Ichthyosis linked to sphingosine 1-phosphate lyase insufficiency is due to aberrant sphingolipid and calcium regulation

Abstract

Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca²⁺ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.

Keywords: calcium signaling; ceramides; differentiation; epidermis; gene set enrichment analysis; keratinocytes; lipid lamellae imbalance; organotypics; pyridoxal 5′-phosphate-dependent aldehyde lyase; sphingolipid accumulation.

Fig. 1

Loss and overexpression of SGPL1 results in a perturbance of sphingolipid species levels and significant changes in growth and migration. A: Schematic of the sphingolipid pathway where SGPL1 performs the final degradative process. B: SGPL1_Control shows a detectable band at 60 KDa, SGPL1_KO show no detectable band and SGPL1_OE show endogenous SGPL1 at 60 KDa and a band at 90 KDa representing SGPL1 linked to GFP. C: Proliferation rate measured of cells grown in serum-free media by MTT assay after 96 h. D, E: Levels of intracellular and extracellular sphingolipid intermediates in SGPL1_Control, SGPL1_KO and SGPL1_OE grown in FBS-containing media. F: Migration assay and quantification of wound gap closure (%) of cell grown in FBS media. (All statistical analyses were conducted using one-way ANOVAs with Tukey’s multiple comparison test, n = 3. Scale bar = 500 μm). G: Proliferation rates measured by MTT assay of cells grown in FBS after 96 h. SGPL1, Sphingosine 1-phosphate lyase.

Fig. 2

RNAseq analysis shows loss and overexpression of SGPL1 causes a dichotomy of gene expression. A: Principal component analysis (PCA plot) displaying the nine samples used for RNAseq analysis with samples clustered by genotype. B: Venn diagram of differentially expressed genes displaying the number of genes present in each comparison SGPL1_Control versus SGPL1_KO, SGPL1_Control versus SGPL1_OE, and SGPL1_KO versus SGPL1_OE. C: Heatmap showing the z-scores of all genes common between all three above comparisons. D, E: The top five enriched pathways in each direction for SGPL1_KO and SGPL1_OE compared to SGPL1_Control. The number of DEGs in each gene set is demonstrated on the x-axis. F: Heatmap showing the z scores of all genes in the KEGG oxidative phosphorylation. G: Dotplot showing the P values and log2Fold change of mitochondrial encoded genes in all comparisons. H: Dotplot showing the P values and log2Fold change of SGPL1_Control versus SGPL1_KO and SGPL1_Control versus SGPL1_OE of genes encoding enzymes involved in the sphingolipid metabolism pathway. I: Sphingolipid metabolism pathway with enzymes colour coded according to log2fold change of SGPL1_Control versus SGPL1_KO. Genes upregulated in SGPL1_KO are red and genes downregulated are blue. SGPL1, Sphingosine 1-phosphate lyase.

Fig. 3

Loss and overexpression of SGPL1 causes a dichotomy in growth rate and overexpression results in a lower differentiated state. A: Heatmap showing the z scores of all genes in the “GO Keratinocyte Differentiation” gene set. B: Western blot of terminal differentiation marker loricrin of cell lysates extracted from cells grown in monolayer in FBS media. C: Densitometry analysis of loricrin Western blot. (One-way ANOVA with Tukey’s multiple comparison test, n = 3). D: Heatmap showing the z scores of all genes in the “KEGG Calcium Pathway” gene set. SGPL1, Sphingosine 1-phosphate lyase.

Fig. 4

SGPL1_KO models present with a thickened stratum corneum and increased differentiation. A: Representative H&E-stained images of SGPL1_Control and SGPL1_KO 3D organotypic models. Total epidermal and stratum corneum thickness were measured in μm using ImageJ. Scale bar = 50 μm. B: Immunofluorescent staining of organotypic models for differentiation markers KRT14, KRT10, involucrin, loricrin, and filaggrin. Images were captured at the same exposure and gain for comparison. Scale bar = 50 μm. (Statistical analysis was conducted using unpaired t test, n = three-fourths, ∗P < 0.05.). SGPL1, Sphingosine 1-phosphate lyase.

Fig. 5

SGPL1_KO organotypic models display abnormal intercellular junctions. Immunofluorescent staining of SGPL1_Control and SGPL1_KO 3D organotypic models for junctional marker E-cadherin. Scale bar = 50 μm. SGPL1, Sphingosine 1-phosphate lyase.

Supplemental Figure S2