Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease
Shivam A. Zaver, … , Johann E. Gudjonsson, Cory L. Simpson
Shivam A. Zaver, … , Johann E. Gudjonsson, Cory L. Simpson
Published August 10, 2023
Citation Information: JCI Insight. 2023;8(18):e170739. https://doi.org/10.1172/jci.insight.170739.
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Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

Abstract

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than 2 decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2-knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomics analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAPK signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings, with lesions showing keratin deficiency, cadherin mislocalization, and ERK hyperphosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multiomic analysis with human organotypic epidermis as a preclinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

Authors

Shivam A. Zaver, Mrinal K. Sarkar, Shaun Egolf, Jonathan Zou, Afua Tiwaa, Brian C. Capell, Johann E. Gudjonsson, Cory L. Simpson

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Figure 3

Proteomics analysis of SERCA2-deficient keratinocytes reveals alterations in key regulators of tissue integrity and skin barrier formation.

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Proteomics analysis of SERCA2-deficient keratinocytes reveals alteration...
(A) Label-free quantitative (LFQ) mass spectrometry–based comparison of the proteomes of control versus HET THEKs differentiated in E-medium for 72 hours. Proteins highlighted in purple were absent or detected in lower abundance in HET cells; proteins highlighted in green were detected in higher abundance in control THEKs; proteins highlighted in black were detected in similar abundance in control and HET cells; raw values and statistical analysis summarized in Supplemental Table 1. (B) Immunoblotting of KRT10 and KRT14 (on a separate blot run in parallel) in lysates from control, HET, and KO THEKs differentiated in E-medium for 72 hours; data representative of 5 independent experiments; β-actin is a loading control. (C) H&E-stained tissue cross sections from organotypic cultures grown from control, HET, or KO THEKs. HET and KO cultures display aberrant differentiation with the upper granular layers exhibiting vacuolization and loss of cohesion along with dyskeratotic cells having deeply pink cytoplasm and condensed nuclei indicative of aberrant cornification (insets); scale bar = 100 μm; insets, original magnification = 40×. (D) Immunostaining of KRT10 and p-ERK in tissue cross sections from organotypic cultures of control or HET THEKs; scale bar = 50 μm; dashed line marks bottom of epidermis. (E) Quantification of tissue immunostaining of KRT10 (relative to Hoechst) and p-ERK (relative to total ERK); data shown as a box plot of the 25th–75th percentile with a line at the median from N ≥ 26 nonoverlapping hpf per condition from 4 experiments using 2 independent HET lines; control mean normalized to 1; P values from 2-tailed unpaired Student’s t test. DSP, desmoplakin; JUP, plakoglobin; IVL, involucrin; TGM1, transglutaminase 1; PKP, plakophilin; p-, phosphorylated.