SERCA2 Dysfunction in Darier Disease Causes Endoplasmic Reticulum Stress and Impaired Cell-to-Cell Adhesion Strength: Rescue by Miglustat  Magali Savignac, Marina Simon, Anissa Edir, Laure Guibbal, Alain Hovnanian  Journal of Investigative Dermatology  Volume 134, Issue 7, Pages 1961-1970 (July 2014) DOI: 10.1038/jid.2014.8 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Endoplasmic reticulum (ER) stress response is increased in Darier keratinocytes (DKs) in basal conditions and after induction by thapsigargin (TG). Keratinocytes were grown in 1.2 mM calcium (Ca2+) and exposed to TG (1 μM). Immunoblotting analysis using phospho-eIF2α and total-eIF2α (a), phospho-IRE1 (inositol-requiring transmembrane kinase and endonuclease 1), and total-IRE1 (b). Reverse-transcriptase–PCR for Xbp1 mRNA (c). Phosphoglycerokinase (PGK) was used as a loading control. a to c are representative of three independent experiments performed with five DKs. (d) Immunofluoresence of Calnexin, ERGIC-53, GM130, and TGN46 in keratinocytes grown in 1.2 mM Ca2+ for 4 hours. Bar=20 μm. (e) Quantification of intracellular compartment volumes. Numbers in brackets correspond to the number of cells analyzed for quantification. **P<0.01 and ***P<0.001. Journal of Investigative Dermatology 2014 134, 1961-1970DOI: (10.1038/jid.2014.8) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Adherens junction (AJ) and desmosome formation is impaired, and adhesion strength is reduced in Darier keratinocytes (DKs). The formation of cell–cell contacts was induced by 1.2 mM Ca2+ for 4 and 6 hours to induce AJ and desmosome formation, respectively. Immunofluorescence of E-cadherin (extracellular domain of E-cadherin, EcadM) (a), β-, α-, p120-catenins, and E-cadherin (green)/F-actin (red) (b), and desmoplakin (DP), desmoglein 3 (Dsg3), and desmocollin 3 (Dsc3) (d). The arrows indicate intercellular space and arrowheads indicate perinuclear staining. Bar=20 μm. The results are representative of three independent experiments. (c) Surface biotinylation assay. The graph shows the intensity of E-cadherin bands. (e) Dispase mechanical dissociation assay. The graphs represent the mean+SEM from three independent experiments. *P<0.05. Journal of Investigative Dermatology 2014 134, 1961-1970DOI: (10.1038/jid.2014.8) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Thapsigargin (TG) treatment of normal keratinocytes (NKs) recapitulates defective adherens junction (AJ) and desmosome formation. NKs were pretreated (TG) or not (-) with 100 nM of TG for 30 minutes before increasing [Ca2+]extra to 1.2 mM for 4 hours (AJ) and 6 hours (desmosomes). (a) Immunofluorescence of Calnexin. Graphs represent the mean of endoplasmic reticulum (ER) volume+SEM from two independent experiments. Numbers in brackets correspond to the number of cells analyzed for quantification. (b) Immunofluorecence of E-cadherin (extracellular domain of E-cadherin, EcadM), β-, α-, and p120-catenin (green)/F-actin (red), and (c) desmoglein 3 (Dsg3), desmocollin 3 (Dsc3), and desmoplakin (DP). The results are representative of two independent experiments. Bar=20 μm. ***P<0.001. Journal of Investigative Dermatology 2014 134, 1961-1970DOI: (10.1038/jid.2014.8) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 E-cadherin, desmoglein 3 (Dsg3), and desmocollin 3 (Dsc3) are retained in the endoplasmic reticulum (ER) in Darier keratinocytes (DKs). The formation of cell–cell contacts was induced by 1.2 mM Ca2+ for 4 and 6 hours to induce adherens junction (AJ) and desmosome formation, respectively. Co-immunofluorescence of E-cadherin (intracellular domain, EcadR) (a) or Dsg3 (b) or Dsc3 (c) and calnexin. Fluorescent signals were detected with a confocal microscope. Pictures represent a single optical section. The lower panel represents the area of overlapping pixels between binary masks for each channel. Bar=20 μm. The graph represents the mean+SEM of surface of colocalized pixel of three (d) and one (e and f) independent experiments. Numbers in brackets correspond to the number of cells analyzed for quantification. **P<0.01 and ***P<0.001. Journal of Investigative Dermatology 2014 134, 1961-1970DOI: (10.1038/jid.2014.8) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Miglustat restores adherens junction (AJ) and desmosome formation and increases cell–cell adhesion strength in Darier keratinocytes (DKs). DKs were treated for 24 hours with Miglustat and its inactive analog (NB-DGJ) before increasing [Ca2+]extra for 4 (a and c) and 6 (b) hours. Immunofluorescence staining of E-cadherin (intracellular domain, EcadR), β-, α-, p120-catenins, E-cadherin (green)/F-actin (red), and desmoplakin (DP), desmoglein 3 (Dsg3), and desmocollin 3 (Dsc3 (a–c). Bar=20 μm. The results shown are from D5 (a), D1 (b), and D1 and D3 (c), and are representative of two independent experiments. Arrows indicate thin and linear staining of the molecules. (d) Surface biotinylation assay. The graph shows the intensity of E-cadherin bands corresponding to E-cadherin detected at the plasma membrane. (e) Dispase mechanical dissociation assay. The graphs represent the mean+SEM from three independent experiments. Journal of Investigative Dermatology 2014 134, 1961-1970DOI: (10.1038/jid.2014.8) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions