Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures

Abstract

Caspase-14 is a nonapoptotic caspase family member whose expression in the epidermis is confined to the suprabasal layers, which consist of differentiating keratinocytes. Proteolytic activation of this caspase is observed in the later stages of epidermal differentiation. In psoriatic skin, a dramatic decrease in caspase-14 expression in the parakeratotic plugs was observed. Topical treatment of psoriatic lesions with a vitamin D3 analogue resulted in a decrease of the psoriatic phenotype and an increase in caspase-14 expression in the parakeratotic plugs. To investigate whether vitamin D3 directly affects caspase-14 expression levels, we used keratinocyte cell cultures. 1alpha,25-Dihydroxycholecalciferol, the biologically active form of vitamin D3, increased caspase-14 expression, whereas retinoic acid inhibited it. Moreover, retinoic acid repressed the vitamin D3-induced caspase-14 expression level. In addition, the use of organotypic skin cultures demonstrated that 1alpha,25-dihydroxycholecalciferol enhanced epidermal differentiation and caspase-14 activation, whereas retinoic acid completely blocked caspase-14 processing. Our data indicate that caspase-14 plays an important role in terminal epidermal differentiation, and its absence may contribute to the psoriatic phenotype.

Figure 1

Vitamin D₃ increases caspase-14 expression in the parakeratotic regions of psoriatic skin. Psoriatic skin was treated with a balm containing a vitamin D₃ analogue, and skin biopsies were used for staining with H&E (A, C, E), or caspase-14 immunodetection (B, D, F). A and B: Normal skin showing orthokeratosis. C and D: Psoriatic skin before treatment; the parakeratotic layer is indicated with double asterisk. E and F: Psoriatic skin after vitamin D₃ treatment; the parakeratotic region is indicated with an asterisk. Original magnifications, ×200.

Figure 2

1α,25-Dihydroxycholecalciferol enhances procaspase-14 protein expression. A: HaCaT cells were treated for 72 hours with different concentrations of 1α,25-dihydroxycholecalciferol as indicated. Cell lysates were used for Western blot analysis. B: HaCaT cells were treated with 10⁻⁷ mol/L 1α,25-dihydroxycholecalciferol for 24, 48, 72, and 96 hours. Protein extracts were analyzed for caspase-14, keratin 10, and caspase-3 expression.

Figure 3

Retinoic acid inhibits vitamin D₃-induced procaspase-14 expression. Western blot analysis of the expression of different caspases by HaCaT cells or primary normal human epidermal keratinocytes treated with different combinations of vitamin D₃, 9-cis-retinoic acid, or all-trans retinoic acid for 72 hours. 9cRA, 9-cis-retinoic acid; atRA, all-trans retinoic acid; vitD₃, 1α,25-dihydroxycholecalciferol.

Figure 4

Vitamin D₃-dependent caspase-14 up-regulation is not a mere consequence of growth arrest. Normal human epidermal keratinocytes were treated with different growth arrest-inducing agents as indicated. The cells were pulsed for 8 hours with ³H-thymidine, harvested, and ³H-radioactivity was measured (A, C, and E). Protein lysates of similarly treated cells were used for immunodetection of caspase-14 and β-actin (B, D, and F). Relative levels of caspase-14, normalized with β-actin levels, are indicated. atRA, all-trans retinoic acid; dex, dexamethasone; mito c, mitomycin c; vitD₃, 1α,25-dihydroxycholecalciferol.

Figure 5

Vitamin D₃ induces and all-trans retinoic acid inhibits caspase-14 processing in organotypic keratinocyte cultures. Organotypic keratinocyte cultures were treated with 1α,25-dihydroxycholecalciferol and/or all-trans retinoic acid for 4 or 8 days. A: Morphological examination of paraffin-embedded sections of skin equivalents. B: Western blot detection of caspase-14 in cell lysates. C: Ki-67 immunodetection on paraffin-embedded sections of skin equivalents. Arrows indicate Ki-67-positive cells. The percentage of Ki-67-positive cells in the basal layer was: 25.5% in control cultures (4 days); 23% in vitamin D₃-treated cultures (4 days); 12.5% in control cultures (8 days); 14.5% in vitamin D₃-treated cultures (8 days). atRA, all-trans retinoic acid; vitD₃, 1α,25-dihydroxycholecalciferol. Original magnifications, ×200 (A); ×400 (C).