Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress

Yoshikazu Uchida,Satoru Murata,Matthias Schmuth,Martin J Behne,Jeong Deuk Lee,Shinichi Ichikawa,Peter M Elias,Yoshio Hirabayashi,Walter M Holleran

doi:10.1194/jlr.m100442-jlr200

Abstract

Ceramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GlcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GlcCer synthesis as a protective mechanism against Cer-induced stress. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50-60% decrease; P < 0.001), and increased Cer levels (6.1- to 6.8-fold; P < 0.001). Proliferation recovered to normal rates by 24 h, in parallel with increased cellular GlcCer. Both GlcCer synthesis and GlcCer synthase activity increased significantly by 8 h following SMase (8.2- and 2.4-fold, respectively; P < 0.01 each vs. control), attributed to antecedent increases in GlcCer synthase mRNA and protein expression. Further evidence that GlcCer production is responsible for normalized CHK proliferation includes: a) attenuation of SMase-induced inhibition of proliferation by exogenous GlcCer; and b) enhancement of the SMase effect in cells cotreated with the GlcCer synthase inhibitor, PDMP (D-threo-1-phenyl-2(decanoylamino)-3-morpholino-1-propanol). Finally, although proliferation in immortalized, nontransformed keratinocytes (HaCaT) also was inhibited by SMase, HaCaT cells that overexpress GlcCer synthase were resistant to this effect. Thus, SMase-induced stress initiates a response in keratinocytes that includes upregulation of GlcCer synthesis which may protect against the deleterious effects of excess Cer.

Highlights

Ceramides (Cers), critical for epidermal barrier function, can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects
Exogenous SMase inhibits DNA synthesis in cultured human keratinocyte (CHK) Previous studies have shown that either exogenous Cer administration or increased endogenous Cer resulting from inhibition of GlcCer synthase, results in a decline in CHK proliferation that reaches its nadir between 18 and 24 h [6]
As the outer integument of the mammalian organism, is exposed to myriad damaging and/or inflammatory stimuli, including many that alter Cer levels, we investigated here the role of GlcCer synthesis in the response of human keratinocytes to exogenous SMase, as a standard Cer stress model

Summary

Introduction

Ceramides (Cers), critical for epidermal barrier function, can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50–60% decrease; P Ͻ 0.001), and increased Cer levels (6.1- to 6.8-fold; P Ͻ 0.001). SMase-induced stress initiates a response in keratinocytes that includes upregulation of GlcCer synthesis which may protect against the deleterious effects of excess Cer.—Uchida, Y., S. Administration of exogenous GlcCer directly into either liver [27] or skin [29, 30] stimulates growth These studies suggest either: 1) that Cer and GlcCer exert opposing effects on cellular. Growth; or 2) that GlcCer synthesis protects against the negative consequences of increased Cer. Hirabayashi et al showed that exogenous SMase-induced increases in cellular Cer inhibit proliferation in UDP-glucose:Cer ␤-d-glucosyltransferase (GlcT-1 or GlcCer synthase)-deficient GM-95 cells, but not in GlcCer synthasereplete B16 melanoma cells [32]. Overexpression of GlcCer synthase in Jurkat cells failed to protect cells from apoptosis induced by CD95, etoposide, or ␥ irradiation [34]

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