Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 in cultured keratinocytes

Abstract

Mutations in the ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mm) or high (1.2 mm) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO-1 did not, however, follow similar expression patterns. Protein levels of occludin, beta-catenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1-deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1-inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD.

Figure 1

Inhibition of ATP2C1 gene expression in keratinocytes cultured in low (0.06mM) or high calcium concentration (1.2mM). (a) Western analysis shows a clear decrease of the expression of SPCA1 protein in siRNA treated samples. (b) Quantification of Western analyses shows significant reduction of the SPCA1 protein by both siRNAs used.

Figure 2

The effect of SPCA1 inhibition on intercellular junction proteins. SiRNA inhibition (72 h) leads to significant changes in the protein expression of claudins 1 and 4. E-cadherin showed no changes after inhibition of SPCA1. Desmoplakin, desmogleins 1-3, desmocollins 2 and 3 and plakoglobin did not display marked changes.

Figure 3

The influence of the calcium switch on mRNA expression of selected genes encoding intercellular junction components as studied using whole human genome DNA microarrays. The mRNA levels of claudin 1, E-cadherin, occludin, ZO-1 and desmoplakin increased during the first 4–24 hours after the switch to high calcium. Thereafter, the expression levels decreased. Betacatenin did not follow the same pattern.

Figure 4

Indirect immunofluorescence labeling of control (a)–(h) and SPCA1 inhibited keratinocytes (a′)–(h′) for intercellular junction proteins. Panels (a)–(e′) were visualized using fluorescence microscope, panels (h)–(h′) using STED microscope and panels (f)–(g′) were photographed with confocal laser scanning microscope. Localization of desmoplakin (a)–(b′) and desmoglein 3 (c)–(c′) to intercellular junctions was delayed in SPCA1 inhibited cells after switch to high calcium medium. Instead, the cytoplasmic labeling of desmoplakin and desmoglein 3 was increased in SPCA deficient cells compared to controls. (d)–(d′) Desmomocollin 2/3 localizes to desmosomes in 72h incubation in high calcium medium. (e)–(e′) SPCA1 inhibition increased intracellular betacatenin in keratinocytes cultured in low or high calcium. Labeling for occludin (f)–(g′) in SPCA1 inhibited keratinocytes cultured in low calcium show a more intense immunosignal for intracellular occludin than control cells. (g)–(g′) Occludin localizes to the intercellular junctions in cells cultured in high calcium (arrows). (g) and (h) show colocalization of ZO-1 and occludin in zipper-like TJs as demonstrated by confocal and STED microscopy. (g′) and (h′) reveal the finger-like TJ sites (arrows). Scale bars 50 μm in (a)–(e′). Scale bars 10 μm in (f)–(h′).