Differentiation-dependent expression of NADP(H):quinone oxidoreductase-1 via NF-E2 related factor-2 activation in human epidermal keratinocytes

Background

NADP(H):quinone oxidoreductase-1 (NQO-1) is known for its protective role in skin carcinogenesis, but the expression of NQO-1 during keratinocyte (KC) differentiation has not been studied.

Objective

The purpose of the current study was to evaluate modulation of NQO-1 and NF-E2-related factor-2 (Nrf2) during KC differentiation.

Methods

Normal human epidermal keratinocytes (NHEKs) were induced to differentiation by prolonged culture after confluency (postconfluence).

Results

NQO-1 was induced at the late stage of differentiation of NHEKs (7th day of postconfluence). The expression of postconfluence-induced NQO-1 was stimulated by 0.1 mM H₂O₂, but attenuated by 5 mM N-acetylcysteine, implying that reactive oxygen species (ROS) are implicated in the expression of NQO-1 in differentiated KCs. Nrf2 was up-regulated at the earlier than NQO-1 induction (3rd day of postconfluence). The Nrf2-dependent expression of NQO-1 was further supported by Nrf2-siRNA experiments. A confocal study confirmed the differentiation-dependent induction and activation of NOQ-1 and Nrf-2 in NHEKs. Immunohistochemistry showed that NQO-1 was accentuated in the upper epidermal layers, supporting the notion that differentiation-dependent NQO-1 expression is functional in human skin in vivo.

Conclusion

These results demonstrate that NQO-1 is modulated during KC differentiation via Nrf2 pathway, suggesting the active role of NQO-1 in the differentiating epidermis.     

Comparison of activities dependent on glutathione S-transferase and cytochrome P-450 IA1 in cultured keratinocytes and reconstructed epidermal models

There is an increasing need for in vitro testing of compounds for topical application. Reconstructed epidermal models may provide a suitable and relevant model for screening compounds that may affect the activities of phase I and II enzymes involved in epidermal detoxification. In this study, we measured the activity of a phase I enzyme, cytochrome P450 IA1, i.e. 7-ethoxyresorufin-O-deethylase (EROD) and 7-ethoxycoumarin-O-deethylase (ECOD) activities, and that of a phase II enzyme, glutathione S-transferase (GST). The enzyme activities were determined in cultured keratinocytes, reconstructed epidermal models and samples of human epidermis or hair follicle. EROD activity was detected in cultured keratinocytes and was induced by 3-methylcholanthrene (3-MC) and beta-naphthoflavone. The level of induction increased with increasing confluence. Induced EROD activity could be inhibited by clotrimazole in a dose-dependent manner. However, EROD activity was not detected in either hair follicles or untreated epidermal models but could be induced by 3-MC. The ability to induce EROD activity in epidermal models was batch dependent, and clotrimazole was able to inhibit the induced EROD activity. ECOD activity was detected in untreated models and paralleled EROD activity. GST activity was detected in cultured keratinocytes and all epidermal models. GST activity in models was equal or higher than the activity in epidermal samples. Reconstructed skin models may be useful to study the effects of non-water-soluble topical formulations on xenobiotic metabolism.     

Expression of microfibril-associated glycoprotein-1 (MAGP-1) in human epidermal keratinocytes

Abstract Microfibril-associated glycoprotein (MAGP) is a major structural component of connective tissue microfibrils. We studied the expression of MAGP-1 in cultured human keratinocytes and its modulation during Ca⁺⁺-induced differentiation. RT-PCR and Western blot assays demonstrated the presence of mRNA and the polypeptide of MAGP-1 in cultured keratinocytes. MAGP-1 mRNA levels in cultured keratinocytes during Ca⁺⁺-induced differentiation were enhanced eightfold with a concomitant increase in involucrin (a marker of terminal differentiation) mRNA levels. Double immunofluorescence labeling of cultured keratinocytes demonstrated that both anti-MAGP-1 and anti-involucrin antibodies reacted with the identical cells. The population of MAGP-1-producing cells in cultured keratinocytes significantly increased during Ca⁺⁺-induced differentiation. These results indicate that MAGP-1 expressed by cultured keratinocytes reaches maximum levels at the stage of terminal differentiation in vitro. Double immunostaining of normal human skin with anti-MAGP-1 and anti-elastin antibodies demonstrated the colocalization of MAGP-1-positive and elastin-positive fibers in the superficial and mid-dermis. MAGP-1 produced by keratinocytes may play some functional role in the formation of dermal matrix organization in the dermis. Received: 22 July 1999 / Received after revision: 20 September 1999 / Accepted: 14 October 1999     

Characterization of multiple P2X receptors in cultured normal human epidermal keratinocytes

ATP-gated ion channels (P2X) are expressed in human epidermis and cultured keratinocytes. The aim of this study was to characterize native P2X receptors in normal human epidermal keratinocytes (NHEK) using whole-cell patch clamp technique, RT-PCR, and determination of intracellular Ca(2+) concentration ([Ca(2+)](i)). Application of ATP resulted in an inward current with a reversal potential of 0 mV. Response to ATP showed two types of currents: the slowly desensitizing response and the rapidly desensitizing response. The slowly desensitizing response was blocked by iso-pyridocaphosphate-6-azophenyl-2', 5' disulfonic acid (PPADS), a P2X receptor antagonist. We found that the expression of multiple P2X(2), P2X(3), P2X(5), and P2X(7) receptor subtype mRNA was increased in differentiated cells. On the other hand, the expression of G-protein-coupled P2Y(2) mRNA was downregulated in differentiated cells. Increases in [Ca(2+)](i) evoked by alphabeta-methylene ATP (alphabeta-meATP) and 2', 3'-O-(4-benzoylbenzoyl) ATP (BzATP) were elevated, whereas elevation of [Ca(2+)](i) evoked by uridine 5'-triphosphate (UTP) was decreased in differentiated cells. Application of ATP or UVB radiation increased the expression of P2X(1), P2X(2), P2X(3), and P2X(7) receptors in NHEK. Changes in the expression levels and cation influx via multiple P2X receptors might be involved in the regulation of differentiation and one of the epidermal external sensors.     

Glutathione reductase in human epidermal tumors

Summary Glutathione reductase activities, both with NADPH and NADH, were determined in basal and squamous cell epitheliomas, in verrucae seborrhoeicae and in human epidermis. Significantly elevated activities were measured in basal cell epitheliomas and in verrucae seborrhoeicae. Some properties of the enzyme were also investigated.     

Effects of ozone in normal human epidermal keratinocytes

Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL‐1α levels both increased while ATP levels decreased. We found a decrease in the NAD‐dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure.     

Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin

De novo dermal epidermal junction morphogenesis was studied in a skin model including dermal fibroblasts and epidermal keratinocytes. Sequential gene expression, protein deposition, and localization of basement membrane zone components were studied during 15 days. The morphogenesis of dermal epidermal junction is characterized by an implementation of the different components and then a subsequent plateau phase occurring at day 11. Three groups of genes were identified depending on cellular origin and expression profile: 1/genes of fibroblastic origin (col I alpha1, col III alpha1, nidogen, and fibrillin 1); 2/genes expressed in fibroblasts and keratinocytes with symmetrical expression pattern between both cell types (col IV alpha1, col VII alpha1, and tenascin C); 3/laminin beta3 only expressed in keratinocytes. Use of modified organotypic models excluding one cell type revealed a tight interplay between fibroblasts and keratinocytes for synthesis and localization of the components of dermal epidermal junction. Keratinocytes downregulated mRNA and proteins of fibroblastic origin, upregulated col VII in fibroblasts and were absolutely required for dermal-epidermal junction localization of fibroblastic proteins. Fibroblasts downregulated mRNA of keratinocytes and were needed for extracellular secretion and correct localization of type VII collagen and laminin 5.     

Substance P induces inositol 1,4,5-trisphosphate and intracellular free calcium increase in cultured normal human epidermal keratinocytes

Abstract Substance P is a neuropeptide which is present in peripheral C nerve endings and released from them. Free nerve endings of C nerve are present in human epidermis. The effects of substance P on the transmembrane signaling system of pig epidermal sheets were previously reported. In these studies, a small amount of cells other than keratinocytes contaminated the epidermal sheets and the species difference from human was also noticed. Therefore we investigated the effects of substance P on cultured normal human epidermal keratinocytes. Alteration of intracellular free calcium (Ca²⁺) in single living keratinocytes was studied using an inverted fluorescence microscope and Ca²⁺ -sensitive dye, Fura 2-AM. Treatment of normal human epidermal kertinocytes with substance P resulted in an increase in inositol 1,4,5-trisphosphate and in intracellular Ca²⁺. Substance P inhibited DNA synthesis of the keratinocytes in a dose-dependent manner. These results are consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate hydrolysis of human keratinocytes, resulting in inositol 1,4,5-trisphosphate-Ca²⁺ signal.     

Aminoisobutyric acid uptake in normal and transformed human epidermal keratinocytes

α-Aminoisobutyric acid transport has been demonstrated in cultured human epidermal keratinocytes as well as in the transformed state of these cells. The concentrative uptake is sodium-dependent and may be ascribed to one Michaelis-Menten component whose maximal velocity is 6.9 nmol/min × 10⁶ cells, with an apparent affinity of 3.8 mM. These parameters may be modified, depending upon the nature of malignant transformation. In SVK14 SV40-virus-transformed cells, there is no change of affinity but the maximal velocity is 1.5 fold less than in normal cells. In the spontaneous epidermoid carcinoma-derived A431 line, this phenomenon is inverted; the maximal velocity is unmodified but the system affinity is 2.2-fold higher than in normal cells. The unresponsiveness of this transport system to insulin and epidermal growth factor (EGF) shows that it behaves differently from those of many other cell types.     

Immunoreactive analogues of erythrocyte ankyrin in human epidermal keratinocytes

Using immunoblot and immunofluorescence analysis, we demonstrated the presence and localization of an immunoreactive form of erythrocyte ankyrin in human epidermal keratinocytes. Immunoblot analysis revealed that both human epidermis and cultured epidermal keratinocytes contained ankyrin-like proteins of molecular mass 210 kDa that crossreacted with antihuman erythrocyte ankyrin antibodies. Immunofluorescence microscopy revealed that the plasma membrane of epidermal keratinocytes was stained. Eccrine sweat gland cells and ductal cells were also stained. These results indicate that in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells, an ankyrin-like protein is present as one of the membrane proteins. The present findings and our recent previous studies showing the presence of a spectrin-like protein (fodrin) and 4.1-like proteins in these cells enable us to suggest that a membrane skeletal protein lattice may exist in these cells.     

Immunoreactive analogues of erythrocyte ankyrin in human epidermal keratinocytes

Using immunoblot and immunofluorescence analysis, we demonstrated the presence and localization of an immunoreactive form of erythrocyte an-kyrin in human epidermal keratinocytes. Immunoblot analysis revealed that both human epidermis and cultured epidermal keratinocytes contained ankyrin-like proteins of molecular mass 210 kDa that crossreacted with antihuman erythrocyte ankyrin antibodies. Immunofluorescence microscopy revealed that the plasma membrane of epidermal keratinocytes was stained. Eccrine sweat gland cells and ductal cells were also stained. These results indicate that in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells, an ankyrin-like protein is present as one of the membrane proteins. The present findings and our recent previous studies showing the presence of a spectrin-like protein (fodrin) and 4.1-like proteins in these cells enable us to suggest that a membrane skeletal protein lattice may exist in these cells. Received: 14 November 1994     

表皮黑素细胞与毛囊角质形成细胞接触性共培养方法的建立

目的研究表皮黑素细胞与毛囊角质形成细胞在接触性共培养体系中的相互作用。方法胰酶消化游离毛囊和包皮的表皮，分别获取纯化的角质形成细胞和黑素细胞，第3代毛囊角质形成细胞接种于6孔板中，48h后以表皮黑素细胞：毛囊角质形成细胞为1：2、1：10和1：20的比例接种黑素细胞。在1：10接种体系中，加α-黑素细胞刺激素和烟酰胺，干预6d后，NKI／beteb单抗染色。结果毛囊角质形成细胞定植后接种黑素细胞，两种细胞均增殖迅速。以1：10比例接种更能模拟生理状态。1：20接种可清晰观察到黑素细胞树突与角质形成细胞的接触方式。1：2接种可获大量黑素细胞。在单纯黑素细胞和共培养体系中，α-黑素细胞刺激素对黑素细胞增殖无明显影响，但树突明显增多、伸长。烟酰胺对单纯黑素细胞培养无影响，共培养状态下，黑素细胞树突收缩，黑素体聚集在胞体周围。结论毛囊角质形成细胞与皮黑素细胞接触性共培养．适用于人色素系统的研究。