Shedding light on the effects of 1,25-dihydroxyvitamin D3 on epidermal lipid barrier formation in three-dimensional human skin equivalents

Abstract

Human skin equivalents (HSEs) are three dimensional models resembling native human skin (NHS) in manyaspects. Despite the manifold similarities to NHS, a restriction in its applications is the altered in vitro lipidbarrier formation, which compromises the barrier functionality. This could be induced by suboptimal cell culturingconditions, which amongst others is the diminished activation of the vitamin D receptor (VDR) signallingpathway. The active metabolite of this signalling pathway is 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). An interactingrole in the formation of the skin barrier has been ascribed to this pathway, although it remains unresolvedto which extent this pathway contributes to the (mal-)formation of the epidermal barrier in HSEs. Ouraim is to study whether cell culture medium enriched with 1,25(OH)2D3 affects epidermal morphogenesis andlipid barrier formation in HSEs. Addition of 20 nM 1,25(OH)2D3 resulted in activation of the VDR signallingpathway by inducing transcription of VDR target genes (CYP24A and LL37) in keratinocyte monocultures and inHSEs. Characterization of HSEs supplemented with 1,25(OH)2D3 using immunohistochemical analyses revealeda high similarity in epidermal morphogenesis and in expression of lipid processing enzymes. The barrier formationwas assessed using state-of-the art techniques analysing lipid composition and organization. Addition of1,25(OH)2D3 did not alter the composition of ceramides. Additionally, the lateral and lamellar organization ofthe lipids was similar, irrespective of supplementation. In conclusion, epidermal morphogenesis and barrierformation in HSEs generated in presence or absence of 1,25(OH)2D3 leads to a similar morphogenesis andcomparable barrier formation in vitro.