紫外线对皮肤角质形成细胞和成纤维细胞产生MMP-1和MMP-3的影响

目的:研究中波紫外线辐射对体外培养的表皮角质形成细胞和真皮成纤维细胞产生基质金属蛋白酶-1(MMP-1)和MMP-3的直接和间接影响,研究绿茶中的主要活性成分表没食子儿茶酚没食子酸酯(EGCG)对此影响的保护作用。方法:体外培养角质形成细胞株HaCaT和真皮成纤维细胞,中波紫外线辐射、不同浓度IL-6刺激及EGCG处理后,ELISA方法测定上清液中Pro-MMP-1和MMP-3蛋白含量,半定量RT-PCR方法测细胞中mRNA含量。结果:UVB30mJ/cm2辐射后角质形成细胞分泌的pro-MMP-1和MMP-3并未增加(P>0.05),真皮成纤维细胞合成和分泌MMP-1和MMP-3mRNA含量和蛋白水平均显著增加(P<0.05),IL-6(8、16、24pg/mL)可显著增加成纤维细胞产生MMP-1和MMP-3(P<0.05)。EGCG(0.15、0.3mM)能够显著抑制紫外线诱导成纤维细胞产生MMP含量的增加(P<0.05),但IL-6刺激成纤维细胞所产生的MMP-1和MMP-3的增加不受EGCG的影响(P>0.05)。结论:中波紫外线辐射并不能直接导致角质形成细胞分泌MMP-1和MMP-3增加,但紫外线辐射后角质形成细胞分泌的IL-6可促进成纤维细胞产生MMP。EGCG对IL-6刺激成纤维细胞产生MMP增加没有影响,但它可以显著抑制紫外线辐射直接导致的成纤维细胞产生MMP-1和MMP-3的增加,对防治皮肤光老化可能有一定作用。     

An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes

One of the critical challenges for cellular genetic studies in primary human skin cells is lack of a gene delivery system that provides efficient transduction and sustained expression of the transgenes. Due to the limited time of survival in culture, the processes of drug selection and clonal expansion for establishing gene stably expressing cell lines are not a realistic option for primary skin cells. We have examined various gene transduction techniques in primary dermal fibroblasts and epidermal keratinocytes of human skin. We report here that vectors based on the human immunodeficiency virus (HIV, lentivirus) offer more than 90% gene transduction efficiency and sustained expression of transgenes in both human skin cell types. In contrast, most of the commonly used techniques have at best 30% transduction efficiency in these cells. Using two previously reported migration control genes, protein kinase Cdelta and p38alpha-MAPK, as examples, we provide evidence that the unprecedented efficiency of the lentiviral system enables a clear detection of the genes' dominant negative effects, which are otherwise greatly compromised by ordinary transfection techniques. We believe that a wide application of this gene transduction system will greatly benefit studies of gene function in human skin cells.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

Basal and UV-induced MMP-1 expression are inhibited by p53 in human dermal fibroblasts

Ultraviolet (UV) triggers induction of matrix metalloproteinase-1 (MMP-1) and also induces the rapid appearance of p53 protein in the epidermis and dermal fibroblasts. However, little is known about the role of p53 on MMP-1 expression in human skin. Here, we investigated the effects of p53 on basal and UV-induced MMP-1 in human dermal fibroblasts. To verify the role of p53 on MMP-1 expression, adenoviral p53 (Ad-p53) gene was infected to human dermal fibroblasts. Our results showed that basal and UV-induced MMP-1 expression was prevented by Ad-p53. In contrast, p53 inhibitor, pifithrin-alpha augmented the UV-induced MMP-1 expression. On the other hand, p53 activator, etoposide, decreased the MMP-1 expression in both normal and p53-overexpressed cells. In addition, MMP-1 expression was not changed by etoposide and/or pifithrin-alpha in HaCaT cells, which have mutation of p53. Taken together, we suggest that p53 inhibits basal and UV-induced MMP-1 expression in human dermal fibroblasts.     

Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.     

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.     

Epidermolysis bullosa acquisita antigen is synthesized by both human keratinocytes and human dermal fibroblasts

In order to determine the source of epidermolysis bullosa acquisita (EBA) antigen, we studied its synthesis by human keratinocytes and fibroblasts in culture. To identify the antigen, we used the sera of 5 patients with EBA. These sera had antibodies directed against the epidermal basement membrane zone (BMZ) at titers of 5-80. The 5 sera were further characterized by immunoblotting on extracts of the epidermal BMZ. In concert with previous reports, 4 sera stained a 290 kD polypeptide, 3 sera weakly stained a 145 kD polypeptide, and 1 serum did not bind either polypeptide. To study the synthesis of the EBA antigen, cultured human keratinocytes and fibroblasts, derived from neonatal foreskins, were metabolically labeled with 14C-labeled amino acids. Radiolabeled newly synthesized proteins that were extracted from these cultures with nonionic detergent were used in an immunoprecipitation assay. The precipitated proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. All 5 EBA sera, but none of 3 bullous pemphigoid or 4 normal human sera, precipitated a 290 kD polypeptide from extracts of both keratinocytes and fibroblasts. Approximately equal amounts of this 290 kD polypeptide were precipitated from equivalent amounts of extracts from either cell type. The 290 kD polypeptide was also specifically precipitated by EBA sera from extracts of a human squamous cell carcinoma line, SCC-15. The 145 kD polypeptide was not detected in the newly synthesized proteins of any of these cell cultures. This finding suggests that the 145 kD polypeptide is not a precursor of the 290 kD polypeptide. Taken together these results demonstrate that the EBA antigen is synthesized by both human keratinocytes and fibroblasts, and is not a tissue (epidermal)-specific product.     

Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine factors important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte growth and differentiation and for use in pharmacotoxicology.     

Calcium/calmodulin regulation of the proliferation of human epidermal keratinocytes, dermal fibroblasts and mouse B16 melanoma cells in culture

We have investigated the relationship between extracellular calcium, intracellular calmodulin and proliferation in normal keratinocytes. Keratinocyte proliferation and its sensitivity to calmodulin antagonists was compared with that of normal human dermal fibroblasts and neoplastic mouse B16 melanoma cells. Keratinocytes were similar to fibroblasts in showing reduced proliferation in low (0.15 mM) calcium medium and unlike B16 cells which continued to proliferate until calcium was reduced to submicromolar levels. Intracellular calmodulin was significantly higher in rapidly dividing keratinocytes in normal (1.15 mM) calcium medium than in slower dividing cells in low (0.15 mM) calcium. Fibroblasts and B16 cells maintained similar calmodulin levels in both low and normal calcium media. Calmodulin antagonists inhibited proliferation of all three cell types equally. Thus, keratinocyte calmodulin seems related to the proliferative state of the cell (unlike fibroblast calmodulin) and calmodulin antagonists may be of use in controlling the hyperproliferation of the psoriatic epidermis.     

Epigallocatechin-3-gallate prevents heat shock-induced MMP-1 expression by inhibiting AP-1 activity in human dermal fibroblasts

The anti-skin aging effects of epigallocatechin-3-gallate (EGCG) have been studied extensively in vitro and in vivo models. Accumulating data suggest that EGCG possesses important antioxidant and photoprotective properties. Our previous study demonstrated that heat exposure induces cutaneous angiogenesis and inflammatory cellular infiltration, disrupts the dermal extracellular matrix by inducing matrix metalloproteinases, and alters dermal structural proteins, thereby causing premature skin aging. In the present study, we examined whether EGCG may inhibit expression of MMP-1 in heat-stimulated human dermal fibroblasts. Furthermore, we investigated the inhibitory mechanism of EGCG on heat-induced MMP-1 expression. Western blot analysis and MMP-1 promoter assay revealed that EGCG markedly inhibited heat shock-induced MMP-1 expression in human dermal fibroblasts. In addition, we found that heat shock increased AP-1 DNA binding activity, and c-Jun was found to be increased mostly by heat stimulation in a supershift assay, which were suppressed by EGCG treatment. Also, in Western blotting, EGCG significantly inhibited the heat-induced expression of AP-1 constituent proteins, c-Jun, JunB and c-Fos. These results demonstrated that EGCG has abilities to inhibit heat-induced collagenolytic MMP-1 production via interfering with AP-1 pathways. Therefore, we propose that EGCG may be a potential agent for the prevention and treatment for heat shock-induced skin aging (thermal skin aging).     

UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo

Summary We report the effect of UVA irradiation on collagen metabolism of fibroblasts, including both synthesis of the collagen degrading enzyme collagenase and de novo synthesis of type I collagen as the major structural component of the dermis. For this purpose confluent fibroblast monolayers were irradiated under standardized conditions (5, 15, 35, 60 J/cm² using UVASUN 3000, Mutzhas, Munich, FRG, and UV source Sellas sunlight type 2.001, Sellas, Gevelsberg, FRG). Subsequently, total RNA was isolated and subjected to dot blot and northern blot analysis using oligolabelled cDNA clones for human type I collagen, collagenase and -actin. Collagen type I and -actin mRNA levels remained unaltered following irradiation, suggesting that the synthetic pathway of collagen metabolism at the pretranslational level is not affected by short-term UVA irradiation. However, collagenase mRNA was found to be dose-dependently induced in fibroblasts after irradiation, thus probably contributing to the actinic damage to the dermis. These in vitro data were confirmed in vivo using in situ hybridization on frozen sections of biopsy material obtained from UVA irradiated patients.     

Myeloid differentiation factor 88 regulates basal and UV-induced expressions of IL-6 and MMP-1 in human epidermal keratinocytes

Myeloid differentiation factor 88 (MyD88) is known as an adaptor protein for the Toll-like receptor (TLR) family and participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated TLR. In this study, we demonstrated that expression of MyD88 is increased in photoaged skin compared with intrinsic aged human skin of the same elderly individuals, and that acute UV irradiation increases MyD88 expression in human skin in vivo. To investigate the effects of these high levels of MyD88 in photoaged skin and acutely UV-irradiated skin, human epidermal keratinocytes were infected with adenovirus expressing wild-type (MyD88wt), dominant-positive (MyD88DeltaC), and dominant-negative (MyD88DeltaN) MyD88 forms. Overexpression of MyD88wt and MyD88DeltaC, but not of MyD88DeltaN, increased the basal expressions of IL-6 and matrix metalloproteinase-1 (MMP-1) in human epidermal keratinocytes. Moreover, overexpression of MyD88DeltaN prevented UV-induced expressions of IL-6 and MMP-1 by inhibiting UV-induced activation of NF-kappaB and activating protein-1. These results suggest that MyD88 is important in IL-6 and MMP-1 expressions in both acutely UV-irradiated skin and in chronically sun-exposed human skin.     

The effects of epidermal keratinocytes and dermal fibroblasts on the formation of cutaneous basement membrane in three-dimensional culture systems

The cutaneous basement membrane (BM) plays an important role in normal and pathological conditions. However, few studies have addressed the formation of the cutaneous BM using three-dimensional culture systems. In this study, to elucidate the effects of human epidermal keratinocytes and dermal fibroblasts on the formation of the cutaneous BM, keratinocytes were cultured on several dermal substrates in the presence or absence of fibroblasts at the air–liquid interface. After 2 weeks of culture, immunohistochemical stainings for the components of the BM and electron microscopic studies of the BM zone (BMZ) were performed. In cultures of keratinocytes alone on dead reticular dermis or collagen gel without fibroblasts, 4 integrin chain, laminin, type IV and VII collagens were all expressed. However, ultrastructurally, BMZ was not formed. In cultures of keratinocytes on fibroblast-populated collagen matrix, laminin, and type IV and VII collagens were expressed more strongly than in the absence of fibroblasts. In addition, elements of the BMZ such as hemidesmosomes, lamina lucida, lamina densa and anchoring fibrils were formed, although it was still incomplete. In the culture of keratinocytes alone on de-epidermized dermis (DED) (surface up), 4 integrin chain, laminin, and type IV and VII collagens were strongly expressed. Also, the BMZ appeared similar to that in normal skin. In cocultures of keratinocytes and fibroblasts on DED or cultures of keratinocytes on DED combined with fibroblast-populated collagen matrix, type IV collagen was expressed more strongly than in cultures of keratinocytes alone. Ultrastructurally, similar findings to those of cultures of keratinocytes alone on DED were seen. Interestingly, when keratinocytes and fibroblasts were cocultured on DED, some fibroblasts were seen in the upper dermis as a result of migration into the dermis through partial loss of the lamina densa. These results show that keratinocytes produce most of the components of the BM such as laminin, and type IV and VII collagens. In addition, fibroblasts stimulate the expression of the components of the BM and the formation of a BMZ, suggesting that fibroblasts may produce laminin, and type IV and VII collagens or influence the effects of keratinocytes on the formation of the BM through a keratinocyte–fibroblast interaction.This investigation was supported by a grant (04-2001-027) from the Seoul National University Hospital Research Fund and partly by the Pacific Corporation.     

Studies on guinea pig skin cell cultures. VI. Growth kinetics of epidermal keratinocytes and dermal fibroblasts.

The growth kinetics of epidermal keratinocytes (EK) and dermal fibroblasts (DF) have been determined by four different methods: incorporation of 3H-thymidine into DNA (3H-microgram DNA ratio); 3H-thymidine/14C amino acid incorporation ratio (3H:14C ratio); 3H-thymidine labelled nuclei, and colchicine-blocked metaphase counts. The growth curve of EK was no different when plotted with the 3H:14C ratio than with the 3H-microgram DNA ratio. However, this was not true for DF. The replacement of sodium bicarbonate with Hepes buffer in the culture medium did not greatly affect the shape of the EK growth curve, whereas the DF growth curve became diphasic instead of monophasic. The elimination of mature (differentiated) keratinocytes from the very onset of EK culture had a profound effect on the EK growth curve. DNA synthesis peaked at day 1 in cultures without, instead of day 9 in cultures with differentiated cells. Furthermore, mitotic activity did not show up before day 6. This suggests that (i) EK in culture are sensitive to the G1 inhibitor released by differentiated epidermal cells, and (ii) they remain in G2 for about 5 days. Thus, EK in culture seem to continue to be susceptible, as in vivo, to homeostatic regulation through the action of G1-G2 inhibitors.