Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

Upregulation of miR-203 and miR-210 affect growth and differentiation of keratinocytes after exposure to sulfur mustard in normoxia and hypoxia.

Exposure of the skin to sulfur mustard (SM) results in long-term complications such as impaired tissue regeneration. Previous own studies in normal human epidermal keratinocytes (NHEK) treated with SM demonstrated reduced proliferation, premature differentiation and a restricted functionality of hypoxia-mediated signaling in the cells. Here, we investigated the involvement of microRNAs, miR-203 and miR-210, in these mechanisms. SM significantly upregulated the expression of miR-203 in NHEK when cultivated under normoxic and hypoxic conditions. SM had no effect on miR-210 under normoxia. However, miR-210 levels were greatly increased in NHEK when grown in hypoxia and further elevated upon exposure of the cells to SM. In normoxia and hypoxia, inhibition of miR-203 by transfection of NHEK with complementary oligonucleotides, anti-miR-203, attenuated the SM-induced impairment of metabolic activity and proliferation, and counteracted SM-promoted keratin-1 expression in these cells. Consistent ameliorating effects on dysregulated metabolic activity, proliferation and keratin-1 expression in SM-treated NHEK were obtained upon inhibition of miR-210 in these cells grown in hypoxia. Our findings provide evidence that miR-203 and miR-210 are key regulators in normal and SM-impaired keratinocyte functionality, and suggest potential usefulness of inhibitors against miR-203 and miR-210 for target-directed therapeutical intervention to improve re-epithelialization of SM-injured skin.

Wnt5a/ß-catenin signaling drives calcium-induced differentiation of human primary keratinocytes.

It is well established that a gradient of extracellular calcium within the epidermis regulates the differentiation of keratinocytes. However, the molecular mechanisms implicated in this process are not fully understood. RNA interference of the calcium-sensing receptor (CaSR) showed that CaSR is essential in calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs) by increasing the levels of free intracellular calcium, which upregulates the expression of Wnt5a but not Wnt3a, Wnt4, and Dkk-1 in the cells. Subsequently, autocrine Wnt5a promotes the differentiation of NHEKs, determined by increased biosynthesis of keratin-1 and loricrin, whereas proliferation is suppressed. Addition of both Wnt5a and calcium to NHEKs activated the Wnt/ß-catenin signaling pathway as indicated by (i) increased stability of ß-catenin in the cells, (ii) enhanced ß-catenin transcriptional activity, demonstrated by a luciferase-based ß-catenin-activated reporter assay, and (iii) augmented Wnt/ß-catenin target gene expression. NHEKs depleted for ß-catenin had a significantly reduced susceptibility to calcium-induced differentiation. Knockdown of axin 2, an antagonist of ß-catenin stability, enhanced the biosynthesis of keratin-1 and loricrin in the cells. Our findings establish a directional crosstalk between CaSR and Wnt/ß-catenin signaling in keratinocyte differentiation via Wnt5a that acts as an autocrine stimulus in this process.

Nile red: Alternative to physical developer for the detection of latent fingermarks on wet porous surfaces?

This paper describes the application of a luminescent lipid stain, nile red, for the development of latent fingermarks on porous surfaces. An optimised formulation is presented that provides rapid development of latent fingermarks on porous surfaces that are or have been wet. A comparison with physical developer (PD), the method of choice to enhance such fingermarks, indicated that nile red was a simpler and more stable technique for the development of fingermarks. The nile red formulation showed similar performance to PD across a range of substrates and ageing conditions, although PD still showed greater sensitivity on five-year-old examination booklets used in a pseudo-operational study. The pseudo-operational trial also indicated that nile red consistently developed different fingermarks to those enhanced by PD, suggesting that it preferentially targets a different fraction of the latent fingermark deposit. Significantly, the compatibility of nile red in a detection sequence with indanedione-zinc, ninhydrin and PD is reported.