Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes

Abstract

ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.

FIGURE 1.

The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.

FIGURE 2.

Exogenous ceramide stimulates ABCA12 mRNA. Primary cultured human keratinocytes were incubated in the presence or absence of C₆-Cer, glucosylceramide (GlcC), sphingosine (Sph), sphinganine (Sphinga), or ceramide 1-phosphate (C1P) for 16 h. ABCA12 and cyclophilin mRNA levels were then measured as described under “Experimental Procedures.” Data are expressed as a percentage of control (100%) and presented as mean ± S.E. (n = 3–4). Similar results were obtained when the experiment was repeated with a different batch of cells. **, p < 0.01; C: C₆-Cer.

FIGURE 3.

Ceramide stimulates ABCA12 mRNA expression in a time- and dose-dependent manner. Cultured human keratinocytes were incubated with C₆-Cer (5 μm) for various periods of time (0, 9, 16, 24, and 48 h) (A). Alternatively, cells were incubated with various doses of C₆-Cer (0–10 μm) for 16 h (B). Messenger RNA levels of full-length ABCA12 or cyclophilin were measured as described. Data are expressed as a percentage of vehicle control (100%) and presented as mean ± S.E. (n = 3–5). C, ABCA12 and β-actin protein levels were measured by Western blot following C₆-Cer treatment for 16 h. Similar results were obtained when the experiment was repeated with a different batch of cells. D, messenger RNA levels of ABCA12 variants (ABCA12-L and -S) were measured, expressed as a percentage of vehicle control (100%), and presented as mean ± S.E. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001; C: C₆-Cer.

FIGURE 4.

Glucosyltransferase inhibitors increase endogenous ceramide levels and induce ABCA12 mRNA. A, cultured human keratinocytes were incubated with vehicle control (open bars) or d-PPMP (10 μm; solid bars) for 6 or 24 h, and ceramide levels were determined as described under “Experimental Procedures.” Data are expressed as mean ± S.D. (n = 4). B, cells were incubated with vehicle, d-PPMP (5 μm; dotted bar), C₆-Cer-5 μm (as positive control; hatched bar), or d-PPMP plus C₆-Cer (solid bar) for 16 h. C, ceramide levels were determined after cells were incubated with vehicle, d-PPMP, C₆-Cer, or d-PPMP plus C₆-Cer for 6 h. Another set of cells were incubated with vehicle, C₆-Cer (5 μm), P4 (10 μm), or P4 plus C₆-Cer for 16 h (D). ABCA12 mRNA levels (B and D) were measured as described. Data are expressed as a percentage of control (100%) and presented as mean ± S.D. (n = 3–4). Experiments were repeated at least once with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001; C6: C₆-Cer.

FIGURE 5.

Sphingomyelin synthase inhibitors increase endogenous ceramide levels and induce ABCA12 mRNA. Cultured human keratinocytes were incubated with vehicle (open bars) or d-609 (25 μm; solid bars) for 6 or 24 h, and ceramide levels were determined (n = 4) (A). Alternatively, cells were incubated with vehicle, D609 (25 μm; dotted bar), C6–5 μm (hatched bar), or D609 plus C₆-Cer (solid bar) for 16 h. ABCA12 mRNA levels were measured as described (B). Data are expressed as a percentage of control (100%) and presented as mean ± S.E. (n = 3). Experiments were repeated at least once with similar results. *, p < 0.05; **, p < 0.01; C6: C₆-Cer.

FIGURE 6.

Ceramidase inhibitors increase endogenous ceramide levels and induce ABCA12 mRNA. A, cultured human keratinocytes were incubated with vehicle (open bars) or d-MAPP (10 μm; solid bars) for 6 or 24 h, and ceramide levels were determined (n = 4). B, cells were incubated with vehicle, d-MAPP (10 μm; dotted bar), C₆-Cer (5 μm; hatched bar), or d-MAPP plus C₆-Cer for 16 h (n = 3). C, cells were transiently transfected with specific alkaline ceramidase-1 siRNA (solid bar) or control siRNA (open bar) (n = 4). D, cells were incubated with vehicle control, B13 (5 μm), C₆-Cer (5 μm), or B13 plus C₆-Cer for 16 h (n = 3). E, cells were incubated with vehicle, B13 (2.5 μm), d-MAPP (2.5 μm), or B13 plus d-MAPP for 16 h. ABCA12 mRNA levels were measured in cells (B–E), and data are expressed as a percentage of control (100%) and presented as mean ± S.E. Experiment was repeated at least once with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001; C6: C₆-Cer.

FIGURE 7.

Exogenous C₆-Cer preferentially stimulates PPARδ expression. Cultured human keratinocytes were incubated with C₆-Cer (5 μm) or vehicle for 16 h, and mRNA levels of PPARα, PPARδ, PPARγ, LXR-α, and LXR-β were measured (A). Alternatively, cells were treated with vehicle or various doses (0, 4, 6, and 8 μm) of C₆-Cer for 24 h (B), and PPARδ mRNA levels were measured, expressed as a percentage of control (100%) and presented as mean ± S.E. (n = 4). In an another set of cells, following C₆-Cer (5 μm) or vehicle treatment for 24 or 48 h, PPARδ protein levels (C) were measured as described under “Experimental Procedures.” Experiments were repeated at least once with similar results. *, p < 0.05; **, p < 0.01. Con, control; C6: C₆-Cer.

FIGURE 8.

Increasing endogenous ceramides by inhibiting glucosylceramide synthase or ceramidase stimulate PPARδ expression. Cultured human keratinocytes were incubated with vehicle, d-PPMP (5 μm), C₆-Cer (5 μm), or d-PPMP plus C₆-Cer for 16 h (A). Alternatively, cells were incubated with vehicle, P4 (10 μm), C₆-Cer (5 μm), or P4 plus C₆-Cer in the same medium for 16 h (B). In another set of experiments, cells were incubated with vehicle, d-MAPP (10 μm), C₆-Cer (5 μm), or d-MAPP plus C₆-Cer in the same medium for 16 h (C). PPARδ mRNA levels were measured, expressed as a percentage of control (100%), and presented as mean ± S.E. (n = 3). Experiments were repeated at least once with similar results. *, p < 0.05; **, p < 0.01; C: C₆-Cer.

FIGURE 9.

Decreasing PPARδ by siRNA specifically attenuates C₆-Cer-induced ABCA12 gene expression. Cultured human keratinocytes were transiently transfected with a specific human PPARδ siRNA or negative control siRNA, followed by treatment with either vehicle or C₆-Cer (5 μm) for 24 h. Messenger RNA levels of PPARδ (A), ABCA12 (B), and involucrin (C) were measured (n = 4) as described under “Experimental Procedures.” Data are expressed as a percentage of negative control (100%) and presented as mean ± S.E. Experiments were repeated twice with similar results. **, p < 0.01; ***, p < 0.001 (versus negative control). ##, p < 0.01 (versus vehicle). NC, negative control; PPARd-si, PPARδ siRNA; INV, involucrin; C6: C₆-Cer.