Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis

Abstract

Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.

Figure 1. Role of keratinocyte-intrinsic Rabgef1 in health and skin homeostasis.

(A) Specificity and efficiency of Cre-mediated Rabgef1 deletion assessed by single-cell PCR; numerals indicate the number of single cells analyzed, and results are pooled from 2 separate experiments. (B–G) Comparison between Rabgef1^K-KO and control mice. (B) Representative RABGEF1 staining of back skin sections from adult mice. (C) Survival (n = 9 and 29, respectively) and (D) body weight (n = 8 and 18, respectively) over time in Rabgef1^K-KO and control mice. (E) Representative photographs at day 1–2, day 7–9, and day 49–56 after birth. (F) Representative H&E staining of back skin sections. (G) Bars show quantification of dermal and epidermal thickness (n = 5–7 mice per group). (H) Experimental outline for tamoxifen-induced Rabgef1 deletion in Rabgef1^KERT2-KO mice. (I–L) Comparison between tamoxifen-treated Rabgef1^fl/fl and Rabgef1^KERT2-KO mice. Representative H&E staining (I) and RABGEF1 staining (J) of back skin sections. (K) Bars show quantification of dermal and epidermal thickness (n = 7–8 mice per group, 2 independent experiments). (L) Bacterial CFU counts per cm² of skin area (n = 9–12 per group, 3 independent experiments). (B, E, F, and I) Pictures are representative of more than 5 samples analyzed per group, and (I and J) 3 independent experiments, each giving similar results. (G, K, and L) Data shown are mean ± SEM, as well as individual values. (B, F, I, and J) Dashed lines identify the dermal-epidermal junction. P values were calculated by Mantel-Cox test (C), 1-way ANOVA (D), 2-way ANOVA with Bonferroni’s test for multiple comparisons (G and K), or Mann-Whitney test (L). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm; original magnification in B and I, ×20; NS, not significant.

Figure 2. Epidermal abnormalities and barrier dysfunction in mice with deletion of RABGEF1 in keratinocytes.

(A–E) Comparison of Rabgef1^K-KO and control mice. (A) Representative H&E staining of back skin sections. Arrowheads indicate spongiosis. (B and D) Representative confocal microscopy pictures of back skin sections with the indicated staining. (C and E) Bars show protein and mRNA levels of the indicated molecules whose staining is depicted in B and D, as assessed by confocal microscopy and RT-qPCR analyses, respectively (n = 4–6 mice per group). (F) Experimental outline for assessment of epidermal barrier function in tamoxifen-treated Rabgef1^KERT2-KO mice. (G) TEWL measurements; results are pooled from 2 independent experiments (n = 6–12 per group). (H) Percentage of dermal DCs containing processed OVA (as assessed by fluorescence in green and red) 30 hours after OVA-DQ skin patching; results are pooled from 3 independent experiments (n = 6–8 per group). (A, B, and D) Pictures are representative of 4 or 5 samples (from 4–5 different mice) per group, each giving similar results. (C, E, G, and H) Results are shown as mean ± SEM, and G and H also show individual values. (A, B, and D) Dashed lines identify the dermal-epidermal junction. P values were calculated by 2-tailed unpaired Student’s t test (C and E) and 1-way ANOVA followed by Tukey’s multiple comparison tests (G and H). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm; original magnification, ×20 (B) or ×63 (D); TEWL, transepidermal water loss; NS, not significant.

Figure 3. Type 2 cutaneous inflammation and elevated serum IgE in adult Rabgef1^K-KO mice.

(A–H) Rabgef1^K-KO and control mice are compared. (A) Representative TSLP staining of back skin sections. (B) Assessment of dermal MCs by toluidine blue (left panel) or avidin staining (right panel) (82). (C) Representative H&E staining of back skin sections (different areas of the sections in Figure 2A); arrows indicate eosinophils. (D) Representative dot plot of dermal CD45⁺ leukocytes, CD4⁺ T cells, SSC^hi eosinophils (also positive for SiglecF), and Ly6G(Gr-1)⁺CD11b⁺ neutrophils; bar graphs show quantification of the percentage of cells (n = 6–8 per group). (E) Representative pictures of skin-draining lymph nodes (DLNs); bar graphs show quantification of LN cell numbers (n = 8–20 mice per group). (F and G) Assessment of IgE levels in the serum (n = 4–10 mice per group) (F) and on the surface of blood basophils (G); bar graph in G shows quantification of the percentage increase in mean fluorescence intensity (MFI) versus control mice (n = 4–10 per group). (H) RT-qPCR analysis of the selected genes (n = 3 mice per group). (I) Experimental outline. (J) Representative dot plots depicting intracellular protein expression of GATA-3, T-bet, Foxp3, and ROR-γt within living CD4⁺ T cells. Insets show percentage of cells in the gated subpopulation. (K) Bars show quantification of the numbers of CD4⁺ T cell subsets in the dermis of the ear pinnae (left) and in the DLN (right) (n = 5–6 mice per group, 2 independent experiments). (A–C) Pictures are representative of 4 or 5 samples per group, each giving similar results. (D–H and K) Results are shown as mean ± SEM, and F, G, and K also show individual values. (A–C) Dashed lines identify the dermal-epidermal junction. P values were calculated by 2-tailed unpaired Student’s t test (E–G) and 2-way ANOVA followed by Bonferroni’s test for multiple comparisons (H and K). **P < 0.01; ***P < 0.001. Scale bars: 50 μm; original magnification in A and B, ×20; NS, not significant.

Figure 4. Increased activation of the IL-1R/MYD88/NF-κB pathway in RABGEF1-deficient keratinocytes.

(A) Differentially expressed (DE) genes between adult Rabgef1^K-KO and control mouse back skin specimens (n = 2 analyses per group) showing DE genes in red/blue. Volcano plot: 760 = total number of DE genes; direction of differential expression is indicated for all DE and highly DE (fold change [FC] >4) genes. (B) RT-qPCR analysis of the selected genes (n = 4 mice per group). (C) Expression of DE genes between back skin specimens from adult Rabgef1^K-KO and control mice, indicating (on right) names and FC of DE genes whose transcript expression was confirmed by RT-qPCR. (D) MYD88 staining of back skin sections from adult mice. Dashed lines: dermal-epidermal junction. (E) Bar graphs show quantification of epidermal MYD88 levels (n = 5–10 mice per group). (F–H) Mouse PDV keratinocyte cells were transfected with control (Ctrl) shRNA or shRNA against Rabgef1 (Rabgef1 shRNA). (F) Western blot showing RABGEF1 and GAPDH (control) expression following shRNA transfection. The sh Ctrl and sh Rabgef1 lanes were run on the same gel but were noncontiguous. (G) Levels of the indicated phospho-proteins and total proteins at different times following recombinant IL-1β (rIL-1β)stimulation (results representative of 1 of 2 independent experiments performed with 2 separate batches of cells). (H) Baseline IL-1β secretion of RABGEF1-deficient keratinocytes in vitro as assessed by ELISA (n = 6 per group, 2 independent experiments performed with 2 different batches of cells). (B, E, and H) Data are mean ± SEM; E and H also show individual values. P values were calculated using a 2-way ANOVA test followed by Sidak’s multiple comparison test (C) and a 2-tailed Student’s t test (E and H). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm; original magnification in D, ×20; NS, not significant.

Figure 5. GSEA between Rabgef1^K-KO and defined gene sets.

Gene set enrichment analysis (GSEA) comparing the transcriptome of Rabgef1^K-KO skin with defined gene sets. Circle size is proportional to the normalized enrichment score (NES), whose numeric value is provided.

Figure 6. MYD88-dependent actin abnormalities and loss of tight junctions in RABGEF1-deficient keratinocytes.

Cells from the mouse PDV keratinocyte cell line were transfected with control (Ctrl) shRNA or shRNA directed against Rabgef1 (Rabgef1 shRNA). (A) Representative confocal microscopy pictures of phalloidin (red) and E-cadherin (green) staining merged with DAPI (cyan) and 3D reconstruction of phalloidin fluorescence enrichment (gray pseudocolor scale). Middle and right panels show higher-magnification photographs of the areas depicted by dashed white lines in the left panels. (B) Bars show quantification of actin content, as assessed by phalloidin staining in A. (C) 3D reconstruction of phalloidin fluorescence enrichment (gray pseudocolor scale) of Rabgef1 shRNA keratinocytes incubated for 24 hours with 100 μM of MYD88 inhibitory or control peptides. (D) Bars show quantification of actin content, as assessed by phalloidin staining in C. (E) Representative transmission electron microscopy pictures; red arrowheads indicate TJs (identified as electron-dense structures with close apposition of adjacent membranes), and blue areas are intercellular spaces. Insets depict higher-magnification photographs of the areas depicted by black squares. (F and G) Bars show quantification of the intercellular spaces (F) and TJ numbers (G). (B, D, F, and G) Results shown are mean ± SEM, as well as individual values, and are pooled from 2 (B) or 3 (D, F, and G) independent experiments. P values were calculated using a Mann-Whitney nonparametric test. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm (A and C); 250 nm (E); original magnification, ×20 (A and C) or ×6,300 (E).

Figure 7. Critical role of keratinocyte-intrinsic MYD88 in the phenotype of Rabgef1^K-KO mice.

Comparison of Rabgef1/Myd88^K-KO and control mice. (A) Specificity and efficiency of Rabgef1 gene deletion assessed by single-cell PCR; numerals indicate the number of cells analyzed for each cell type; results are pooled from 2 sorting experiments. (B) Representative RABGEF1 and MYD88 staining of back skin sections. (C and D) Survival (n = 8–29 mice per group) (C) and body weight (n = 3–6 mice per group) (D) over time. (E) Representative photographs of mice from the indicated genotype at day 49–56 and 1 year after birth. (F) Representative H&E staining of back skin sections. (G) Bars show quantification of dermal and epidermal thickness (n = 5 mice per group). (H and J) Representative confocal microscopy pictures of back skin sections with the indicated staining. (I and K) Bars show protein and mRNA levels of the indicated molecules whose staining is depicted in H and J, as assessed by confocal microscopy and RT-qPCR analyses, respectively (n = 3–4 per group). (B and F) Pictures are representative of 4 or 5 samples per group, each giving similar results. (B, H, and J) Dashed lines identify the dermal-epidermal junction. P values were calculated by Mantel-Cox test (C), 1-way ANOVA (D), 2-way ANOVA with Bonferroni’s test for multiple comparisons (G), and 2-tailed unpaired Student’s t test (I and K). Scale bars: 50 μm; original magnification, ×20 (B and I) or ×63 (J); NS, not significant.

Figure 8. Contribution of global and cell-specific IL-1R expression to the development of ear skin lesions in Rabgef1^KERT2-KO mice following topical tamoxifen treatment.

(A) Experimental outline for data shown in B–F. (B) Quantification of ear thickness over time. (C and D) Representative photographs of ear pinnae (C) and representative H&E staining of ear pinna sections (D). (E and F) Bars show quantification of epidermal and dermal thickness (E) and inflammatory cell infiltrates in the dermis (F). (G) Experimental outline for data shown in H and I. (H) RT-qPCR analysis of Il1r1 gene expression in peripheral blood mononuclear cells (PBMCs) isolated from the indicated groups of mice. (I) Quantification of ear thickness over time. (J) Representative H&E staining of ear pinna sections. (K and L) Bars show quantification of epidermal and dermal thickness (K) and inflammatory cell infiltrates in the dermis (L). Results are shown as mean ± SEM (as well as individual values in E, F, H, K, and L), and are pooled from 3 independent experiments (n = 6–12 mice per group), each of them giving similar results. (D and J) Dashed lines identify the dermal-epidermal junction. P values were calculated by 2-tailed unpaired Student’s t test (comparison of Il1r1^+/+ Rabgef1^KERT-KO vs. Il1r1^–/– Rabgef1^KERT-KO mice) (B); 1-way ANOVA followed by Tukey’s tests for multiple comparisons (E, F, K, and L); or 2-way ANOVA followed by Sidak’s tests for multiple comparisons (I). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm; NS, not significant.

Figure 9. RABGEF1 quantification in HDM/SEB–induced skin inflammatory lesions.

(A) Experimental mouse model of HDM/SEB–induced AD used in B–D. (B) Representative H&E staining of back skin sections from vehicle- or HDM/SEB–treated mice. Arrowheads and arrows indicate spongiosis and dermal leukocytes, respectively. (C) Representative confocal microscopy pictures of RABGEF1 staining. (D) Quantification of RABGEF1 MFI in a randomly defined region (shown in the white boxes) in back skin specimens from vehicle- or HDM/SEB–treated mice (n = 12 or 20 mice per group, respectively). (B and C) Dashed lines identify the dermal-epidermal junction. (D) Results are shown as mean ± SEM. P values were calculated by 2-tailed unpaired Student’s t test. **P < 0.01. Scale bars: 50 μm; original magnification in C, ×20; AD, atopic dermatitis; HDM, house dust mite; SEB, staphylococcus enterotoxin B; MFI, mean fluorescence intensity.

Figure 10. GSEA between Rabgef1^K-KO and HDM/SEB mouse models.

Gene set enrichment analysis (GSEA) comparing the HDM/SEB–induced AD model (GSE53132) with gene sets defined as upregulated genes in back skin samples from Rabgef1^K-KO mice or control mice (P < 0.01; fold change [FC] > 2; see Figure 4A). The size of the circle is proportional to the normalized enrichment score, whose numeric value is also indicated. HDM, house dust mite; SEB, staphylococcal enterotoxin B; MFI, mean fluorescence intensity.

Figure 11. RABGEF1 detection and quantification in human skin lesions.

(A) Representative H&E-stained sections of normal human skin (left panels) and lesional human skin with spongiotic dermatitis from AD (middle panels) or ACD (right panels) patients; arrowheads and asterisks indicate spongiosis and dermal infiltrates of leukocytes, respectively. (B and C) Representative confocal microscopy pictures of RABGEF1 (B) or MYD88 (C) staining merged with DAPI. Right panels show higher-magnification photographs of the areas depicted by white lines in the left panels. (D and E) Quantification of keratinocyte RABGEF1 (D) or MYD88 (E) protein expression in a randomly defined region (shown in the boxes delimited by white dashed lines) in normal skin and lesional skin with spongiotic dermatitis from patients with AD or ACD (n = 20, 12, and 8, respectively). See Supplemental Table 4 for patient characteristics and diagnostic criteria. (A–C) Dashed lines identify the dermal-epidermal junction. (D and E) Results are shown as mean ± SEM, as well as individual values. P values were calculated by 1-way ANOVA followed by Tukey’s tests for multiple comparisons. **P < 0.01; ***P < 0.001. Scale bars: 50 μm; original magnification in B and C, ×20; AD, atopic dermatitis; ACD, allergic contact dermatitis; NS, not significant.

Figure 12. GSEA between Rabgef1^K-KO mouse models and AD skin samples.

Gene set enrichment analysis (GSEA) comparing publicly available mRNA expression analyses of human atopic dermatitis (AD) skin lesions (also see Supplemental Table 3) with gene sets defined as upregulated genes in back skin samples from Rabgef1^K-KO mice or control mice (P < 0.01; fold change [FC] > 2; see Figure 4A). The size of the circle is proportional to the NES, whose numeric value is also indicated.