Suppression of proliferation of human epidermal keratinocytes by 1,25-dihydroxyvitamin D3Analysis of its effect on psoriatic lesion and of its mechanism using human keratinocytes in culture

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

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Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

Molecules ◽

10.3390/molecules26082153 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2153

Author(s):

Raffaella Marina Lecci ◽

Isabella D’Antuono ◽

Angela Cardinali ◽

Antonella Garbetta ◽

Vito Linsalata ◽

...

Keyword(s):

Antioxidant Activities ◽

Biological Activities ◽

Human Keratinocytes ◽

Ldl Oxidation ◽

Trolox Equivalent Antioxidant Capacity ◽

Epidermal Keratinocytes ◽

Olive Cultivar ◽

Human Epidermal Keratinocytes ◽

Apoptotic Effect

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.

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β1 Integrins Regulate Keratinocyte Adhesion and Differentiation by Distinct Mechanisms

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.453 ◽

2000 ◽

Vol 11 (2) ◽

pp. 453-466 ◽

Cited By ~ 98

Author(s):

Laurence Levy ◽

Simon Broad ◽

Dagmar Diekmann ◽

Richard D. Evans ◽

Fiona M. Watt

Keyword(s):

Point Mutations ◽

Terminal Differentiation ◽

Focal Adhesions ◽

Human Keratinocytes ◽

Proximal Part ◽

Epidermal Keratinocytes ◽

Β1 Integrin ◽

Primary Human Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Β1 Integrins

In keratinocytes, the β1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick β1 integrin subunits. We examined the ability of adhesion-blocking chick β1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick β1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by β1 integrins in normal keratinocytes was “do not differentiate” (transduced by ligand-occupied receptors) as opposed to “do differentiate” (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the β1 subunit. We conclude that distinct signaling pathways are involved in β1 integrin–mediated adhesion and differentiation control in keratinocytes.

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Apoptosis is not required for acantholysis in pemphigus vulgaris

AJP Cell Physiology ◽

10.1152/ajpcell.00161.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. C162-C172 ◽

Cited By ~ 32

Author(s):

Enno Schmidt ◽

Judith Gutberlet ◽

Daniela Siegmund ◽

Daniela Berg ◽

Harald Wajant ◽

...

Keyword(s):

Caspase 3 ◽

Pemphigus Vulgaris ◽

Human Keratinocytes ◽

Skin Lesions ◽

Terminal Deoxynucleotidyl Transferase ◽

Epidermal Keratinocytes ◽

Death Domain ◽

Human Epidermal Keratinocytes ◽

Desmosomal Cadherins ◽

Normal Human

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE)-like inhibitory proteins FLIPL and FLIPS to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.

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Mangostanin, a Xanthone Derived from Garcinia mangostana Fruit, Exerts Protective and Reparative Effects on Oxidative Damage in Human Keratinocytes

Pharmaceuticals ◽

10.3390/ph15010084 ◽

2022 ◽

Vol 15 (1) ◽

pp. 84

Author(s):

Mario Abate ◽

Cristina Pagano ◽

Milena Masullo ◽

Marianna Citro ◽

Simona Pisanti ◽

...

Keyword(s):

Oxidative Damage ◽

Cell Damage ◽

Human Keratinocytes ◽

Skin Aging ◽

Epidermal Keratinocytes ◽

Anticancer Activities ◽

Garcinia Mangostana ◽

Human Epidermal Keratinocytes ◽

Extracellular Signal

The fruit of Garcinia mangostana (mangosteen) is known in ancient traditional Asian medicine for its antioxidant, anti-inflammatory, immunomodulatory and anticancer activities. These effects are mainly due to the action of polyphenols known as xanthones, which are contained in the pericarp of the fruit. In recent years, there has been a growing interest from pharmaceutical companies in formulating new topicals based on mangosteen full extracts to prevent skin aging. However, the molecules responsible for these effects and the mechanisms involved have not been investigated so far. Here, the arils and shells of Garcinia mangostana were extracted with chloroform and methanol, and the extracts were further purified to yield 12 xanthone derivatives. Their effects were evaluated using in vitro cultures of human epidermal keratinocytes. After confirming the absence of cytotoxicity, we evaluated the antioxidant potential of these compounds, identifying mangostanin as capable of both protecting and restoring oxidative damage induced by H2O2. We showed how mangostanin, by reducing the generation of intracellular reactive oxygen species (ROS), prevents the activation of AKT (protein kinase B), ERK (extracellular signal-regulated kinase), p53, and other cellular pathways underlying cell damage and apoptosis activation. In conclusion, our study is the first to demonstrate that mangostanin is effective in protecting the skin from the action of free radicals, thus preventing skin aging, confirming a potential toward its development in the nutraceutical and cosmeceutical fields.

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Decreased expression of fibronectin and the alpha 5 beta 1 integrin during terminal differentiation of human keratinocytes

Journal of Cell Science ◽

10.1242/jcs.98.2.225 ◽

1991 ◽

Vol 98 (2) ◽

pp. 225-232 ◽

Cited By ~ 5

Author(s):

L.J. Nicholson ◽

F.M. Watt

Keyword(s):

Messenger Rna ◽

Basal Layer ◽

Terminal Differentiation ◽

Human Keratinocytes ◽

Epidermal Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Fibronectin Receptor ◽

Beta 1 Integrin ◽

Differentiation Marker ◽

Unit Gravity Sedimentation

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

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Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.

The Journal of Cell Biology ◽

10.1083/jcb.103.1.41 ◽

1986 ◽

Vol 103 (1) ◽

pp. 41-48 ◽

Cited By ~ 40

Author(s):

A S Ma ◽

T T Sun

Keyword(s):

Human Keratinocytes ◽

Epidermal Differentiation ◽

Immunofluorescent Staining ◽

Soluble Form ◽

Epidermal Keratinocytes ◽

Cell Periphery ◽

Human Epidermal Keratinocytes ◽

Intracellular Location ◽

Differentiated Cells ◽

Insoluble Form

We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.

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S100C/A11 is a key mediator of Ca2+-induced growth inhibition of human epidermal keratinocytes

The Journal of Cell Biology ◽

10.1083/jcb.200304017 ◽

2003 ◽

Vol 163 (4) ◽

pp. 825-835 ◽

Cited By ~ 80

Author(s):

Masakiyo Sakaguchi ◽

Masahiro Miyazaki ◽

Mikiro Takaishi ◽

Yoshihiko Sakaguchi ◽

Eiichi Makino ◽

...

Keyword(s):

Growth Inhibition ◽

Growth Regulation ◽

Nuclear Translocation ◽

Basal Layer ◽

Human Keratinocytes ◽

Negative Regulator ◽

Epidermal Keratinocytes ◽

Proliferating Cells ◽

Human Epidermal Keratinocytes

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

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Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0147 ◽

2017 ◽

Vol 95 (1) ◽

pp. 64-68 ◽

Cited By ~ 4

Author(s):

Ryo Uchida ◽

Reiji Aoki ◽

Ayako Aoki-Yoshida ◽

Atsushi Tajima ◽

Yoshiharu Takayama

Keyword(s):

P38 Mapk ◽

Barrier Function ◽

Regulatory Element ◽

Human Keratinocytes ◽

Epithelial Differentiation ◽

Mitogen Activated Protein Kinase ◽

Keratinocyte Differentiation ◽

Epidermal Keratinocytes ◽

Bovine Lactoferrin ◽

Human Epidermal Keratinocytes

The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

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TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.

Cell Regulation ◽

10.1091/mbc.1.11.791 ◽

1990 ◽

Vol 1 (11) ◽

pp. 791-809 ◽

Cited By ~ 83

Author(s):

Y Choi ◽

E Fuchs

Keyword(s):

Retinoic Acid ◽

Environmental Changes ◽

Human Keratinocytes ◽

Epidermal Keratinocytes ◽

Basal Cells ◽

Tgf Beta ◽

Human Epidermal Keratinocytes ◽

Human Epidermal Cells ◽

Epidermal Growth

In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.

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