Conditional KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis

Abstract

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-β1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-β1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

Fig 1

A) Plasmid construct for generation of Kcnn4 transgenic mice and induction (gene product: KCa3.1) in epithelial tissues. B) Induction of KCa3.1 transgene expression by 2-weeks DOX-treatment over basal levels in various tissues as measured by qRT-PCR. Data (% of control (-DOX)) are given as means +/- SEM; *P<0.01, Student T test; Br, brain (DOX, n = 4; -DOX, n = 2); Int, small intestine (DOX, n = 7; -DOX, n = 6); Lu, lung (DOX, n = 11; -DOX, n = 10); Sk, skin (DOX, n = 7; -DOX, n = 6); Skin-Epi, skin epidermis (DOX, n = 7; -DOX, n = 2); Sp, spleen (DOX, n = 4; -DOX, n = 2). C) Whole-cell patch-clamp on freshly isolated keratinocytes from tail skin. Representative recordings of large KCa3.1 currents in keratinocytes (+DOX) from DOX-treated mice and currents in keratinocytes from untreated Ctrls (-DOX). Note: For an additional recording of small KCa3.1 currents in a keratinocyte from untreated Ctrl see S1A Fig. Inhibition of KCa3.1 currents by RA-2 at 1 μM. Inset: Summary data of KCa3.1-outward currents at a clamp potential of 0 mV. Data (pA/pF) are given as means +/- SEM (-DOX, n = 4; DOX n = 5); *P<0.01, Student’s T test. D). Immune histochemical detection of KCa3.1 protein in the epidermis of DOX-treated mice (+DOX, n = 2) and an untreated mouse (-DOX): panels a and b with primary AB against KCa3.1; c and d without primary AB against KCa3.1. Inserts: 3 x zoom into epidermal layer.

Fig 2. Macroscopic skin pathology DOX-treated KCa3.1+ mice.

Photographs of A) untreated Ctrl (-DOX), B) DOX-treated KCa3.1+, C) higher magnification of the neck shown in B, D) patchy erythematous and scaly skin with ulcerative areas of a DOX-treated mouse. Note: Videos of DOX-treated mice showing severe scratching behavior and of Ctrls are found in the supplement.

Fig 3. Histological evaluation of skin pathology in KCa3.1+ mice.

H&E-stained sections of normal skin of the neck region from an untreated mouse (A) and a skin of the same region from a DOX-treated KCa3.1+ (B) with severe hyperplasia and hyperkeratosis. C) Summary of pathology scores. Data are given as means +/- SEM, n = 4 (1 week DOX), n = 26 (2 weeks DOX), n = 15 (Ctrls); *P < 0.05 vs. 1 week DOX, Student’s T test. Note that the scores for Ctrl skin are 0. D) Higher magnification of the hyperplastic epidermis of DOX-treated KCa3.1+. Note the presence of intra-epidermal edema with enlarged intra-cellular space (indicated by white arrow). E) Immune histological stains of the proliferation marker, PCNA, in the hyperplastic epidermis of DOX-treated KCa3.1+. Note the intense staining of the basal layer (white arrow) that becomes weaker when approaching the stratum corneum (representative image from 3 mice). F). The TUNEL assay detected no apoptotic keratinocytes in the hyperplastic epidermis.

Fig 4. Cytokine mRNA-expression profile.

(A) Alterations of cytokine mRNA-expression profile in DOX-treated KCa3.1+ mice (DOX/-DOX). (B) Cytokines with higher expression in the epidermis than in the underlying skin tissue (epidermis/ skin w/o epidermis). EREG, epiregulin (dox, n = 5; -DOX, n = 5); HB-EGF, Heparin-binding EGF-like growth factor (DOX, n = 5; -DOX, n = 5); HGF, Hepatocyte growth factor (DOX, n = 5; -DOX, n = 5), Interleukin(IL)-1β (DOX, n = 7; -DOX, n = 7), IL-6 (DOX, n = 7; -DOX, n = 7), TGFα, transforming growth factor α (DOX, n = 5; -DOX, n = 5); TNFα; tumor necrosis factor-α (DOX, n = 5; -DOX, n = 5). Data (DOX/-DOX; Epidermis (n = 4/Skin w/o Epidermis (n = 4)) are given as means +/- SEM; *P<0.05, Student’s T test.

Fig 5

Senicapoc suppressed skin pathology (A) and induction of IL-1β and IL-6 in DOX-treated KCa3.1+ mice (B). Data (Senicapoc/Ctrl) are given as means +/- SEM, n = 5 each, *P<0.05, **P<0.01 Student’s T test. C) Examples of skin pathology in DOX-treated KCa3.1+ mice receiving vehicle or Senicapoc. Inserts: 2.7-digital zoom-into epidermis showing lower grade of intra-epidermal edema in Senicapoc-treated mice.