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Notes: Oxysterols stimulate keratinocyte differentiation, which involves the formation of the cornified envelope on the inner plasma membrane by transglutaminase crosslinking of several constituent proteins. Oxysterols increase the expression of one of these crosslinked proteins, involucrin, which can be abolished by mutations of the distal activator protein (AP)-1 response element in the involucrin promoter. Here, we show that oxysterols increase AP-1 binding in electrophoretic gel mobility shift assays and that oxysterols induce the expression of an AP-1 reporter. We further describe the individual components of the AP-1 complex, which are involved in the oxysterol-mediated AP-1 activation and stimulation of keratinocyte differentiation. We identified Fra-1 within the AP-1 DNA binding complex by super shift analysis of nuclear extracts from oxysterol-treated, cultured keratinocytes. Western blot analysis demonstrated that oxysterol treatment increased the levels of Fra-1 and Jun-D, while Northern analysis revealed that oxysterols increased mRNA levels for Fra-1, c-Fos and Jun-D. Together these data indicate that oxysterols stimulate involucrin expression by increasing the levels of specific proteins of the AP-1 complex, indicating that oxysterols regulate keratinocyte differentiation by inducing the AP-1 factors, which in turn activate the genes required for epidermal differentiation. The presence of LXREs within the promoter regions of individual AP-1 proteins suggests that these oxysterol effects may be mediated by LXR.

Notes: Abstract The signalling mechanisms that regulate epidermal permeability barrier homeostasis arc not known. Previous Northern blot analysis showed that both acute and chronic barrier disruption increase mRNA levels of several cytokines in murine epidermis. To further characterize the epidermal response to barrier abrogation, we used more sensitive, multi-probe RNasc protection assays to measure the mRNA levels of additional cytokines. as well as cytokine receptors in acute and chronic models of barrier disruption. Normal mouse epidermis expressed interleukin (IL)-lα, interferon-γ (IFN-γ). tumor necrosis factor-α (TNF-α) and IL-6 mRNAs. Following tape-stripping, only the mRNA levels for TNF-α, IL-lα, IL-lβ and IL-6 increased at 2.5 and 7 h, and returned toward normal levels by IX h. No mRNAs encoding TNF-β. IL-2, IL-3. IL-4 or IL-5, were detected in the epidermis either under basal conditions or after tape-stripping. Similarly, in a chronic model, essential fatty acid deficiency, epidermal levels of TNF-α, IL-lα, IL-lβ and IL-6 mRNAs, but not IFN-γ mRNA. were elevated over controls; and again, mRNAs for the remaining probed cytokines were not detected. In contrast, in the dermis. only IL-Iβ mRNA levels increased 2.5 h after tape-stripping, and remained elevated at I8 h. mRNAs encoding the IL-1 (p60), IFN-γ ami IL-6 receptors were present in epidermis, but their levels remained unchanged following either acute or chronic barrier disruption. In contrast, epidermal TNF (p55) receptor mRNA levels were increased by 87% (P〈0.01) at 2.5 h, returned to control levels at 7 h and were increased by 68% (p〈0.03) at 18 h after tape-stripping. The increase at 2 h was confirmed by Northern blot analysis and was not prevented by latex occlusion performed immediately after tape-stripping. mRNAs for the IL-I (p80) receptor and TNF (p75) receptor were not detected in epidermis. Low levels of TNF (p55) receptor mRNA were present in the dermis. and they remained unchanged after tape-stripping. The presence of specific receptor mRNAs in the epidermis and dermis suggests that these tissues are capable of responding in an autocrine and/or paracrine fashion to the cognate cytokines. These results suggest that epidermal cytokines produced after barrier disruption may initiate a cytokine cascade which could regulate cytokine and cytokine receptor production and/or inflammatory responses.

Notes: Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.

Notes: Abstract:  With the exception of vitamin D production, virtually all epidermal functions can be considered as protective, or more specifically, as defensive in nature (1). Yet, the term “barrier function” of the stratum corneum (SC) is often used synonymously with only one such defensive function, although arguably its most important, i.e., permeability barrier homeostasis. Regardless of their hierarchy of relative importance, these critical protective functions largely reside in the SC. In this short review, we explore the ways in which the multiple defensive functions of the SC are linked and interrelated, either by their shared localization or by common biochemical processes.

Notes: Abstract Disruption of the cutaneous permeability barrier increases mRNA levels for TNF, GM-CSF, FL-1 alpha, and IL-1 beta in the epidermis. We have hypothesized that the cylokines mediate the changes in lipid and DNA synthesis which occur following barrier disruption. To further characterize the cytokine response to barrier abrogation, we examined the levels of epidermal IL-Ira mRNA in two acute models and one chronic model in the hairless mouse. IL-Ira mRNA levels increased shortly after acute disruption of the barrier with acetone, reached a peak at 3–4 h after treatment, and returned to control levels by 8h. These changes in mRNA levels parallel those which occur for IL-1 alpha and beta. Furthermore, IL-Ira mRNA levels were elevated 5-fold and 4-fold, at 2.5 h and 4 h, respectively, following tape-stripping, a second acute model of barrier disruption. Finally, IL-Ira mRNA levels were elevated 2.5-fold in the epidermis of EFAD mice, which have a chronic barrier defect. Thus, the cutaneous response to barrier disruption includes mechanisms which increase IL-I and IL-Ira mRNA levels in a coordinate manner. The net result provides a regulatory mechanism for controlling the biological effects of increased IL-1 production.

Notes: A system is described for the study of the in vitro synthesis of bovine keratohyalin for periods of up to 24 h. Keratohyalin granules appeared morphologically unaltered during culture, although histo-chetnical stains for RNA indicate a markedly diminished RNA content by 6 h. Incorporation of tritiated histidine began slowly, then became linear between 4 and 24 h in serum-containing media. However, following pre-incubation in serum-free media, increased incorporation occurred from time zero. The internal variation of the system using a standard 6 h pulse was ±10, ±24, ±22, ±18%, in four separate experiments. Measurements of modulations in bovine keratohyalin synthesis in vitro may prove useful in the development of drugs of therapeutic potential in dermatology.