Endogenous β-glucocerebrosidase activity in Abca12⁻/⁻epidermis elevates ceramide levels after topical lipid application but does not restore barrier function

Abstract

ABCA12 mutations disrupt the skin barrier and cause harlequin ichthyosis. We previously showed Abca12(-/-) skin has increased glucosylceramide (GlcCer) and correspondingly lower amounts of ceramide (Cer). To examine why loss of ABCA12 leads to accumulation of GlcCer, de novo sphingolipid synthesis was assayed using [(14)C]serine labeling in ex vivo skin cultures. A defect was found in β-glucocerebrosidase (GCase) processing of newly synthesized GlcCer species. This was not due to a decline in GCase function. Abca12(-/-) epidermis had 5-fold more GCase protein (n = 4, P < 0.01), and a 5-fold increase in GCase activity (n = 3, P < 0.05). As with Abca12(+/+) epidermis, immunostaining in null skin showed a typical interstitial distribution of the GCase protein in the Abca12(-/-) stratum corneum. Hence, we tested whether the block in GlcCer conversion could be circumvented by topically providing GlcCer. This approach restored up to 15% of the lost Cer products of GCase activity in the Abca12(-/-) epidermis. However, this level of barrier ceramide replacement did not significantly reduce trans-epidermal water loss function. Our results indicate loss of ABCA12 function results in a failure of precursor GlcCer substrate to productively interact with an intact GCase enzyme, and they support a model of ABCA12 function that is critical for transporting GlcCer into lamellar bodies.

Keywords: ABCA12 antibody; glucosylceramide; harlequin ichthyosis; skin permeability barrier.

Fig. 1.

Synthesis and metabolic conversion of ceramides and glucosylceramides in the epidermis. Nucleated keratinocytes synthesize the precursors GlcCer and SM and store these lipids in lamellar bodies. Upon release into the interstices of the stratum corneum, they are hydrolyzed to Cer by GCase or sphingomyelinase as part of the process that forms the interstitial lamellar lipid domains that surround the corneocytes of the stratum corneum. GlcCer synthase, glucosylceramide synthase.

Fig. 2.

ABCA12 protein is largely restricted to the epidermis. A: Tissue expression of ABCA12 mRNA in the adult mouse as determined by reverse transcription and quantitative PCR (RT-QPCR) (± SD, n = 3). B: A newly produced rabbit anti-ABCA12 polyclonal antibody specifically recognizes ABCA12 in the epidermis of neonatal mice and does not cross-react with other ABCA transporters transiently expressed in HEK 293 cells, including ABCA1, ABCA2, ABCA3, and ABCA7. Panels show signals derived from one immune-blot sequentially probed for the indicated transporters, involucrin and β-actin. C, ABCA12 expression in E18.5 embryos is largely restricted to the epidermis, though expression in the stomach was detected at markedly lower levels (13). D: Histological skin section stained with H and E, depicting the epidermal structures of E18.5 embryos used in our experiments. E: Histological section of the stomach stained with H and E identifying the hyperkeratosis of the gastric mucosa in the forestomach region in Abca12^−/− animals (arrowheads point to the keratosis in the Abca12^−/− tissues. L, lumen; SB, stratum basale; SQ, stratified squamous epithelium; SS, stratum spinosum).

Fig. 3.

Alteration of endogenous sphingolipid levels in the Abca12^−/− epidermis. A: Shown are endogenous lipids levels detected in extracts of Abca12^+/+ and Abca12^−/− day 18.5 embryonic skin cultures analyzed by copper charring of TLC plates. Lipids labeled “a” to “d” mark the most altered sphingolipid species between the genotypes. These were isolated by preparative TLC from additional Abca12^+/+ and Abca12^−/− day 18.5 embryonic epidermis and subjected to quantitative mass spectrometry. B–D: Graphed are the three most predominate species identified for each of the analyzed samples (n = 5, ± SD, *P < 0.05). The nomenclature, e.g., d18:1(50:2 ester), indicates a sphingosine base (d18:1) in an amide linkage to a very long chain fatty acid, which is ester-linked to another fatty acid; these two acyl chains contain a total of 50 carbons and two bonds. Previous data indicate that 50:2 is a 32-carbon saturated fatty amide linked to linoleic acid (18:2) (13).

Fig. 4.

Loss of ABCA12 activity causes a defect in the GCase-mediated conversion of de novo synthesized GlcCer to Cer. Abca12^+/+ and Abca12^−/− day 18.5 embryonic skin cultured in the presence of [¹⁴C]serine were untreated or treated with either an inhibitor of GCase (CBE, 100 µM) or glucosylceramide synthase (PDMP, 30 µM). Lipids from the isolated epidermis were separated on TLC plates. A: TLC plates of [¹⁴C] serine-labeled lipids are representative samples. B: Graphed is the amount of labeled lipid quantitated by phosphor imaging and normalized to the amount of labeled phosphatidylethanolamine (n = 3, ± SD, *P < 0.05 control versus CBE- or PDMP-treated, ^%P < 0.05 control Abca12^+/+ versus control Abca12^−/−).

Fig. 5.

GCase protein levels and activity are increased in the Abca12^−/− epidermis. A: GCase protein levels are significantly elevated in the epidermis of Abca12^−/− day 18.5 embryos. Left panels show immunoblots of GCase, β-actin, and serine palmitoyltransferase (SPTLC1) of a representative sample; the graph depicts the GCase/β-actin ratio of the Abca12^+/+ and Abca12^−/− samples (n = 4, ± SD, P < 0.01). B: Gba1 mRNA levels in the epidermis of Abca12^−/− day 18.5 embryos are not significantly different from those of Abca12^+/+ mice as determined by RT-QPCR; graphed is the GCase/β-actin ratio expressed relative to that of the Abca12^+/+ samples (n = 4, ± SD, P = 0.7). C: Immunostaining of GCase protein in day 18.5 embryo skin cross-sections. The brown GCase staining is markedly increased in the Abca12^−/− epidermis, particularly in the upper SC (bar = 30 µm; SB, stratum basale; SS, stratum spinosum). D: GCase activity measured in situ on whole-skin samples (n = 3, ± SD, P < 0.01). E: GCase-specific activity measured in vitro using SC lysates (Abca12^+/+ n = 4, Abca12^−/− n = 3, ± SD, P < 0.05).

Fig. 6.

Topical application of GlcCer precursors to the SC restores ceramide production. [¹⁴C]GlcCer was applied onto the SC of Abca12^+/+ and Abca12^−/− whole-skin samples obtained from day 18.5 mouse embryos. After 1 h incubation at room temperature followed by an overnight incubation at 4°C, total lipids were extracted. Ceramides were separated from their unhydrolyzed precursors using a silica gel column. An aliquot was quantitated by liquid scintillation counting (A). Values at the top of the bars represent the average percentage of substrate converted into ceramides (n = 3, ± SD, P < 0.05). B: Shown are the converted ceramides and unhydrolyzed precursors resolved on a TLC plate and visualized by phosphor imaging. The “Ctrl” lane shows a wild-type skin which was processed immediately after addition of the radioactive substrate to control for hydrolysis of the substrate during the lipid extraction. C: TEWL measurements of E18.5 Abca12^−/− skins grown on an air/liquid interface and treated with an acute dose of GlcCer for the indicated periods did not show a significant improvement of the skin permeability barrier function (n = 6 for times 12 and 24 h, n = 2 for times 6 and 48 h).