Briefly, following RNA isolation, cDNA was synthesized to measure the relative mRNA levels of hABCA12 (full length), alternative transcripts (hABCA12-L and -S). The primer sequences for PCR are listed in Table 1. A mixture of (20 l) individual PCR reaction contains 30 ng cDNA, 450–600 nM forward or reverse primers and 10 l of 2 SYBR Green quantitative PCR Master Mix (BIO-RAD). The PCR reaction was performed at 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of amplification of melting at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds, respectively. The PCR reaction was performed in duplicate, with 3–4 samples in each group (n=3–4). Experiments were repeated at least once using a different batch of cells to ensure reproducibility. Gel electrophoresis and melting curve analyses were performed to confirm accurate PCR product sizes and absence of nonspecific bands. The expression levels of each gene were normalized against cyclophilin (an invariant transcript) using the comparative C_t method, and expressed as percentage of control, with the control as 100%.

Western blot analysis

Western blots were carried out according to the manufacturer's protocol. Briefly, the whole cell extract was prepared from CHK and 80–100 g proteins were fractionated on pre-cast gels (3–8%) and transferred to polyvinylidine difluoride membranes, overnight at 4°C. The proteins on the membrane were subsequently probed with polyclonal primary antibodies against human ABCA12 (1:1000), and then visualized by horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:7500) using the Enhanced Chemiluminescence (ECL) Western Blotting Detection System Kit. Membranes were then exposed to CL-XPosure film. An identical blot was probed with anti-GAPDH antibody to verify the equal loading of protein (data not shown).

Statistical analysis

All data are expressed as meanSEM. Comparison between two groups is undertaken using two-tail and unpaired t-test. Differences in values are considered significant if P<0.05.

Institutional approval

The use of human keratinocytes and all experimental procedures were approved by the appropriate committees including the Committees of Human Research at the University of California, San Francisco and the San Francisco Veterans Affairs Medical Center.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

This study was supported by National Institutes of Health grants: AR050629, AR39448, and the Research Service, Department of Veterans Administration at San Francisco. We thank Ms Sally Pennypacker for her excellent assistance on cell culture.

Supplementary Material

Figure S1. Activation of PPAR-/ increases ABCA12 mRNA levels in a time- and dose-dependent manner.

Figure S2. PPAR-/ activation also increases ABCA1 protein expression.