SERCA2-controlled Ca²+-dependent keratinocyte adhesion and differentiation is mediated via the sphingolipid pathway: a therapeutic target for Darier's disease

Abstract

Darier's disease (DD), caused by mutations in the endoplasmic reticulum (ER) Ca(2+) ATPase ATP2A2 (SERCA2b), is a skin disease that exhibits impaired epidermal cell-to-cell adhesion and altered differentiation. Although previous studies have shown that keratinocyte Ca(2+) sequestration and fluxes are controlled by sphingolipid signaling, the role of this signaling pathway in DD previously has not been investigated. We show here that sphingosine levels increase and sphingosine kinase (SPHK1) expression decreases after inactivating SERCA2b with the specific SERCA2 inhibitors thapsigargin (TG) or small interfering RNA to SERCA2b. Conversely, inhibiting sphingosine lyase rescues the defects in keratinocyte differentiation, E-cadherin localization, desmoplakin (DP) translocation, and ER Ca(2+) sequestration seen in TG-treated keratinocytes. Here, we report early evidence that the keratinocyte sphingolipid and Ca(2+) signaling pathways intersect in ATP2A2-controlled ER Ca(2+) sequestration, E-cadherin and DP localization, and Ca(2+)-controlled differentiation, and thus may be important mediators in DD.

Figure 1. Sphingosine Metabolism is Controlled by SERCA2

A. Sphingosine metabolism in keratinocytes. B. SERCA2 Inactivation Increases Sphingosine. Normal human keratinocytes were treated with TG 100 nM for 2 hours. Sphingosine levels were measured with HPLC (see Materials and Methods) 24 and 48 hours after TG treatment. Data are presented as the mean ± SD. N=3 for each data point. * = p<0.05 C. SPHK1 Expression is Decreased After SERCA2 Inhibition. Normal human keratinocytes were treated with 10 and 100 nM TG for 2 hours, washed, recultured and harvested at 24 (grey bars) and 48 (black bars) hours. In separate experiments, normal human keratinocytes were treated with siRNA to SERCA2b, and then harvested at 48 hours (marble bars). Control keratinocytes were treated with TG vehicle or scrambled siRNA (marble bar) and normalized to 1. SPHK1 expression was assessed with RT-PCR (see Materials and Methods).

Figure 2. Defects in Keratinocyte Morphology, Induced by SERCA2 Inhibition, are Normalized by Inhibiting SGPL1

Human Keratinocytes were cultured in growth media containing 0.06 mM Ca²⁺. When 80% confluent, some of the cells were transfected with siRNA against SGPL1 for 8 hours, then 10 nM (final concentration) TG was added. After two hours exposure to TG cells were washed and fed with growth media containing 1.2 mM Ca²⁺. Images were acquired 48 hours after raising extracellular Ca^2+. A. Vehicle treated cells showing normal morphology: cells are tightly packed and flattened after 48 hours in high calcium; B and C) cells treated with scrambled siRNA or siRNA against SGPL1 show no morphological difference from untreated cells. D) 10 nM TG treatment produces larger keratinocytes that have not flattened. E) Treatment with scrambled siRNA has no effect on TG treated cells F) cells treated with both TG and siRNA to SGPL1 demonstrate a morphology similar to control keratinocytes. All images were acquired at the same magnification. The scale bar in panel A indicates 50μm.

Figure 3. Defects in Involucrin and E-cadherin synthesis and Processing Are Normalized by Inhibiting SGPL1

Human Keratinocytes were cultured and treated as in Figure 2, above. Cells were harvested 48 hours after raising extracellular Ca²⁺. E-cadherin and involucrin protein levels were assessed using Western blotting (see Methods).

Figure 4. Defects in E-cadherin localization Are Normalized by Inhibiting SGPL1

A-D) Keratinocytes cultured in 1.2 mM Ca²⁺ for 48 hours were stained with an E-cadherin antibody that recognizes an extracellular epitope. E-H) Keratinocytes cultured in 1.2 mM Ca²⁺ for 48 hours stained with an E-cadherin antibody that recognizes an intracellular epitope. (A and E) Control keratinocytes. (B and F) After treatment with 10 nM TG, extracellular E-cadherin staining (B) is discontinuous, with filamentous strands (arrows). (F) Perinuclear accumulation of E-cadherin is seen (arrows). (C and G) Scrambled siRNA does not rescue intracellular or extracellular E-cadherin localization (arrows). (D and H) SGPL1 inhibition with siRNA restores continuous, fine extracellular E-cadherin localization (D) and reverses the E-cadherin intracellular retention (H) seen after SERCA2 inhibition with TG.

Figure 5. Defects in E-cadherin localization following downregulation of SERCA2b expression with siRNA Are Normalized by Inhibiting SGPL1

A-F) Extracellular E-cadherin staining. A-C) Untreated, SGPL1 siRNA, and scrambled siRNA treated control cells show linear, smooth staining at the cell-to-cell borders; D) Treatment with SERCA2 siRNA results in rough, discontinuous, filamentous borders, and larger cells similar to TG treated cells (compare to Figure 4, above); E) SGPL1 inhibition with siRNA restores normal E-cadherin localization; F) Scrambled siRNA has no effect. G-L) Intracellular E-cadherin staining; G-I) Untreated, SGPL1 siRNA, and scrambled siRNA treated control cells show normal, minimal intracellular retention of E-cadherin; J) Cells treated with siRNA to SERCA2b demonstrate more diffuse intracellular staining, irregular cell borders and larger cells; K) SGPL1 siRNA treatment restores normal E-cadherin localization; L) scrambled siRNA has no effect.

Figure 6. Defects in Desmoplakin (DP) localization, Caused by SERCA2 inactivation with TG, Are Normalized by Inhibiting SGPL1

Desmoplakin staining of human keratinocytes treated with 10nM TG, 3hours (A-F) and 48 hours (G-L) after raising extracellular Ca²⁺. In control (A), SGPL1 (B) and scrambled siRNA-treated cells (C), DP accumulation at cell-to-cell borders is evident within 3 hours after raising extracellular Ca^2+. (G-I) 48 hours after raising extracellular Ca²⁺, DP localizes exclusively at the cell to cell borders in control (G) , SGPL1 siRNA (H) and scrambled siRNA-treated (I) cells. Treatment with 10 nM TG reduces DP staining at the cell borders three hours after raising extracellular Ca²⁺ (D) and intracellular retention of DP is evident at 48 hours (J); SGPL1 inhibition with siRNA improves DP localization at 3 hrs (E), but complete normalization of DP localization is seen only at 48 hours (K). Scrambled siRNA had no effect (F and L).

Figure 7. Defects in ER Ca²⁺ Sequestration are Induced by ER SERCA2 Inhibition, and are Normalized by Inhibiting SGPL1

Primary normal human keratinocytes were transfected with the ER-targeted Ca²⁺ sensor D1ER (see Methods). Cells also were transfected with SGPL1 siRNA or scrambled siRNA as a control. 8 hours after siRNA transfection, cells were treated with 10 nM thapsigargin for two hours. The cells then were washed, and incubated in high calcium media for an additional 24 hours. A) average FRET ratio; B) average ER Ca²⁺ concentration; C) Relative Ca²⁺ changes between control (black bars) and TG treated cells (grey bars) in untreated, scrambled siRNA, and SGPL1 siRNA treated samples. Data are presented as the mean +/− s.e.m. N=15–29 cells from two independent sets of experiments in each group. Significance was calculated using a one-way ANOVA. Distributions with p<0.05 were assumed as statistically different.