Characterization of Tight Junctions and Their Disruption by UVB in Human Epidermis and Cultured Keratinocytes

It has not been confirmed whether tight junctions (TJs) function as a paracellular permeability barrier in adult human skin. To clarify this issue, we performed a TJ permeability assay using human skin obtained from abdominal plastic surgery. Occludin, a marker protein of TJs, was expressed in the granular layer, in which a subcutaneously injected paracellular tracer, Sulfo-NHS-LC-Biotin (556.59Da), was halted. Incubation with ochratoxin A decreased the expression of claudin-4, an integral membrane protein of TJs, and the diffusion of paracellular tracer was no longer prevented at the TJs. These results demonstrate that human epidermis possesses TJs that function as an intercellular permeability barrier at least against small molecules (∼550Da). UVB irradiation of human skin xenografts and human skin equivalents (HSEs) resulted in functional deterioration of TJs. Immunocytochemical staining of cultured keratinocytes showed that occludin was localized into dot-like shapes and formed a discontinuous network when exposed to UVB irradiation. Furthermore, UVB irradiation downregulated the active forms of Rac1 and atypical protein kinase C, suggesting that their inactivation caused functional deterioration of TJs. In conclusion, TJs function as a paracellular barrier against small molecules (∼550Da) in human epidermis and are functionally deteriorated by UVB irradiation

Characterization of hair follicle development in engineered skin substitutes.

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes

Absence of pilosebaceous units in chimeric ESS.

<p>Nile red and ALP stainings were performed on epidermis and dermis of ESS controls and ESS with mDPC-GFP (a). In ESS controls, epithelium was thin enough to visualize the skin surface lipids on the serosal side of the epidermis (a). Contrary to host skin containing sebaceous glands (arrowheads), no sebaceous glands were detected in ESS controls and ESS with mDPC-GFP. ALP in the dermis corresponded to the area where the hair follicles were situated (a, arrows). Close examination confirmed that neofollicles were deficient of sebaceous glands (b). On the other hand, sebaceous glands (white arrowheads) were observed above the bulge regions of pelage hairs. Immunohistochemistry confirmed the human origin (HuNu) and demonstrated no co-localization between Mel-5 and GFP in the bulb (c). Skin barrier integrity was evaluated by TEWL (d). Significantly higher TEWL was observed in ESS with mDPC-GFP compared to host skin, but not different from human volunteers (NHS). Scale bars in (a) = 500 µm; (b) = 100 µm and (c) = 50 µm.</p

Comparison of ESS models.

<p>Gene expression analyses of selected genes involved in Wnt/β-catenin pathway, fatty acid metabolism and skin cornification, including <i>LEF1, WNT10B, LOR, INV, SOX9, PRDM1, SCD</i> and <i>FABP3</i>, were compared between ESS controls and ESS with mDPC-GFP after normalization to NHS (a). Asterisks represent statistically significant differences between the ESS groups (<i>p</i><0.05). Morphological comparison of regenerated hair follicles in ESS with mDPC-GFP (b–d) and in newborn murine ESS was demonstrated (e–h). Scale bars = 50 µm, except in (b, e) = 100 µm and in (f) = 1 mm.</p

Immunostaining of regenerated hair follicles.

<p>Indirect immunohistochemistry was performed on frozen sections of microdissected hair follicles using antibodies against (a) CD34, (b) SOX9, (c) LHX2, (d) CD200 and (e) K15, respectively. Alexa Fluor 488 (green) was used to localize selected molecular markers before counterstaining of nuclei with DAPI (blue). Scale bars = 50 µm.</p

Characteristic features of hair induction <i>in vivo</i>.

<p>Compared to epithelium of ESS controls, which has fewer Ki67+ cells and no K6 staining (a), epithelium of ESS with mDPC-GFP contains numerous Ki67 with K6 expression (b). K10 stained positively in the suprabasal layer of ESS controls (c) and ESS with mDPC-GFP (d), but not the invaginating epidermis in ESS with mDPC-GFP (d). Similarly to embryonic development, LEF1 was restricted to the actively growing hair bulb (f, arrowhead) and placode (g, arrowheads). No nuclear LEF1 was observed in ESS controls (e). Corroborating these results, GFP+ dermal condensation was detected beneath the developing placode (h, arrowhead) and within the bulb (i, arrowhead). Dotted lines represent dermal-epidermal junctions. Scale bars = 100 µm.</p