The calcium-activated chloride channel-associated protein rCLCA2 is expressed throughout rat epidermis, facilitates apoptosis and is downmodulated by UVB

Abstract

The rodent chloride channel regulatory proteins mCLCA2 and its porcine and human homologues pCLCA2 and hCLCA2 are expressed in keratinocytes but their localization and significance in the epidermis have remained elusive. hCLCA2 regulates cancer cell migration, invasion and apoptosis, and its loss predicts poor prognosis in many tumors. Here, we studied the influences of epidermal maturation and UV-irradiation (UVR) on rCLCA2 (previous rCLCA5) expression in cultured rat epidermal keratinocytes (REK) and correlated the results with mCLCA2 expression in mouse skin in vivo. Furthermore, we explored the influence of rCLCA2 silencing on UVR-induced apoptosis. rClca2 mRNA was strongly expressed in REK cells, and its level in organotypic cultures remained unchanged during the epidermal maturation process from a single cell layer to fully differentiated, stratified cultures. Immunostaining confirmed its uniform localization throughout the epidermal layers in REK cultures and in rat skin. A single dose of UVR modestly downregulated rClca2 expression in organotypic REK cultures. The immunohistochemical staining showed that CLCA2 localized in basal and spinous layers also in mouse skin, and repeated UVR induced its partial loss. Interestingly, silencing of rCLCA2 reduced the number of apoptotic cells induced by UVR, suggesting that by facilitating apoptosis, CLCA2 may protect keratinocytes against the risk of malignancy posed by UVB-induced corrupt DNA.

Keywords: Apoptosis; Epidermis; REK; UVR; rCLCA2.

Fig. 1

Rat epidermal keratinocytes express CLCA2. REK cells were grown in organotypic cultures (ROC) where they stratify and differentiate (a, d, e, f). a Samples were collected for qRT-PCR at the indicated time points (3 independent experiments, each with duplicate cultures). d, e, f Immunostaining with an anti-CLCA2 antibody or non-immune IgG at day 4 (d) and day 12 (e, f) (3 independent experiments). b, c REK cells grown as monolayers were transfected with control (b) and rClca2-specific siRNA (c) and stained for rCLCA2. The experiment was repeated 4 times. In all experiments, CLCA2 immunostaining intensity was lower in cultures treated with Clca2 siRNA as compared to those with control siRNA. g Adult rat skin skin of 3 animals was stained for rCLCA2

Fig. 2

CLCA2 shows a partial colocalization with trans-golgi network (TGN). a–c Monolayer REK cell cultures were incubated with Texas Red-transferrin for 30 min, fixed and stained with an anti-rCLCA2 antibody. In panels d–f REK cells were stained for TGN (green) and CLCA2 (red). The data represent 3 individual experiments, each performed with duplicates. Magnification bar represents 50 µm

Fig. 3

Clca2 silencing reduces UVR-induced cell death in REKs. CLCA2 expression was silenced in REK cells in monolayer cultures. 2 days after the silencing, the cells were either exposed to UVB (10 mJ/cm²) or sham exposed. a The cultures were analyzed for caspase 3/7 expression using Incucyte imaging system (3 independent experiments, each with 4 replicate wells). The difference between Cntr siRNA + UVB and Clca2 siRNA + UVB was statistically significant (p < 0.001, linear mixed model). b The membrane integrity of the cultures was analyzed using Cytotoxicity assay (2 independent experiments, each with 3 replicate wells) as described in “Materials and methods”

Fig. 4

Clca2 is downmodulated by UVR. a REK organotypic cultures (9 days) were either sham-treated or UVB-exposed, and samples were collected at the indicated time points and analyzed for rClca2 mRNA. The Ct-value of the sham-treated culture was set as 1 at each time point and the mRNA level of the UV-exposed culture was compared to it. The data represent means and SE from 3 individual experiments, each performed with duplicate cultures. The statistical differences were tested using 2-way ANOVA with Bonferroni’s Post Hoc test. **p < 0.01. b–f Histological sections from 14 sham (b) or 14 UVR (c–f)-exposed mouse back skins were stained with anti-CLCA2 antibody. b The sham-exposed skin with normal epidermal morphology shows intense, regular immunostaining in the epidermis. Both basal and the suprabasal cells are CLCA2 positive (b, inset) as well as the hair follicles and sebaceous glands. c A UVR-exposed specimen with strong epidermal hyperplasia showing irregular, focally reduced CLCA2 staining (arrowhead). d–f A UVR-exposed specimen with SCC. d Overview, e an area with focally reduced CLCA2 immunostaining, f an area with intense regular staining. Similar irregular pattern was observed in all 5 SCCs and 8 hyperplastic areas found in the specimens. Magnification bars 50 µm