Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis

Chujor S.N Chujor,Kenneth R Feingold,Peter M Elias,Walter M Holleran

doi:10.1016/s0022-2275(20)33889-x

Chujor S.N Chujor, Kenneth R Feingold + Show 2 more

Open Access

https://doi.org/10.1016/s0022-2275(20)33889-x

Copy DOI

Abstract

Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis. UDP-glucose:ceramide glucosyltransferase (GlcCer synthase) (EC 2.4.1.80) catalyzes the glucosylation of ceramide to form GlcCer. Recently, we demonstrated a progressive increase in GlcCer synthase expression during fetal barrier development, while others have reported increased GlcCer synthase activity with differentiation of cultured human keratinocytes. To further delineate the role of GlcCer synthase in barrier homeostasis, we determined GlcCer synthase activity and localization in hairless mouse epidermis, both under basal conditions and after acute barrier perturbation. Under basal conditions, GlcCer synthase activity localizes predominantly (∼80%) to the dithiothreitol-separated outer epidermis; i.e., 6.2 ± 0.6 versus 1.2 ± 0.1 pmol/min/mg for outer vs. lower epidermis, respectively (P < 0.0001). Although acute barrier disruption does not up-regulate epidermal GlcCer synthase activity at any time point up to 24 h, GlcCer synthase is required for barrier homeostasis: topical d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4), a specific GlcCer synthase inhibitor, applied immediately after acute barrier disruption, causes a delay in barrier recovery attributable to specific enzyme inhibition. These findings demonstrate first, that GlcCer synthase activity predominates in the outer epidermis, consistent with an increased formation of GlcCer during barrier ontogenesis and maintenance. Second, GlcCer synthase activity is required for normal permeability barrier homeostasis. Third, baseline epidermal GlcCer synthase activity appears to accommodate acute challenges to the barrier.—Chujor, C. S. N., K. R. Feingold, P. M. Elias, and W. M. Holleran. Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis.

Highlights

Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis
These results demonstrate that epidermis is highly enriched in GlcCer synthase, and that a majority of epidermal GlcCer synthase activity is associated with the outer, differentiating layers of epidermis
Two lines of evidence suggest that GlcCer synthase is important for the epidermal differentiation: 1) its activity correlates directly with the extent of differentiation of cultured human keratinocytes [11]; and 2) its activity increases in parallel with early stages of fetal barrier development [12]

Summary

Introduction

Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis. Nothing is known about either the regulation of GlcCer synthase in epidermis in relation to permeability barrier homeostasis, and direct evidence for the requirement of GlcCer synthase for normal epidermal permeability barrier homeostasis is lacking To address these points, we first localized GlcCer synthase activity in murine epidermis in relation to differentiation in vivo; second, we determined whether GlcCer synthase activity is Together with cholesterol and free fatty acids, ceramides (Cer) comprise the predominant lipid species in Abbreviations: Cer, ceramide; GlcCer, glucosylceramide; GlcCer synthase, UDP-glucose:ceramide d-glucosyltransferase; TEWL, transepidermal water loss; SPL, sphingolipids. Our findings show that GlcCer synthase activity increases with epidermal differentiation, but baseline GlcCer synthase activity apparently suffices to accommodate acute challenges to the permeability barrier; and that GlcCer synthase is important for normal permeability barrier homeostasis

Methods

Results

Conclusion