Epac1 contributes to apremilast-mediated rescue of pemphigus autoantibody-induced loss of keratinocyte adhesion

Abstract

In the bullous autoimmune disease pemphigus vulgaris (PV), autoantibodies directed mainly against desmoglein 1 (Dsg1) and Dsg3 cause loss of desmosomal adhesion. We recently showed that intracellular cAMP increase by the phosphodiesterase 4 inhibitor apremilast was protective in different PV models. Thus, we here analyzed the involvement of the cAMP effector exchange factor directly activated by cAMP1 (Epac1). In Epac1-deficient mice pemphigus antibody-induced blistering was ameliorated in vivo while apremilast had no additional effect. Interestingly, augmented protein levels of Dsg1 and Dsg3 as well as increased Dsg1 mRNA levels and higher numbers of Dsg1- and Dsg3-dependent single-molecule interactions were detected in keratinocytes derived from Epac1-deficient mice. This was paralleled by stronger intercellular adhesion under baseline conditions and prevention of pemphigus autoantibody-induced loss of intercellular adhesion. However, the protective effect of apremilast against loss of intercellular adhesion in response to the pathogenic Dsg3 antibody AK23 was attenuated in Epac1-deficient keratinocytes. Similarly, the Epac1 inhibitor Esi09 protected keratinocytes from pemphigus antibody-induced loss of adhesion. Mechanistically, Epac1 deficiency resulted in lack of apremilast-induced Rap1 activation and phosphorylation of Pg at S665. Taken together, these data indicate that Epac1 is involved in the regulation of baseline and cAMP-mediated stabilization of keratinocyte adhesion.

Keywords: Autoimmune diseases; Cell biology; Cell migration/adhesion; Dermatology; Skin.

Figure 1. Epidermis of Epac1-ko mice shows no alterations.

(A) H&E staining of murine epidermis shows no differences between WT and Epac1-ko mice. n = 4. (B–D) Immunostaining for Dsg1, Dsg3, and Dsc3 shows no difference in the expression pattern of WT and Epac1-ko mice (representative of n > 5). (E–G) Quantification of epidermal staining of Dsg1, Dsg3, and Dsc3. (H) Western blot analysis of epidermis obtained from WT and 2 different Epac1-ko mice shows no significant alterations for desmosomal proteins (representative of n > 3). Columns indicate mean value ± SEM, *P < 0.05. 2-tailed Student’s t test. α-Tub, α-Tubulin, CK14, Cytokeratin 14. Scale bars: 12.5 μm (A); 25 μm (B, C, and D, left and middle); 8 μm (B, C, and D, right).

Figure 2. Murine Epac1-ko keratinocytes display upregulated desmosomal proteins and strengthened intercellular adhesion.

Costaining of Dsg3/CK14 (A) and Dsg1/Dp (B) in murine keratinocytes show upregulation of Dsg1 and Dsg3 in Epac1-ko cells (Representative of n > 4). (C) Western blot analysis of Triton fractionation and whole SDS cell lysates of murine keratinocytes shows upregulation of Dsg1 and Dsg3 (representative of n > 3). (D) PCR analysis of WT and Epac1-ko mRNA displays upregulation of Dsg1 and Epac2 mRNA due to missing Epac1 (representative of n = 4). (E) Quantification of data presented in D. Keratinocyte dissociation assays under basal conditions (F) and after treatment with AK23 (G) (representative of n > 4). Adhesion of Epac1-ko keratinocytes is strengthened compared with WT cells. (H) Topography overview images of AFM measurements on living keratinocytes using Dsg1-functionalized tips reveal heightened cell borders. Small areas along the cell borders were chosen for adhesion measurements. Each pixel represents a force-distance curve. In the adhesion panel, each green pixel represents a Dsg1-specific binding event. Cell borders are marked as blue dotted line. (I–K) Quantification of AFM adhesion measurements. Epac1-ko cells displayed higher binding frequencies (I) compared with WT cells, whereas unbinding forces (J) and distribution of binding events (K) were unaltered. (10 cell borders from 4–5 independent experiments with 900 force-distance curves per cell border). Bars indicate mean value ± SEM. *P < 0.05. 2-tailed Student’s t test (E, F, and I–K), 2-way ANOVA with Bonferroni correction (G). α-Tub, α-Tubulin;CK14, cytokeratin 14; Dp, desmoplakin. Scale bars: 10 μm (A); 25 μm (B); 10 μm (H top) 1 μm (H bottom).

Figure 3. Protective effect of apremilast is ameliorated in Epac1-ko keratinocytes.

Keratinocyte dissociation assays in WT or Epac1-ko keratinocytes after apremilast (Apr) (A) or forskolin/rolipram (F/R) (B) treatment followed by AK23 incubation (n > 4). (C) Epac1-ko keratinocytes, show, in contrast to WT cells, no loss of adhesion upon PV1-IgG treatment (n > 4). Bars indicate mean value ± SEM. *P < 0.05. 2-way ANOVA with Bonferroni correction. PV-IgG, Pemphigus vulgaris IgG; ctr-IgG, IgG of healthy volunteers.

Figure 4. Apremilast rescues PV-IgG-induced alteration of keratins Epac1 dependent.

(A) Immunofluorescence of Dsg3/CK14 after pretreatment with either apremilast (Apr) or forskolin/rolipram (F/R). PV1-IgG-induced fragmentation of Dsg3 staining in both cell lines is rescued upon F/R only (representative of n > 3). The protective effect of Apr on keratin alterations after PV1-IgG treatment is diminished in Epac1-ko cells. DAPI (blue) was added to stain nuclei. (B) Quantification of Dsg3 fluorescence intensities. Bars indicate mean value ± SEM. *P < 0.05. 2-way ANOVA with Bonferroni correction. Pemphigus vulgaris IgG (PV-IgG), IgG of healthy volunteers (ctr-IgG), cytokeratin 14 (CK14). Scale bars: 10 μm; 5 μm (insets).

Figure 5. Apremilast induces phosphorylation of Pg and Rap1 activation in a Epac1-dependent manner.

(A) Western blot analysis of WT and Epac1-ko keratinocyte lysates after treatment with apremilast (Apr) or forskolin/rolipram (F/R) for 1 hour (representative of n > 6). Phosphorylation status of desmoplakin (Dp) and plakoglobin (Pg) were analyzed. Epac1 ko was verified and α-tubulin (α-tub) was used as a loading control. Pg is phosphorylated upon F/R treatment independently of Epac1 and upon Apr treatment in WT cells only. Neither phosphorylation on S165 nor S2849 of Dp was affected by Apr or F/R. Quantification of P-Pg (S665) (B), P-Dp (S2849) (C),and P-Dp (S165) (D). (E) Active Rap1 upon Apr treatment in WT and Epac1-ko keratinocytes was pulled down and analyzed by Western blot (representative of n = 3). (F) Quantification of Rap1 activation. Bars indicate mean value ± SEM. *P < 0.05, 2-way ANOVA with Bonferroni (B–D) or Tukey (F) correction.

Figure 6. Epac1 depletion attenuates blistering in the pemphigus mouse model and abrogates the protective effect of apremilast.

(A) H&E staining of neonatal mouse skin after injection of vehicle or apremilast (Apr) prior to injection of AK23 or ctr-IgG (representative of n > 4). AK23 induced suprabasal blistering of WT epidermis, which was rescued by apremilast. Epac1-ko epidermis showed only microblisters (marked with green arrows) after AK23 injection, which were not improved by apremilast. (B) Quantification of blistered epidermis shows a protective effect of apremilast in WT but not in Epac1-ko mice. (C) Quantification of blistered epidermis after PV3-IgG injection shows a protective effect of apremilast in WT but not in Epac1-ko mice (representative of n > 4). Columns indicate mean value ± SEM, *P < 0.05. 2-way ANOVA with Bonferroni correction. PV-IgG, Pemphigus vulgaris IgG, ctr-IgG, IgG of healthy volunteers. Scale bars: 100 μm.