Protective effects of Phytohustil® on primary human keratinocytes in vitro

We compared raw bee-collected pollen (Raw-BCP), spontaneously fermented BCP (Unstarted-BCP), and BCP fermented with selected microbial starters (Started-BCP) to deepen whether fermentation may favorably affect the nutrients bioaccessibility and functional features of BCP. Under in vitro gastrointestinal batches, the highest serum-availability of phenolic compounds was found in Started-BCP, highlighting the positive effect exerted by selected microbial starters. The same effect was not found in spontaneously fermented BCP. In colon adenocarcinoma cell line-2 (Caco-2) cells stressed by a pro-inflammatory stimulus, the treatment with Started-BCP halted the increase of pro-inflammatory mediator’s level. Started-BCP counteracted efficiently the deleterious effects of inflammatory stimuli on the integrity of the Caco-2 cells monolayer and its barrier function. Started-BCP successfully counteracted the H2O2-induced intracellular accumulation of reactive oxygen species (ROS) in Caco-2 cells. A protective role against lipopolysaccharide (LPS)-induced inflammation was exerted by Started-BCP in human keratinocytes. The same protective effects on Caco-2 and keratinocyte cell lines were negligible after treatments with Raw-BCP or Unstarted-BCP.

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HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

Mediators of Inflammation ◽

10.1155/2017/7435621 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 43

Author(s):

Irma Colombo ◽

Enrico Sangiovanni ◽

Roberta Maggio ◽

Carlo Mattozzi ◽

Stefania Zava ◽

...

Keyword(s):

Cell Density ◽

Skin Diseases ◽

Inflammatory Responses ◽

Human Keratinocytes ◽

Suitable Model ◽

Hacat Cells ◽

Primary Human Keratinocytes ◽

Proinflammatory Mediators ◽

Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

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Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.720111 ◽

2021 ◽

Vol 9 ◽

Author(s):

Shuangshuang Wang ◽

Hua Qian ◽

Liwei Zhang ◽

Panpan Liu ◽

Dexuan Zhuang ◽

...

Keyword(s):

Nuclear Translocation ◽

Human Keratinocytes ◽

Primary Human Keratinocytes ◽

Activated T Cells ◽

Ras Pathway ◽

Ras Family ◽

Interfering Rna

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

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A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

10.1101/122580 ◽

2017 ◽

Author(s):

Ajay Mishra ◽

Angela Oliveira Pisco ◽

Benedicte Oules ◽

Tony Ly ◽

Kifayathullah Liakath-Ali ◽

...

Keyword(s):

Stem Cell ◽

Terminal Differentiation ◽

Human Keratinocytes ◽

Cell Renewal ◽

Stable States ◽

Primary Human Keratinocytes ◽

Network Modelling ◽

Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation1,2. While progress has been made in characterising the stem and differentiated cell compartments3, the transition between the two cell states, termed commitment4, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h. We have previously shown that commitment begins at approximately 4h and differentiation is initiated by 8h5. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal differentiation by negatively regulating ERK MAPK and positively regulating key API transcription factors. Their activity is antagonised by concomitant upregulation of the anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of transient positive and negative interactions, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem cell and differentiated cell) via an unstable (committed) state. In addition phosphatase expression is spatially regulated relative to the location of stem cells, both in vivo and in response to topographical cues in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

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Increased In Vitro Lifespan of Primary Human Keratinocytes Correlates with Decreased Migration

Journal of Investigative Dermatology ◽

10.1038/sj.jid.5700205 ◽

2006 ◽

Vol 126 (5) ◽

pp. 1179-1181 ◽

Cited By ~ 2

Author(s):

Jens Hasskarl ◽

Palanivel Velupillai ◽

Karl Münger

Keyword(s):

Human Keratinocytes ◽

Primary Human Keratinocytes

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Plasma exosomal miR-375-3p regulates mitochondria-dependent keratinocyte apoptosis by targeting XIAP in severe drug-induced skin reactions

Science Translational Medicine ◽

10.1126/scitranslmed.aaw6142 ◽

2020 ◽

Vol 12 (574) ◽

pp. eaaw6142

Author(s):

Chen Zhang ◽

ZhenLai Zhu ◽

JiXin Gao ◽

LuTing Yang ◽

ErLe Dang ◽

...

Keyword(s):

Human Keratinocytes ◽

Clinical Severity ◽

Stevens Johnson Syndrome ◽

Primary Keratinocytes ◽

Primary Human Keratinocytes ◽

Drug Induced ◽

Skin Reactions ◽

Apoptosis Protein ◽

Human Primary Keratinocytes

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug-induced cutaneous reactions characterized by keratinocyte apoptosis. Exosomes are nanometer-sized membranous vesicles in body fluids. They contain functional proteins, mRNAs, and miRNAs, which induce immune dysfunction and influence disease progression. However, their roles and mechanisms in SJS/TEN remain unknown. Our results demonstrate that exosomes isolated from the plasma of patients with SJS/TEN were 30 to 200 nm in diameter and expressed CD9, CD63, CD81, and TSG101 exosome marker proteins. miR-375-3p was markedly up-regulated in 35 patients with SJS/TEN and correlated with clinical severity. Plasma exosomes were internalized by human primary keratinocytes and promoted keratinocyte apoptosis in vitro. Furthermore, miR-375-3p overexpression promoted intrinsic (mitochondria-dependent) apoptosis of human primary keratinocytes via down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a key apoptosis regulator in primary human keratinocytes. In sum, our study indicates that the circulating exosomal miR-375-3p enters keratinocytes, down-regulates XIAP, and induces keratinocyte apoptosis in patients with SJS/TEN.

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Downregulation of Bax mRNA expression and protein stability by the E6 protein of human papillomavirus 16

Journal of General Virology ◽

10.1099/vir.0.80453-0 ◽

2005 ◽

Vol 86 (3) ◽

pp. 611-621 ◽

Cited By ~ 27

Author(s):

Sharon Shnitman Magal ◽

Anna Jackman ◽

Shahar Ish-Shalom ◽

Liat Edri Botzer ◽

Pinhas Gonen ◽

...

Keyword(s):

Human Papillomavirus ◽

Protein Stability ◽

Mrna Expression ◽

Human Keratinocytes ◽

Dependent Manner ◽

Primary Human Keratinocytes ◽

Experimental Conditions ◽

Human Papillomavirus 16 ◽

Bax Protein

Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.

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Specific betapapillomaviruses associated with squamous cell carcinoma of the skin inhibit UVB-induced apoptosis of primary human keratinocytes

Journal of General Virology ◽

10.1099/vir.0.83317-0 ◽

2008 ◽

Vol 89 (9) ◽

pp. 2303-2314 ◽

Cited By ~ 51

Author(s):

Linda Struijk ◽

Els van der Meijden ◽

Siamaque Kazem ◽

Jan ter Schegget ◽

Frank R. de Gruijl ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Human Keratinocytes ◽

Epidemiological Studies ◽

Primary Human Keratinocytes ◽

Carcinogenic Effect ◽

Uvb Irradiation ◽

Induced Apoptosis

Epidemiological studies have shown an association between infections by specific betapapillomaviruses, such as human papillomavirus (HPV) types 5 and 8, and cutaneous squamous cell carcinoma (SCC). The role of betapapillomaviruses in the development of cutaneous SCC is, however, still enigmatic. The ability to inhibit UVB-induced apoptosis, as demonstrated for HPV5 in vitro, may be important in this respect, as survival of DNA-damaged and mutated cells increases the risk of transformation. The aim of this study was to assess whether inhibition of UVB-induced apoptosis is a general property of betapapillomaviruses and to identify apoptotic factors that are potentially involved in this process. Primary human keratinocytes transduced with E6 and E7 of selected betapapillomaviruses (HPV5, HPV8, HPV15, HPV20, HPV24 and HPV38) were characterized and subjected to UVB irradiation. HPV8- and HPV20-expressing keratinocytes in particular showed fewer signs of apoptosis, as demonstrated by lower levels of active caspase 3, less enzymic caspase activity and less DNA fragmentation. The observed inhibition of UVB-induced apoptosis was mediated by E6 and coincided with reduced steady-state expression of the pro-apoptotic protein Bax. In conclusion, E6 of HPV8 and HPV20 reduces the apoptotic responses upon UVB irradiation when expressed in primary human keratinocytes. Infections with HPV8 and HPV20 may therefore augment the carcinogenic effect of UV radiation and potentially contribute to oncogenic transformation of the skin.

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C6 Ceramide (d18:1/6:0) as a Novel Treatment of Cutaneous T Cell Lymphoma

Cancers ◽

10.3390/cancers13020270 ◽

2021 ◽

Vol 13 (2) ◽

pp. 270

Author(s):

Raphael Wilhelm ◽

Timon Eckes ◽

Gergely Imre ◽

Stefan Kippenberger ◽

Markus Meissner ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Human Keratinocytes ◽

Protective Effects ◽

Hacat Keratinocytes ◽

Primary Human Keratinocytes ◽

T Cell Lymphomas ◽

Keratinocyte Cell ◽

C6 Ceramide ◽

Apoptosis And Necrosis

Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.

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