Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress

Ceramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GIcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GIcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GIcCer 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 GIcCer. Both GIcCer synthesis and GIcCer 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 GIcCer synthase mRNA and protein expression. Further evidence that GIcCer 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 GIcCer synthase inhibitor, PDMP (D-threo-1-phenyl-2(decanoylamino)-3-morpholinol-propanol). Finally, although proliferation in immortalized, nontransformed keratinocytes (HaCaT) also was inhibited by SMase, HaCaT cells that overexpress GIcCer synthase were resistant to this effect,(jlr) Thus, SMase-induced stress initiates a response in keratinocytes that includes upregulation of GIcCer synthesis which may protect against the deleterious effects of excess Cer.