bromochloroacetic-acid and lucifer-yellow

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Topics: Apoptosis; Cell Differentiation; Cells, Cultured; Cytoplasmic Granules; Diffusion; Epidermal Cells; Epidermis; Fibroblasts; Filaggrin Proteins; Fluorescent Dyes; Humans; Intermediate Filament Proteins; Isoquinolines; Keratin-1; Keratin-10; Keratin-2; Keratinocytes; Keratins; Lipid Metabolism; Microscopy, Electron; Organ Culture Techniques; Permeability; RNA, Small Interfering; Solubility; Ultraviolet Rays; Urocanic Acid

We have characterized cell-to-cell communication (coupling) within intact human skin by microinjecting single keratinocytes with a gap junction-permeant tracer (Lucifer Yellow). 25-50 keratinocytes from different layers of the epidermis were seen to be coupled after most injections (n = 31). A few noncommunicating cells were also microinjected (n = 3) or observed within large territories of coupled keratinocytes. Microinjections of dermal fibroblasts demonstrated an extensive coupling (greater than 100 fibroblasts); however, none of the keratinocyte (n = 34) or fibroblast (n = 3) injections revealed coupling between the epidermal and dermal compartments. Cell coupling was found to be more extensive in epidermal ridges than in suprapapillary plates and, in both regions, was less extensive after injection of the basal layer of the epidermis than after that of the suprabasal layers. This study shows that junctional cell-to-cell communications take place in normal and fully differentiated human tissue. The quantitative data gathered also indicate a regional heterogeneity of keratinocyte-to-keratinocyte communication within intact adult skin and the lack of effect of retinoids on this pattern.

Topics: Adult; Cell Communication; Cell Count; Epidermal Cells; Epidermis; Humans; Isoquinolines; Keratins; Microinjections; Microscopy, Fluorescence; Skin; Skin Physiological Phenomena