Horse-Derived Ceramide Accentuates Glucosylceramide Synthase and Ceramide Synthase 3 by Activating PPARβ/δ and/or PPARγ to Stimulate Ceramide Synthesis

Abstract

Horse-derived ceramide (HC), which contains galactosylceramides as its main component, significantly improves skin symptoms when applied topically to patients with atopic dermatitis. We speculated that efficacy resulted from the amelioration of epidermal ceramide metabolism, and we characterized those effects using reconstructed human epidermal equivalents. Lipid analysis, RT-PCR and Western blotting revealed that HC significantly increased the total ceramide content of the stratum corneum (SC), accompanied by significantly increased gene and/or protein expression levels of ceramide synthase (CERS) 3, fatty acid elongase (ELOVL) 4, glucosylceramide synthase (GCS), β-glucocerebrosidase, sphingomyelin synthase and acid sphingomyelinase. Mechanistic analyses using cultures of primary human keratinocytes revealed the marked stimulatory effects of HC on the mRNA expression levels of CERS3, ELOVL4 and GCS under high calcium-derived differentiation conditions. Signaling analyses demonstrated that an antagonist of PPARβ/δ significantly abrogated the HC-stimulated mRNA expression levels of GCS, CERS3 and ELOVL4. GW9662, an antagonist of PPARγ, significantly abolished the HC-up-regulated mRNA expression levels of GCS and ELOVL4, but not of CERS3. These findings suggest that HC has the distinct potential to accentuate the expression of GCS, CERS3 and ELOVL4 via the activation of PPARβ/δ and/or PPARγ to accelerate ceramide synthesis in the SC.

Keywords: ceramide synthase 3; fatty acid elongase 4; galactosylceramide; glucosylceramide synthase; horse-derived ceramide; keratinocytes; peroxisome proliferator activated receptors; reconstructed human epidermal equivalents; sphingomyelin synthase; β-glucocerebrosidase.

Figure 1

Residual levels of HC on RHEEs. (a) HPTLC chromatogram. (b) Levels of HC. A total of 4% HC glycerin aqueous solution (at 0, 10 and 20 mg/mL) was applied for 3 h on the surface of RHEEs, which were then washed with PBS. The RHEEs were then extracted with chloroform, methanol and PBS as described in the Section 4 Materials and Methods (Section 4.4 Evaluation of the residual level of HC applied on RHEEs) to obtain total lipids with applied and remaining HC. The total lipids were subjected to HPTLC analysis to measure the residual levels of HC in RHEEs. HC contents are expressed as µg/well. Bars represent mean ± standard deviation (SD), n = 3, Dunnett’s multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 2

Effect of HC on SC ceramide levels in RHEEs. (a) HPTLC chromatogram. (b) Levels of total ceramides. (c) Levels of ceramide species. RHEEs were treated daily with HC (at 0, 10 and 20 mg/mL) for 10 days and the SC was separated from the epidermis after which total lipids were extracted as described in the Section 4 Materials and Methods. Total lipids extracted were subjected to HPTLC analysis. Ceramide contents are expressed as µg/mg SC protein. Bars represent mean ± SD, n = 3, Dunnett’s multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 3

Effect of HC on the expression of ceramide synthesis- (a) and keratinization-related (b) genes in RHEEs. RHEEs were treated daily with HC (at 0 and 20 mg/mL) for 2 days and total RNA was extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent mean ± SD, n = 3, Student’s t-test. *: p < 0.05, **: p < 0.01.

Figure 4

Effect of HC on expression levels of ceramide synthesis- and keratinization-related proteins in RHEEs. (a) ELOVL4, (b) CERS3, (c) GCS, (d) GBA, (e) SMS1, (f) ASM, (g) INV, (h) TGM1, (i) TGM3. RHEEs were treated with HC (at 0, 10 and 20 mg/mL) for 4 days and total proteins were extracted. Protein expression levels were determined by Western blotting as described in the Section 4 Materials and Methods and are expressed as each protein level/β-actin. Bars represent means ± SD, n = 3, Dunnett’s multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 5

Effect of HC on the skin barrier function in RHEEs by TEWL analysis. RHEEs were treated with HC (at 0, 1 and 10 mg/mL) and were incubated for 6 days, and TEWL was measured at days 0, 1, 2, 4 and 6 of culturing as described in Section 4 Materials and Methods. During that period, the medium and samples were replaced every other day. Data represent the mean ± SD; n = 4. ** p < 0.01 vs. control.

Figure 6

Effect of HC on the expression of ceramide synthesis- and keratinization-related genes in NHKs under proliferating and differentiating conditions. (a) SPT1, (b) SPT2, (c) ELOVL4, (d) CERS3, (e) DESG1, (f) ASM, (g) SMS1, (h) SMS2, (i) GCS, (j) GBA, (k) aCDase, (l) INV, (m) TGM1. NHKs were treated with HC (at 0 or 200 µg/mL) for 6, 12, 24 and 48 h in low-Ca²⁺ or high-Ca²⁺ conditions, after which total RNAs were extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/GAPDH. Bars represent mean ± SD, n = 3, Tukey–Kramer multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 6

Effect of HC on the expression of ceramide synthesis- and keratinization-related genes in NHKs under proliferating and differentiating conditions. (a) SPT1, (b) SPT2, (c) ELOVL4, (d) CERS3, (e) DESG1, (f) ASM, (g) SMS1, (h) SMS2, (i) GCS, (j) GBA, (k) aCDase, (l) INV, (m) TGM1. NHKs were treated with HC (at 0 or 200 µg/mL) for 6, 12, 24 and 48 h in low-Ca²⁺ or high-Ca²⁺ conditions, after which total RNAs were extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/GAPDH. Bars represent mean ± SD, n = 3, Tukey–Kramer multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 7

Effect of HC on the mRNA expression levels of PPARs under proliferating and differentiating conditions of NHKs. (a) PPARα, (b) PPAR β/δ, (c) PPARγ. NHKs were treated with HC (at 0 or 200 µg/mL) for 6, 12, 24 and 48 h in low-Ca²⁺ or high-Ca²⁺ conditions. Total RNAs were extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/GAPDH. Bars represent mean ± SD, n = 3, Tukey–Kramer multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 8

Effect of agonists or antagonists of PPARs on the HC-stimulated expression levels of ceramide synthesis-related genes under differentiating conditions of NHKs. Agonists/antagonists of (a) PPARα, (b) PPARβ/δ, (c) PPARγ. NHKs were treated with HC (at 0 or 200 µg/mL), with or without an agonist or antagonist of PPARs for 48 h in differentiating conditions and total RNAs were extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/GAPDH. Bars represent mean ± SD, n = 3, Tukey–Kramer multiple comparison test. *: p < 0.05, **: p < 0.01.

Figure 8

Effect of agonists or antagonists of PPARs on the HC-stimulated expression levels of ceramide synthesis-related genes under differentiating conditions of NHKs. Agonists/antagonists of (a) PPARα, (b) PPARβ/δ, (c) PPARγ. NHKs were treated with HC (at 0 or 200 µg/mL), with or without an agonist or antagonist of PPARs for 48 h in differentiating conditions and total RNAs were extracted as described in the Section 4 Materials and Methods. mRNA expression levels were determined by real-time RT-PCR and are expressed as each mRNA level/GAPDH. Bars represent mean ± SD, n = 3, Tukey–Kramer multiple comparison test. *: p < 0.05, **: p < 0.01.