Abstract
β-Sitosterol 3-O-d-glucoside (BSG) is known to act as an agonist by binding to estrogen receptors, and estrogen has been reported to enhance the activity of β-glucocerebrosidase, an epidermal ceramide metabolizing enzyme. In this study, we determined whether BSG up-regulates ceramide levels in the stratum corneum (SC) of a reconstructed human epidermal keratinization (RHEK) model. Treatment with BSG significantly increased the total ceramide content by 1.2-fold compared to that in the control in the SC of the RHEK model, accompanied by a significant increase of the ceramide species, Cer[EOS] by 2.1-fold compared to that in the control. RT-PCR analysis demonstrated that BSG significantly up-regulated the mRNA expression levels of serine palmitoyltransferase (SPT)2, ceramide synthase (CerS)3, glucosylceramide synthase (GCS) and acid sphingomyelinase by 1.41–1.89, 1.35–1.44, 1.19 and 2.06-fold, respectively, compared to that in the control in the RHEK model. Meanwhile, BSG significantly down-regulated the mRNA expression levels of sphingomyelin synthase (SMS)2 by 0.87–0.89-fold. RT-PCR analysis also demonstrated that BSG significantly up-regulated the mRNA expression levels of CerS3 and GCS by 1.19–1.55 and 1.20-fold, respectively, but not of SPT2 and significantly down-regulated that of SMS2 by 0.74-fold in HaCaT keratinocytes. Western blotting analysis revealed that BSG significantly increased the protein expression levels of CerS3 and GCS by 1.78 and 1.28–1.32-fold, respectively, compared to that in the control in HaCaT cells. These findings indicate that BSG stimulates ceramide synthesis via the up-regulated expression levels of CerS3 and GCS in the glucosylceramide pathway, which results in a significantly increased level of total ceramides in the SC accompanied by significantly increased levels of acylceramide species such as Cer[EOS].
Highlights
Ceramides are comprised of sphingolipids consisting of a sphingoid base and a saturated fatty acid moiety
Effects of β-sitosterol 3-O-D-glucoside (BSG) on mRNA levels of ceramide synthesis associated enzymes in reconstructed human epidermal keratinization (RHEK) models When RHEK models were treated with BSG for 2 and 4 days, the mRNA expression levels of SPT2, CerS3, glucosylceramide synthase (GCS) and acid sphingomyelinase (ASM) were significantly increased by BSG at 1 and 10, at 1 and 10, at 10 and at 10 μg/mL after 2 or 4, 4, 4 and 4 days of culture, respectively (Fig 3A, 3B, 3C and 3F)
As for the stratum corneum (SC) ceramide level, we found for the first time that BSG significantly increases the total SC ceramide level in the RHEK model in concert with a significantly increased level of acylceramide type Cer[EOS]
Summary
Ceramides are comprised of sphingolipids consisting of a sphingoid base and a saturated fatty acid moiety. Ceramides are the dominant intercellular lipid in the stratum corneum (SC) and together with other lipids such as free fatty acids and cholesterol ester, they play essential roles as a water reservoir and as a barrier [1]. At this time, 12 major classes of ceramide profiles have been found in human SC [2]. Ceramides in the SC are produced via sequential enzymatic reactions of several sphingolipid metabolizing enzymes such as serine palmitoyltransferase (SPT), ceramide synthase (CerS), glucosylceramide synthase (GCS), sphingomyelin synthases (SMS), β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM). Ceramides are subsequently hydrolyzed by acid ceramidase to yield sphingosine, the deficiency of which is predominantly associated with the Staphylococcus aureus colonization frequently observed in the SC of patients with atopic dermatitis (AD) [9]
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