Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions.

PubMed

Lin, Yin-Ku; Chen, Hsiao-Wen; Leu, Yann-Lii; Yang, Yueh-Lung; Fang, Yu; Su Pang, Jong-Hwei; Hwang, Tsong-Long

2013-01-30

Indigo naturalis is used in traditional Chinese medicine to treat various dermatoses. Our previous clinical studies showed that indigo naturalis is an effective treatment for psoriasis. Herein, the capabilities of indigo naturalis extract and its derivatives to increase claudin-1 expression and tight junction (TJ) function in human keratinocytes and psoriatic lesions were further studied. Claudin-1 expression in psoriatic plaques with or without indigo naturalis treatment was analyzed by immunohistochemical methods. In primary human keratinocytes, the expression of claudin-1 was analyzed by fluorescent immunostaining, a real-time RT-PCR, and Western blot analysis. The effect of indigo naturalis on TJs was evaluated by measuring the transepithelial electrical resistance (TEER) and paracellular tracer flux. The indigo naturalis extract upregulated mRNA and protein expressions of claudin-1 and function of TJs in primary human keratinocytes in concentration-dependent manners. Its main components, indirubin, indigo, and tryptanthrin, exerted synergistic effects on upregulating TJ functions in primary human keratinocytes. In addition, indigo naturalis increased the activity of protein kinase C (PKC), and a known potent PKC inhibitor, Ro318220, attenuated the indigo naturalis-induced claudin-1 expression. Significantly, restoration of claudin-1 was observed in healed psoriatic lesions after indigo naturalis treatment. Indigo naturalis upregulates claudin-1 expression and restores TJ function in keratinocytes. Our data also suggest that indirubin, indigo, and tryptanthrin have a synergistic effect on TJ function. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

PubMed Central

Udasin, Ronald G.; Wen, Xia; Bircsak, Kristin M.; Aleksunes, Lauren M.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2016-01-01

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC50 = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2−/− mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

PubMed

Calapre, Leslie; Gray, Elin S; Kurdykowski, Sandrine; David, Anthony; Descargues, Pascal; Ziman, Mel

2017-06-10

Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

PubMed

Yeung, Bonnie H Y; Wong, Chris K C

2011-01-01

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

PubMed

Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

2014-01-01

MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair

Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

PubMed Central

Dai, Xiuju; Okazaki, Hidenori; Hanakawa, Yasushi; Murakami, Masamoto; Tohyama, Mikiko; Shirakata, Yuji; Sayama, Koji

2013-01-01

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal. PMID:23874436

Hypoxia Regulates mTORC1-Mediated Keratinocyte Motility and Migration via the AMPK Pathway

PubMed Central

Yan, Tiantian; Zhang, Junhui; Tang, Di; Zhang, Xingyue; Jiang, Xupin; Zhao, Liping; Zhang, Qiong; Zhang, Dongxia; Huang, Yuesheng

2017-01-01

Keratinocyte migration, the initial event and rate-limiting step in wound healing, plays a vital role in restoration of the intact skin barrier, also known as re-epithelialization. After acute tissue injury, hypoxic microenvironment gradually develops and acts as an early stimulus to initiate the healing process. Although we have previously found that hypoxia induces keratinocyte migration, the underlying mechanism remains unknown. Here, we first observed that hypoxia increased mTORC1 activity. Recombinant lentivirus vector and Rapamycin were used for silencing mTORC1 in HaCaT cells and primary mouse keratinocytes (MKs). Using cell migration assay and a Zeiss chamber equipped with imaging system, we also demonstrated that mTORC1 downregulation reversed hypoxia-induced keratinocyte motility and lateral migration. Importantly, hypoxia-activated mTORC1 was accompanied by the AMPK downregulation, and we found that the AMPK pathway activators Metformin (Met) and 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) decreased the mTORC1 activity, cell motility and lateral migration. Thus, our results suggest that hypoxia regulates mTORC1-mediated keratinocyte motility and migration via the AMPK pathway. PMID:28068384

SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

2014-06-15

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutatedmore » (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.« less

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

PubMed

Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

2015-02-01

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. © FASEB.

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions

PubMed Central

Leelakanok, Nattawut; Fischer, Carol L.; Bates, Amber M.; Guthmiller, Janet M.; Johnson, Georgia K.; Salem, Aliasger K.; Brogden, Kim A.; Brogden, Nicole K.

2015-01-01

Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM ± std err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2–35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and > 40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. PMID:26367466

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients.

PubMed

Chomiski, Verônica; Gragnani, Alfredo; Bonucci, Jéssica; Correa, Silvana Aparecida Alves; Noronha, Samuel Marcos Ribeiro de; Ferreira, Lydia Masako

2016-08-01

To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

TLR3 mediates release of IL-1β and cell death in keratinocytes in a caspase-4 dependent manner.

PubMed

Grimstad, Øystein; Husebye, Harald; Espevik, Terje

2013-10-01

Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1β through activation of caspase-1 in keratinocytes. TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1β and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1β was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. TLR3 induced release of substantial amounts of pro-IL-1β in keratinocytes. NLRP3 or ASC dependent processing of IL-1β into its cleaved bioactive form was found to be minimal. The release of IL-1β was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1β, but further processing to active IL-1β is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Sumoylation Dynamics During Keratinocyte Differentiation

PubMed Central

Deyrieux, Adeline F.; Rosas-Acosta, Germán; Ozbun, Michelle A.; Wilson, Van G.

2012-01-01

Summary SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several critical keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), calcium-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding critical sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2, and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similar to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Lastly, abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process. PMID:17164289

Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

PubMed Central

Hsu, Mei-Ju; Rixon, Frazer J.; Knebel-Mörsdorf, Dagmar

2011-01-01

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol. PMID:22022400

Effects of Human Mesenchymal Stem Cells Coculture on Calcium-Induced Differentiation of Normal Human Keratinocytes.

PubMed

Sah, Shyam Kishor; Kim, Hae Young; Lee, Ji Hae; Lee, Seong-Wook; Kim, Hyung-Sik; Kim, Yeon-Soo; Kang, Kyung-Sun; Kim, Tae-Yoon

2017-06-01

The influence of mesenchymal stem cells (MSCs) on keratinocytes in altered microenvironments is poorly understood. Here, we cocultured umbilical cord blood-derived MSCs with normal human epidermal keratinocytes to evaluate their paracrine effect in the presence of high extracellular calcium (Ca 2+ ) concentration. High Ca 2+ environment to keratinocytes can disrupt normal skin barrier function due to abnormal/premature differentiation of keratinocytes. Surprisingly, we found that MSCs suppress both proliferation and differentiation of keratinocytes under a high Ca 2+ environment in transforming growth factors β1 (TGFβ1)-dependent manner. Furthermore, we determined that MSCs can regulate the mitogen-activated protein kinases, phosphatidylinositol 3-kinase/protein kinase B, and protein kinase C pathways in Ca 2+ -induced differentiated keratinocytes. Knockdown of TGFβ1 from MSCs results in decreased suppression of differentiation with significantly increased proliferation of keratinocytes compared with control MSCs. MSCs-derived TGFβ1 further induced growth inhibition of keratinocyte in high extracellular Ca 2+ environment as analyzed by a decrease in DNA synthesis, accumulation of phosphorylated retinoblastoma protein, cdc2, and increased mRNA level of p21, and independent of TGFβ1/SMAD pathway. Taken together, we found that MSCs-derived TGFβ1 is a critical regulator of keratinocyte function, and involves multiple proximal signaling cascades. Stem Cells 2017;35:1592-1602. © 2017 AlphaMed Press.

Cyclin D1 localizes in the cytoplasm of keratinocytes during skin differentiation and regulates cell–matrix adhesion

PubMed Central

Fernández-Hernández, Rita; Rafel, Marta; Fusté, Noel P; Aguayo, Rafael S; Casanova, Josep M; Egea, Joaquim; Ferrezuelo, Francisco; Garí, Eloi

2013-01-01

The function of Cyclin D1 (CycD1) has been widely studied in the cell nucleus as a regulatory subunit of the cyclin-dependent kinases Cdk4/6 involved in the control of proliferation and development in mammals. CycD1 has been also localized in the cytoplasm, where its function nevertheless is poorly characterized. In this work we have observed that in normal skin as well as in primary cultures of human keratinocytes, cytoplasmic localization of CycD1 correlated with the degree of differentiation of the keratinocyte. In these conditions, CycD1 co-localized in cytoplasmic foci with exocyst components (Sec6) and regulators (RalA), and with β1 integrin, suggesting a role for CycD1 in the regulation of keratinocyte adhesion during differentiation. Consistent with this hypothesis, CycD1 overexpression increased β1 integrin recycling and drastically reduced the ability of keratinocytes to adhere to the extracellular matrix. We propose that localization of CycD1 in the cytoplasm during skin differentiation could be related to the changes in detachment ability of keratinocytes committed to differentiation. PMID:23839032

EphA2 transmembrane domain is uniquely required for keratinocyte migration by regulating ephrin-A1 levels.

PubMed

Ventrella, Rosa; Kaplan, Nihal; Hoover, Paul; Perez White, Bethany E; Lavker, Robert M; Getsios, Spiro

2018-04-26

EphA2 receptor tyrosine kinase (RTK) is activated by ephrin-A1 ligand, which harbors a GPI-anchor that enhances lipid raft localization. While EphA2 and ephrin-A1 modulate keratinocyte migration and differentiation, the ability of this cell-cell communication complex to localize to different membrane regions in keratinocytes remains unknown. Using a combination of biochemical and imaging approaches, we provide evidence that ephrin-A1 and a ligand-activated form of EphA2 partition outside of lipid raft domains in response to calcium-mediated cell-cell contact stabilization in normal human epidermal keratinocytes (NHEKs). EphA2 transmembrane domain (TMD) swapping with a shorter and molecularly distinct TMD of EphA1 resulted in decreased localization of this RTK at cell-cell junctions and increased expression of ephrin-A1, which is a negative regulator of keratinocyte migration. Accordingly, altered EphA2 membrane distribution at cell-cell contacts limited the ability of keratinocytes to seal linear scratch wounds in vitro in an ephrin-A1-dependent manner. Collectively, these studies highlight a key role for the EphA2 TMD in receptor-ligand membrane distribution at cell-cell contacts that modulates ephrin-A1 levels to allow for efficient keratinocyte migration with relevance for cutaneous wound healing. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes*

PubMed Central

Ko, Eunbi; Choi, Hyun; Kim, Borim; Kim, Minsun; Park, Kkot-Nara; Bae, Il-Hong; Sung, Young Kwan; Lee, Tae Ryong; Shin, Dong Wook; Bae, Yun Soo

2014-01-01

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca2+]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca2+]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1. PMID:25164816

AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

PubMed

Thrash, Barry R; Menges, Craig W; Pierce, Robert H; McCance, Dennis J

2006-04-28

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Cyclic stretch induces upregulation of endothelin-1 with keratinocytes in vitro: Possible role in mechanical stress-induced hyperpigmentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurita, Masakazu, E-mail: masakazukurita@gmail.com; Okazaki, Mutsumi; Fujino, Takashi

2011-05-27

Highlights: {yields} Influence of cyclic stretch on melanogenetic paracrine cytokines was investigated. {yields} Keratinocyte-derived endothelin-1 was upregulated with cyclic stretch. {yields} Degree of upregulation increases dose-dependently. {yields} This upregulation possibly plays a role in the pathogenesis of pigmented disorders. -- Abstract: The aim of this study was to investigate the possible pathological relation between mechanical stress and hyperpigmentation. We did this by investigating the influence of cyclic stretch on the expression of keratinocyte- and fibroblast-derived melanogenetic paracrine cytokines in vitro. Using primary human keratinocytes and fibroblasts, alterations of mRNA expression of melanogenetic paracrine cytokines due to cyclic stretch were investigatedmore » using a real-time polymerase chain reaction (PCR). The cytokines included basic fibroblast growth factor (bFGF), stem cell factor (SCF), granulocyte/macrophage colony-stimulating factor, interleukin-1{alpha}, and endothelin-1 (ET-1) for keratinocytes and bFGF, SCF, and hepatocyte growth factor for fibroblasts. The dose dependence of keratinocyte-derived ET-1 upregulation was further investigated using real-time PCR and an enzyme-linked immunosorbent assay. We also investigated the effects of cyclic stretch on the proliferation and differentiation of keratinocytes. Among the melanogenetic paracrine cytokines investigated, keratinocyte-derived ET-1 was consistently upregulated in all four cell lines. The degree of upregulation increased with the degree of the length and frequency of the stretch; in contrast, cell number and differentiation markers showed no obvious alterations with cyclic stretch. Keratinocyte-derived ET-1 upregulation possibly plays a significant role in the pathogenesis of pigmented disorders, such as friction melanosis, caused by mechanical stress.« less

Investigation of mitomycin-C-treated fibroblasts in 3-D collagen gel and conditioned medium for keratinocyte proliferation.

PubMed

Huang, Yi-Chau; Wang, Tzu-Wei; Sun, Jui-Sheng; Lin, Feng-Huei

2006-03-01

Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. In this study, the paracrine effect of mitomycin-C-treated fibroblasts with various densities in collagen gel for keratinocyte proliferation was investigated from which an optimum cell density and optimum conditioned medium would be determined to expand keratinocyte without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum collected medium preparation will be determined by checking the level of keratinocyte growth factor and granulocyte macrophage colony-stimulating factor in conventional medium. The results showed that the cell density of 1 x 10(5) cells/gel in the feeder gel is better to produce optimum collected medium. The conditioned medium is prepared by mixing together the optimum collected medium and molecular cellular and developmental biology (MCDB) 153 medium in different ratios for keratinocyte growth. The keratinocyte viability will be measured by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed as the better medium for keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to coculture with keratinocytes is also determined as 1 x 10(5) cells/gel. Keratin 10 (K10) and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocytes should not be cultured in postconfluent conditions due to undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays a more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned

Resveratrol induces human keratinocyte damage via the activation of class III histone deacetylase, Sirt1.

PubMed

Lee, Ju-Hee; Kim, Jin-Shang; Park, Sang-Youel; Lee, You-Jin

2016-01-01

Human skin diseases are various and induce chronic inflammatory disorders, including psoriasis, atopic dermatitis and certain forms of ichthyosis. Psoriasis is a chronic inflammatory skin disease characterized by circumscribed, red, thickened plaques. Regulation of the balance between growth, differentiation and death is critical to keratinocytes; when altered, epidermal keratinocytes undergo hyperproliferation, abnormal differentiation and inflammatory infiltration. In the present study, we focused on the effects of resveratrol, found in red wine and peanuts, on the cell death of keratinocytes. We additionally studied the mechanism of resveratrol on Sirt1, a class III histone deacetylase, and Akt phosphorylation. Resveratrol caused apoptosis and increased Sirt1 expression in human HaCaT keratinocytes, following a decrease in the p62 protein level. Inhibition of Sirt1 by Sirt1 inhibitor restored cell viability and protein levels. Furthermore, we showed that resveratrol-induced Sirt1 blocked Akt phosphorylation. The present results indicated that resveratrol inhibited the Akt pathways by inducing Sirt1, thus leading to cell death. These data suggest that resveratrol-mediated activation of Sirt1 histone deacetylase may be a potential therapeutic target for skin diseases including psoriasis.

Melanosome uptake is associated with the proliferation and differentiation of keratinocytes.

PubMed

Choi, Hye-In; Sohn, Kyung-Cheol; Hong, Dong-Kyun; Lee, Young; Kim, Chang Deok; Yoon, Tae-Jin; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Lee, Young Ho

2014-01-01

Melanosomes are synthesized in melanocytes and transferred to neighboring keratinocytes. However, the associations of melanosome uptake with the proliferation and differentiation of keratinocytes are not fully understood. We examined the associations of melanosome uptake with keratinocyte differentiation and proliferation. SV40T-transformed human epidermal keratinocytes (SV-HEKs) were treated with isolated melanosomes. The effects of melanosome uptake on the proliferation and differentiation of the keratinocytes were analyzed by Western blotting and flow cytometry. The relationship between melanosome uptake and keratinocyte differentiation status was verified by determining the melanin content in the cells. Melanosomes reduced the proliferation of SV-HEKs in a dose-dependent manner, but did not induce differentiation. Melanosome uptake was higher in differentiating keratinocytes compared to non-differentiating keratinocytes, and inhibited significantly by PAR-2 inhibitor. Melanosomes inhibit keratinocyte proliferation. Moreover, melanosome uptake is influenced by keratinocyte differentiation status, being highest in mid-stage differentiating keratinocytes in a PAR-2 dependent manner.

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

PubMed

Chang, Wing Y; Bryce, Dawn M; D'Souza, Sudhir J A; Dagnino, Lina

2004-12-03

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes.

PubMed

Okiyama, Naoko; Katz, Stephen I

2014-09-01

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases. Published by Elsevier Ltd.

Heparinoid suppresses Der p-induced IL-1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes.

PubMed

Utsunomiya, Ryo; Dai, Xiuju; Murakami, Masamoto; Okazaki, Hidenori; Tsuda, Teruko; Mori, Hideki; Shiraishi, Ken; Tohyama, Mikiko; Sayama, Koji

2018-05-13

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro-inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen-induced interleukin (IL)-1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1β, but also suppressed IL-1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signaling pathways, the activation of extracellular signal-regulated kinase and p38 pathways, which are required for IL-1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL-1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte-mediated skin inflammation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Hanwen; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistancemore » to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.« less

The Effect of Lipoaspirates on Human Keratinocytes.

PubMed

Kim, Bong-Sung; Gaul, Charel; Paul, Nora E; Dewor, Manfred; Stromps, Jan-Philipp; Hwang, Soo Seok; Nourbakhsh, Mahtab; Bernhagen, Jürgen; Rennekampff, Hans-Oliver; Pallua, Norbert

2016-09-01

One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

PubMed

Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

2013-02-01

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Quantitative analysis of peripheral blood Th0, Th1, Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia

PubMed Central

Saito, S; Sakai, M; Sasaki, Y; Tanebe, K; Tsuda, H; Michimata, T

1999-01-01

We calculated the percentage of Th1, Th2, Th0 cells and the Th1:Th2 cell ratio of peripheral blood from normal pregnant subjects and preeclampsia patients using flow cytometry which can analyse both the surface marker, CD4, and intracellular cytokines, interleukin (IL)-4 and interferon (IFN)-γ. In normal pregnancy, the percentage of Th1 cells was significantly lower in the third trimester, and the ratios of Th1:Th2 were significantly lower in the second and third trimester than in nonpregnant subjects. In contrast, the percentage of Th1 cells and the ratios of Th1:Th2 in preeclampsia were significantly higher than in normal third trimester pregnant subjects. The percentage of Th2 cells in preeclampsia was significantly lower than in third trimester of normal pregnancy. Additionally, peripheral blood mononuclear cells from these subjects and patients were cultured with phytohemagglutinin stimulation, and IL-4 and IFN-γ concentrations were determined in the supernatant by enzymed linked immunosorbent assays. The percentage of Th1 and Th2, and the ratios of Th1:Th2 were correlated with cytokine (IFN-γ and IL-4) secretion level. These results demonstrated that Th2 cells were predominant in the second and third trimesters of normal pregnancy, but Th1 cells predominated in preeclamptic patients. PMID:10469061

Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease.

PubMed

Scorza, Breanna M; Wacker, Mark A; Messingham, Kelly; Kim, Peter; Klingelhutz, Aloysius; Fairley, Janet; Wilson, Mary E

2017-10-01

All Leishmania species parasites are introduced into mammalian skin through a sand fly bite, but different species cause distinct clinical outcomes. Mouse studies suggest that early responses are critical determinants of subsequent adaptive immunity in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant cell in the epidermis. We hypothesized that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with Leishmania infantum or Leishmania major, which cause visceral or cutaneous leishmaniasis, respectively, elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of proinflammatory genes for IL-6, IL-8, tumor necrosis factor, and IL-1B, whereas keratinocytes exposed to several L. major isolates did not. Furthermore, keratinocyte-monocyte co-incubation studies across a 4 µM semipermeable membrane suggested that L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication (P < 0.01). L. major-exposed keratinocytes had no comparable effect. These data suggest that L. infantum and L. major differentially activate keratinocytes to release factors that limit infection in monocytes. We propose that keratinocytes initiate or withhold a proinflammatory response at the site of infection, generating a microenvironment uniquely tailored to each Leishmania species that may affect the course of disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

NASA Technical Reports Server (NTRS)

Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

1996-01-01

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes

PubMed Central

Hiroyasu, Sho; Colburn, Zachary T.; Jones, Jonathan C. R.

2016-01-01

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.—Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes. PMID:26936359

Transcription of thymic stromal lymphopoietin via Toll-like receptor 2 in canine keratinocytes: a possible association of Staphylococcus spp. in the deterioration of allergic inflammation in canine atopic dermatitis.

PubMed

Sakamoto, Mayu; Asahina, Ryota; Kamishina, Hiroaki; Maeda, Sadatoshi

2016-06-01

Colonization, overgrowth and subsequent infection by Staphylococcus spp. is frequently observed in canine atopic dermatitis (CAD), where it contributes to the intensity of cutaneous inflammation. The mechanisms by which staphylococci contribute to the pathogenesis of CAD are unclear. Studies suggest that thymic stromal lymphopoietin (TSLP), a cytokine induced by a cell wall component of Staphylococcus spp., may play a critical role in Th2 responses including the pathogenesis of CAD. To determine if synthetic triacylated lipopeptide (TLR1/2 ligand), a cell wall component of Staphylococcus spp., induces the transcription of TSLP via TLR2 in canine keratinocytes. Transcription of TSLP was quantified in a canine keratinocyte cell line after stimulation with synthetic triacylated lipopeptide, and again after inhibition of TLR2 by a targeted small interfering RNA. The transcription of TSLP was enhanced 6 h after stimulation with the synthetic triacylated lipopeptide; it was completely suppressed by knockdown of TLR2. The results demonstrated that a synthetic cell wall component of Staphylococcus spp. induced transcription of TSLP via TLR2 in canine keratinocytes. Additional studies will be required to investigate whether Staphylococcus spp. contributes to Th2 responses in CAD through TLR2-mediated TSLP production. © 2016 ESVD and ACVD.

Differential Utilization and Localization of ErbB Receptor Tyrosine Kinases in Skin Compared to Normal and Malignant Keratinocytes1

PubMed Central

Stoll, Stefan W; Kansra, Sanjay; Peshick, Scott; Fry, David W; Leopold, Wilbur R; Wiesen, Jane F; Sibilia, Maria; Zhang, Tong; Werb, Zena; Derynck, Rik; Wagner, Erwin F; Elder, James T

2001-01-01

Abstract Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals. PMID:11571634

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

PubMed

Akaza, Narifumi; Akamatsu, Hirohiko; Takeoka, Shiori; Mizutani, Hiroshi; Nakata, Satoru; Matsunaga, Kayoko

2012-11-01

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and β-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not β-1,3-glucan expression.

Cyr61/CCN1 induces CCL20 production by keratinocyte via activating p38 and JNK/AP-1 pathway in psoriasis.

PubMed

Li, Huidan; Li, Haichuan; Huo, Rongfen; Wu, Pinru; Shen, Zhengyu; Xu, Hui; Shen, Baihua; Li, Ningli

2017-10-01

Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) has recently been implicated in psoriasis pathogenesis by promoting keratinocyte activation. However, the mechanisms by which CCN1 enhances cutaneous inflammation are not fully understood. In this study, we investigated the role of CCN1 on the expression of CCL20 in human keratinocyte. By double-label immunofluorescence staining, we first identified that the expression of CCN1 colocalized well with CCL20 production in the epidermis of psoriasis skin lesion. Furthermore, in vivo, blocking or knockdown CCN1 expression ameliorated skin inflammation and reduced the expression of CCL20 in both imiquimod and IL-23-induced psoriasis-like mouse models, which indicated that CCN1 might be involved in the regulation of CCL20 production in psoriasis. Next, in vitro, we stimulated primary normal human epidermal keratinocyte (NHEK) with exogenous protein CCN1 and found that CCN1 directly upregulated CCL20 production independent of TNF-α, IL-22 and IL-17 pathway. Lastly, the signaling pathway study showed that CCN1 enhanced the binding of AP-1 to the CCL20 promoter via crosstalk with p38 and JNK. Our study demonstrates that CCN1 stimulates CCL20 production in vitro and in vivo, and thus supports the notion that overexpressed CCN1 in hyperproliferating keratinocyte is functionally involved in the recruitment of inflammatory cells to skin lesions affected by psoriasis. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

PubMed

Li, Jian; Tang, Lu-Yan; Fu, Wen-Wen; Yuan, Jin; Sheng, You-Yu; Yang, Qin-Ping

2016-12-01

Hydrogen peroxide (H 2 O 2 ) may have a biphasic effect on melanin synthesis and melanosome transfer. High H 2 O 2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H 2 O 2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H 2 O 2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H 2 O 2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H 2 O 2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H 2 O 2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H 2 O 2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H 2 O 2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H 2 O 2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

Keratinocyte-melanocyte interactions during melanosome transfer.

PubMed

Seiberg, M

2001-08-01

The epidermal-melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte-melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation ('injection'), keratinocyte-melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease-activated receptor-2 (PAR-2) support the phagocytosis theory. PAR-2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR-2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte-melanocyte contact. Moreover, PAR-2 is induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of UVB-induced tanning. The role of PAR-2 in mediating UV-induced responses remains to be elucidated.

Melanosome transfer to and translocation in the keratinocyte.

PubMed

Boissy, Raymond E

2003-01-01

Complexion coloration in humans is primarily regulated by the amount and type of melanin synthesized by the epidermal melanocyte. However, additional and equally contributing factors consist of (1) efficient transfer of melanin from the melanocytes to the neighboring keratinocytes and (2) distribution and degradation of the transferred melanosomes by the recipient keratinocytes. Once synthesized in the cell body of the epidermal melanocyte, pigmented melanosomes are translocated down the dendrites and captured at the dendritic tips via various cytoskeletal elements. Molecules recently identified that participate in this process consist of Rab27a, myosin-Va and melanophilin. Eventually, these peripherally localized melanosomes are transferred to keratinocytes by a presently undefined mechanism. The protease-activated receptor-2 (PAR-2) and unidentified surface lectins and glycoproteins facilitate this transfer process. Once incorporated into the keratinocytes, melanosomes are distributed individually or as clusters, aggregated towards the apical pole of the nucleus, and degraded as the keratinocytes undergo terminal differentiation and desquamation. Ultraviolet irradiation (UVR) can modulate the process of melanosome transfer from the melanocytes to the keratinocytes. UVR can upregulate expression of PAR-2 and lectin-binding receptors and increase phagocytic activity of cultured keratinocytes. Therefore, many cellular and molecular events that occur after melanogenesis contribute to skin color.

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients.

PubMed

Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Lanziani, Larissa Elias; Ipolito, Michele Zampieri; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.

Transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to gelatin.

PubMed

Kubo, Miyoko; Clark, Richard A F; Katz, Anne B; Taichman, Lorne B; Jin, Zaishun; Zhao, Ying; Moriguchi, Takahiko

2007-04-01

alphavbeta3 is a multiligand integrin receptor that interacts with fibrinogen (FG), fibrin (FB), fibronectin (FN), vitronectin (VN), and denatured collagen. We previously reported that cultured normal human keratinocytes, like in vivo keratinocytes, do not express alphavbeta3 on the cell surface, and do not adhere to and migrate on FG and FB. Furthermore, we reported that human keratinocytes transduced with beta3 integrin subunit cDNA by a retrovirus-mediated transduction method express alphavbeta3 on the cell surface and adhere to FG, FB, FN, and VN significantly compared with beta-galactosidase (beta-gal) cDNA-transduced keratinocytes (control). In this study, we determined whether these beta3 integrin subunit cDNA-transduced keratinocytes or normal human keratinocytes adhere to denatured collagen (gelatin) using a 1 h cell adhesion assay. beta3 cDNA-transduced keratinocytes adhered to gelatin, whereas no significant adhesion was observed with the control cells (beta-gal cDNA-transduced keratinocytes and normal human keratinocytes). The adhesion to gelatin was inhibited by LM609, a monoclonal antibody to alphavbeta3, and RGD peptides but not by normal mouse IgG1 nor RGE peptides. Thus, transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to denatured collagen (gelatin) as well as to FG, FB, VN, and FN. Otherwise, normal human keratinocytes do not adhere to gelatin. These data support the idea that beta3 cDNA-transduced human keratinocytes can be a good material for cultured epithelium to achieve better take rate with acute or chronic wounds, in which FG, FB, and denatured collagen are abundantly present.

Antioxidant Opuntia ficus-indica Extract Activates AHR-NRF2 Signaling and Upregulates Filaggrin and Loricrin Expression in Human Keratinocytes.

PubMed

Nakahara, Takeshi; Mitoma, Chikage; Hashimoto-Hachiya, Akiko; Takahara, Masakazu; Tsuji, Gaku; Uchi, Hiroshi; Yan, Xianghong; Hachisuka, Junichi; Chiba, Takahito; Esaki, Hitokazu; Kido-Nakahara, Makiko; Furue, Masutaka

2015-10-01

Opuntia ficus-indica (OFI) is a cactus species widely used as an anti-inflammatory, antilipidemic, and hypoglycemic agent. It has been shown that OFI extract (OFIE) inhibits oxidative stress in animal models of diabetes and hepatic disease; however, its antioxidant mechanism remains largely unknown. In this study, we demonstrated that OFIE exhibited potent antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the downstream antioxidant enzyme quinone oxidoreductase 1 (NQO1), which inhibited the generation of reactive oxygen species in keratinocytes challenged with tumor necrosis factor α or benzo[α]pyrene. The antioxidant capacity of OFIE was canceled in NRF2 knockdown keratinocytes. OFIE exerted this NRF2-NQO1 upregulation through activation of the aryl hydrocarbon receptor (AHR). Moreover, the ligation of AHR by OFIE upregulated the expression of epidermal barrier proteins: filaggrin and loricrin. OFIE also prevented TH2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHR-dependent manner because it was canceled in AHR knockdown keratinocytes. Antioxidant OFIE is a potent activator of AHR-NRF2-NQO1 signaling and may be beneficial in treating barrier-disrupted skin disorders.

Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells.

PubMed

Mohan, Girish C; Zhang, Huayi; Bao, Lei; Many, Benjamin; Chan, Lawrence S

2017-01-01

We investigated IL-1-induced regulation of genes related to inflammation and atherogenesis in human keratinocytes and endothelial cells, and if 'diacerein', an oral IL-1 inhibiting drug currently approved for use in osteoarthritis, would reverse IL-1's effects on these cells. Primary human keratinocytes and coronary artery endothelial cells were treated with either IL-1α or IL-1β, with and without diacerein. Using PCR-array, we assessed differential gene-expression regulated by IL-1 and diacerein. We identified 34 pro-atherogenic genes in endothelial cells and 68 pro-inflammatory genes in keratinocytes significantly (p<0.05) regulated at least 2-fold by IL-1, in comparison to control. Diacerein completely or partially reversed this regulation on almost all genes. Using ELISA, we confirmed diacerein's ability to reverse IL-1-driven gene-regulation of 11 selected factors, at the protein level. The results support a novel idea that diacerein acts as an inhibitor of the pro-atherogenic and pro-inflammatory effects of IL-1. Diacerein may have therapeutic applications to diminish IL-1-induced skin inflammation in psoriasis and attenuate IL-1-induced development of atherosclerosis. Further investigation into diacerein's effect on skin inflammation, atherogenesis and cardiovascular risk in animal models or humans is warranted.

Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells

PubMed Central

Bao, Lei; Many, Benjamin; Chan, Lawrence S.

2017-01-01

We investigated IL-1-induced regulation of genes related to inflammation and atherogenesis in human keratinocytes and endothelial cells, and if ‘diacerein’, an oral IL-1 inhibiting drug currently approved for use in osteoarthritis, would reverse IL-1’s effects on these cells. Primary human keratinocytes and coronary artery endothelial cells were treated with either IL-1α or IL-1β, with and without diacerein. Using PCR-array, we assessed differential gene-expression regulated by IL-1 and diacerein. We identified 34 pro-atherogenic genes in endothelial cells and 68 pro-inflammatory genes in keratinocytes significantly (p<0.05) regulated at least 2-fold by IL-1, in comparison to control. Diacerein completely or partially reversed this regulation on almost all genes. Using ELISA, we confirmed diacerein’s ability to reverse IL-1-driven gene-regulation of 11 selected factors, at the protein level. The results support a novel idea that diacerein acts as an inhibitor of the pro-atherogenic and pro-inflammatory effects of IL-1. Diacerein may have therapeutic applications to diminish IL-1-induced skin inflammation in psoriasis and attenuate IL-1-induced development of atherosclerosis. Further investigation into diacerein’s effect on skin inflammation, atherogenesis and cardiovascular risk in animal models or humans is warranted. PMID:28323859

Delayed Activation Kinetics of Th2- and Th17 Cells Compared to Th1 Cells.

PubMed

Duechting, Andrea; Przybyla, Anna; Kuerten, Stefanie; Lehmann, Paul V

2017-09-12

During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.

Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herbert, Katharine J.; Holloway, Adele; Cook, Anthony L.

2014-11-15

Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylationmore » of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

PubMed Central

Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo. PMID:28808357

Human Atopic Dermatitis Skin-derived T Cells can Induce a Reaction in Mouse Keratinocytes in vivo.

PubMed

Martel, B C; Blom, L; Dyring-Andersen, B; Skov, L; Thestrup-Pedersen, K; Skov, S; Skak, K; Poulsen, L K

2015-08-01

In atopic dermatitis (AD), the inflammatory response between skin-infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice through keratinocyte activation and consequently cause the development of eczematous lesions. Punch biopsies of the lesional skin from AD patients were used to establish skin-derived T cell cultures, which were transferred to NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that the subcutaneous injection of the human AD skin-derived T cells resulted in the migration of the human T cells from subcutis to the papillary dermis followed by the development of erythema and oedema in the mouse skin. Furthermore, the human T cells induced a transient proliferative response in the mouse keratinocytes shown as increased numbers of Ki-67(+) keratinocytes and increased epidermal thickness. Out of six established AD skin-derived T cell cultures, two were superior at inducing a skin reaction in the mice, and these cultures were found to contain >10% CCR10(+) T cells compared to <2% for the other cultures. In comparison, blood-derived in vitro-differentiated Th2 cells only induced a weak response in a few of the mice. Thus, we conclude that human AD skin-derived T cells can induce a reaction in the mouse skin through the induction of a proliferative response in the mouse keratinocytes. © 2015 The Foundation for the Scandinavian Journal of Immunology.

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair.

PubMed

Yang, Leilei; Li, Wenlong; Wang, Shaoxia; Wang, Lijuan; Li, Yang; Yang, Xiao; Peng, Ruiyun

2012-10-01

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.

Th1, Th2, and Th17 Cytokine Involvement in Thyroid Associated Ophthalmopathy

PubMed Central

Shen, Jie; Li, Zhangfang; Li, Wenting; Ge, Ying; Xie, Min; Lv, Meng; Fan, Yanfei; Chen, Zhi; Zhao, Defu; Han, Yajuan

2015-01-01

To determine serum cytokine profiles in Graves' disease (GD) patients with or without active and inactive thyroid associated ophthalmopathy (TAO), we recruited 65 subjects: 10 GD only (without TAO), 25 GD + active TAO, 20 GD + TAO, and 10 healthy controls. Liquid chip assay was used to measure serum Th1/Th2/Th17 cytokines including IFN-γ (interferon-gamma), TNF-α (tumor necrosis factor-alpha), IL-1α (interleukin-1 alpha), IL-1Ra (IL-1 receptor antagonist), IL-2, IL-4, IL-6, and IL-17 and two chemokines: RANTES (regulated upon activation, normal T cell expressed and secreted) and IP-10 (IFN-γ-induced protein 10). Serum levels of TSH (thyroid stimulating hormone) receptor autoantibodies (TRAb) were measured using an enzyme linked immunosorbent assay. Compared with healthy controls, TAO patients showed significantly elevated serum levels of IFN-γ, TNF-α, IL-1α, IL-4, IL-6, IL-17, and IP-10. Comparing active and inactive TAO, serum Th1 cytokines IFN-γ and TNF-α were elevated in active TAO, while serum Th2 cytokine IL-4 was elevated in inactive TAO. Serum Th17 cytokine IL-17 was elevated in GD but reduced in both active and inactive TAO. A positive correlation was found between TRAb and IFN-γ, TNF-α, IL-1α, IL-2, IL-4, and IL-6. Taken together, serum Th1/Th2/Th17 cytokines and chemokines reflect TAO disease activity and may be implicated in TAO pathogenesis. PMID:26089587

Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.

PubMed

Lai-Cheong, J E; Ussar, S; Arita, K; Hart, I R; McGrath, J A

2008-09-01

Kindler syndrome (KS) results from pathogenic loss-of-function mutations in the KIND1 gene, which encodes kindlin-1, a focal adhesion and actin cytoskeleton-related protein. How and why abnormalities in kindlin-1 disrupt keratinocyte cell biology in KS, however, is not yet known. In this study, we identified two previously unreported binding proteins of kindlin-1: kindlin-2 and migfilin. Co-immunoprecipitation and confocal microscopy studies show that these three proteins bind to each other and colocalize at focal adhesion in HaCaT cells and normal human keratinocytes. Moreover, loss-of-function mutations in KIND1 result in marked variability in kindlin-1 immunolabeling in KS skin, which is mirrored by similar changes in kindlin-2 and migfilin immunoreactivity. Kindlin-1, however, may function independently of kindlin-2 and migfilin, as loss of kindlin-1 expression in HaCaT keratinocytes by RNA interference and in KS keratinocytes does not affect KIND2 or FBLIM1 (migfilin) gene expression or kindlin-2 and migfilin protein localization. In addition to identifying protein-binding partners for kindlin-1, this study also highlights that KIND1 gene expression and kindlin-1 protein labeling are not always reduced in KS, findings that are relevant to the accurate laboratory diagnosis of this genodermatosis by skin immunohistochemistry.

Effect of gamma-hydroxybutyrate on keratinocytes proliferation: A preliminary prospective controlled study in severe burn patients.

PubMed

Rousseau, Anne-Françoise; Bargues, Laurent; Bever, Hervé Le; Vest, Philippe; Cavalier, Etienne; Ledoux, Didier; Piérard, Gérald E; Damas, Pierre

2014-04-01

Hypermetabolism and hyposomatotropism related to severe burns lead to impaired wound healing. Growth hormone (GH) boosts wound healing notably following stimulation of the production of insulin-like growth factor-1 (IGF1), a mitogen factor for keratinocytes. Gamma-hydroxybutyrate (GHB) stimulates endogenous GH secretion. To assess effects of GHB sedation on keratinocytes proliferation (based on immunohistochemical techniques). Monocentric, prospective, controlled trial. Patients (aging 18-65 years, burn surface area >30%, expected to be sedated for at least one month) were alternately allocated, at the 5(th) day following injury, in three groups according to the intravenous GHB dose administered for 21 days: Evening bolus of 50 mg/kg (Group B), continuous infusion at the rate of 10 mg/kg/h (Group C), or absence of GHB (Group P). They all received local standard cares. Immunohistochemistry (Ki67/MIB-1, Ulex europaeus agglutinin-1 and Mac 387 antibodies) was performed at D21 on adjacent unburned skin sample for assessing any keratinocyte activation. Serum IGF1 levels were measured at initiation and completion of the protocol. Categorical variables were compared with Chi-square test. Comparisons of medians were made using Kruskal-Wallis test. Post hoc analyses were performed using Mann-Whitney test with Bonferroni correction for multiple comparisons. A P < 0.05 was considered to be statistically significant. A total of 14 patients completed the study (Group B: n = 5, Group C: n = 5, Group P: n = 4). Continuous administration of GHB was associated with a significant higher Ki67 immunolabeling at D21 (P = 0.049) and with a significant higher increase in the IGF1 concentrations at D21 (P = 0.024). No adverse effects were disclosed. Our preliminary data support a positive effect of GHB on keratinocyte proliferation and are encouraging enough to warrant large prospective studies.

Effect of gamma-hydroxybutyrate on keratinocytes proliferation: A preliminary prospective controlled study in severe burn patients

PubMed Central

Rousseau, Anne-Françoise; Bargues, Laurent; Bever, Hervé Le; Vest, Philippe; Cavalier, Etienne; Ledoux, Didier; Piérard, Gérald E.; Damas, Pierre

2014-01-01

Background: Hypermetabolism and hyposomatotropism related to severe burns lead to impaired wound healing. Growth hormone (GH) boosts wound healing notably following stimulation of the production of insulin-like growth factor-1 (IGF1), a mitogen factor for keratinocytes. Gamma-hydroxybutyrate (GHB) stimulates endogenous GH secretion. Aim: To assess effects of GHB sedation on keratinocytes proliferation (based on immunohistochemical techniques). Design: Monocentric, prospective, controlled trial. Materials and Methods: Patients (aging 18-65 years, burn surface area >30%, expected to be sedated for at least one month) were alternately allocated, at the 5th day following injury, in three groups according to the intravenous GHB dose administered for 21 days: Evening bolus of 50 mg/kg (Group B), continuous infusion at the rate of 10 mg/kg/h (Group C), or absence of GHB (Group P). They all received local standard cares. Immunohistochemistry (Ki67/MIB-1, Ulex europaeus agglutinin-1 and Mac 387 antibodies) was performed at D21 on adjacent unburned skin sample for assessing any keratinocyte activation. Serum IGF1 levels were measured at initiation and completion of the protocol. Statistical Analysis: Categorical variables were compared with Chi-square test. Comparisons of medians were made using Kruskal-Wallis test. Post hoc analyses were performed using Mann-Whitney test with Bonferroni correction for multiple comparisons. A P < 0.05 was considered to be statistically significant. Results: A total of 14 patients completed the study (Group B: n = 5, Group C: n = 5, Group P: n = 4). Continuous administration of GHB was associated with a significant higher Ki67 immunolabeling at D21 (P = 0.049) and with a significant higher increase in the IGF1 concentrations at D21 (P = 0.024). No adverse effects were disclosed. Conclusions: Our preliminary data support a positive effect of GHB on keratinocyte proliferation and are encouraging enough to warrant large prospective studies. PMID

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

PubMed

McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Stimulatory effect of boron and manganese salts on keratinocyte migration.

PubMed

Chebassier, Nathalie; Ouijja, El Houssein; Viegas, Isabelle; Dreno, Brigitte

2004-01-01

Keratinocyte proliferation and migration are essential for the reconstruction of the cutaneous barrier after skin injury. Interestingly, thermal waters which are rich in trace elements (e.g. boron and manganese), are known to be able to improve wound healing. In order to understand the mechanism of action of this effect, our study investigated the in vitro modulation of keratinocyte migration and proliferation by boron and manganese salts, which are present in high concentrations in a thermal water (Saint Gervais). Our in vitro study demonstrated that incubating keratinocytes for 24 h with boron salts at concentrations between 0.5 and 10 microg/ml or manganese salts at concentrations between 0.1 and 1.5 microg/ml accelerated wound closure compared with control medium (+20%). As this acceleration was not related to an increase in keratinocyte proliferation we suggest that boron and manganese act on wound healing mainly by increasing the migration of keratinocytes.

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

PubMed

Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

2017-08-01

CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative

Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xin; Dai, Hui; Zhuang, Binyu

The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagicmore » vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.« less

Direct Measurement of Initial 230TH/ 232TH Ratios in Central Texas Speleothems for More Accurate Age Determination

NASA Astrophysics Data System (ADS)

Wortham, B. E.; Banner, J. L.; James, E.; Loewy, S. L.

2013-12-01

Speleothems, calcite deposits in caves, preserve a record of climate in their growth rate, isotope ratios and trace element concentrations. These variables must be tied to precise ages to produce pre-instrumental records of climate. The 238U-234U- 230Th disequilibrium method of dating can yield precise ages if the amount of 230Th from the decay of radiogenic 238U can be constrained. 230Th in a speleothem calcite growth layer has two potential sources - 1) decay of radioactive 238U since the time of growth of the calcite layer; and 2) initial detrital 230Th, incorporated along with detrital 232Th, into the calcite layer at the time it grew. Although the calcite lattice does not typically incorporate Th, samples can contain impurities with relatively high Th contents. Initial 230Th/232Th is commonly estimated by assuming a source with bulk-Earth U/Th values in a state of secular equilibrium in the 238U-decay chain. The uncertainty in this 230Th/232Th estimate is also assumed, typically at +/-100%. Both assumptions contribute to uncertainty in ages determined for young speleothems. If the amount of initial detrital 230Th can be better quantified for samples or sites, then U-series ages will have smaller uncertainties and more precisely define the time series of climate proxies. This study determined the initial 230Th/232Th of modern calcite to provide more precise dates for central Texas speleothems. Calcite was grown on glass-plate substrates placed under active drips in central Texas caves. The 230Th/232Th of this modern calcite was determined using thermal ionization mass spectrometry. Results show that: 1) initial 230Th/232Th ratios can be accurately determined in these young samples and 2) measuring 230Th/232Th reduces the uncertainties in previously-determined ages on stalagmites from under the same drips. For example, measured initial 230Th/232Th in calcite collected on substrates from different locations in the cave at Westcave Preserve are 15.3 × 0.67 ppm

Intermittent pressure decreases human keratinocyte proliferation in vitro.

PubMed

Nasca, Maria R; Shih, Alan T; West, Dennis P; Martinez, Wanda M; Micali, Giuseppe; Landsman, Adam S

2007-01-01

The aim of this study was to investigate the correlation between pressure changes and keratinocyte proliferation by determining whether keratinocytes exposed to altered mechanical pressures would proliferate at different rates compared to control cells not subjected to pressure changes. Tissue culture flasks of human keratinocytes plated at an approximate density of 15,000 cells/cm(2) undergoing an intermittent cyclic pressure of 362 mm Hg at a frequency of 2.28 or 5.16 cycles/min (0.038 or 0.086 Hz) for 8 h were compared to control flasks grown at ambient room pressure. An in-line pressure transducer was used to monitor and adjust pressure within the cell chambers, using a solenoid valve. A thymidine incorporation assay assessed the amount of cell proliferation in each set of experiments. Differences in proliferation between keratinocytes subjected to cyclic pressure changes and control cells were found to be statistically significant (p < 0.05) in 4 out of 5 proliferation assays. Also, a higher frequency of pressure changes consistently generated a reduced proliferation rate compared to that seen in cells exposed to a lower frequency of pressure changes. These data indicate that keratinocytes undergoing intermittent pressure changes exhibit decreased proliferation rates compared to controls. Furthermore, an increased frequency rate seems to have a greater effect on proliferation than low-frequency rate pressure changes, suggesting that the stress caused by frequently changed pressure may play a greater role in reducing keratinocyte proliferation than the actual magnitude of load applied to the cells. Our results support the current treatment protocol of reducing speed and duration of walking on the site of the wound to promote healing of foot ulcers. (c) 2007 S. Karger AG, Basel.

Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury

PubMed Central

Gauglitz, Gerd G.; Zedler, Siegfried; v. Spiegel, Felix; Fuhr, Jasmin; v. Donnersmarck, Guido Henkel; Faist, Eugen

2012-01-01

Background In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu. Methods In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. Results Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß. Conclusion Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be

Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

PubMed

Rahmoun, Massilva; Molès, Jean-Pierre; Pedretti, Nathalie; Mathieu, Marc; Fremaux, Isabelle; Raison-Peyron, Nadia; Lecron, Jean-Claude; Yssel, Hans; Pène, Jérôme

2009-03-01

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell-surface glycoprotein, belonging to the carcinoembryonic antigen family, expressed by human neutrophils, epithelial cells, activated T and NK cells. CEACAM1 is expressed as a cell-surface molecule with different isoforms or can be secreted as a soluble protein. Here, we show that keratinocytes in the outer epidermal layer of psoriatic skin express CEACAM1, unlike those in healthy skin or in cutaneous lesions of patients with atopic or nummular dermatitis. Stimulation of primary human keratinocytes or in vitro reconstituted epidermis with culture supernatants of activated psoriatic lesion-infiltrating T cells, IFN-gamma or oncostatin M, but not IL-17, induced the expression of transcripts for the CEACAM1-long and -short isoforms and cell-surface CEACAM1, whereas soluble CEACAM1 was not produced. The uppermost layers of the epidermis in psoriatic lesions also contain neutrophils, a cell type with inflammatory and antimicrobial properties. Coculture of CEACAM1-expressing keratinocytes or CHO transfectants with neutrophils delayed spontaneous apoptosis of the latter cells. These results show that cytokine-induced cell-surface expression of CEACAM1 by keratinocytes in the context of a psoriatic environment might contribute to the persistence of neutrophils and thus to ongoing inflammation and the decreased propensity for skin infection, typical for patients with psoriasis.

Reciprocal role of vitamin D receptor on β-catenin regulated keratinocyte proliferation and differentiation.

PubMed

Hu, Lizhi; Bikle, Daniel D; Oda, Yuko

2014-10-01

promote keratinocyte proliferation, 1,25(OH)2D3/VDR promotes the ability of β-catenin to stimulate hair follicle differentiation. This article is part of a Special Issue entitled '16th Vitamin D Workshop'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Th-1, Th-2 Cytokines Profile among Madurella mycetomatis Eumycetoma Patients.

PubMed

Nasr, Amre; Abushouk, Amir; Hamza, Anhar; Siddig, Emmanuel; Fahal, Ahmed H

2016-07-01

Eumycetoma is a progressive and destructive chronic granulomatous subcutaneous inflammatory disease caused by certain fungi, the most common being Madurella mycetomatis. The host defence mechanisms against fungi usually range from an early non-specific immune response to activation and induction of specific adaptive immune responses by the production of Th-1 and Th-2 cytokines. The aim of this study is to determine the levels of Th-1 and Th-2 cytokines in patients infected with Madurella mycetomatis, and the association between their levels and disease prognosis. This is a descriptive cross-sectional study conducted at the Mycetoma Research Centre, University of Khartoum, Sudan, where 70 patients with confirmed M. mycetomatis eumycetoma were enrolled; 35 with, and 35 without surgical excision. 70 healthy individuals from mycetoma endemic areas were selected as controls. The levels of serum cytokines were determined by cytometric bead array technique. Significantly higher levels of the Th-1 cytokines (IFN-γ, TNF-α, IL-1β and IL-2) were recorded in patients treated with surgical excision, compared to those treated without surgical excision. In contrast, the Th-2 cytokines (IL-4, IL-5, IL-6 and IL-10) were significantly lower in patients treated with surgical excision compared to those treated without surgical excision. In conclusion, the results of this study suggest that cell-mediated immunity can have a role to play in the pathogenesis of eumycetoma.

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir

2014-03-01

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependentmore » increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

Coexistence of Th1/Th2 and Th17/Treg imbalances in patients with allergic asthma.

PubMed

Shi, Yu-heng; Shi, Guo-chao; Wan, Huan-ying; Jiang, Li-hua; Ai, Xiang-yan; Zhu, Hai-xing; Tang, Wei; Ma, Jia-yun; Jin, Xiao-yan; Zhang, Bo-ying

2011-07-05

Recent recognition is that Th2 response is insufficient to fully explain the aetiology of asthma. Other CD4(+) T cells subsets might play a role in asthma. We investigated the relative abundance and activities of Th1, Th2, Th17 and CD4(+)CD25(+) Treg cells in patients with allergic asthma. Twenty-two patients with mild asthma, 17 patients with moderate to severe asthma and 20 healthy donors were enrolled. All patients were allergic to house dust mites. Plasma total IgE, pulmonary function and Asthma Control Questionnaire were assessed. The proportions of peripheral blood Th1, Th2, Th17 and CD4(+)CD25(+) Treg cells were determined by flow cytometry. The expression of cytokines in plasma and in the culture supernatant of peripheral blood mononuclear cells was determined by enzyme linked, immunosorbent assay. The frequency of blood Th2 cells and IL-4 levels in plasma and culture supernatant of peripheral blood mononuclear cells were increased in all patients with allergic asthma. The frequency of Th17 cells and the plasma and culture supernatant levels of IL-17 were increased, whereas the frequency of CD4(+)CD25(+) Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. Dermatophagoides pteronyssinus specific IgE levels were positively correlated with the percentage of blood Th2 cells and plasma IL-4 levels. Forced expiratory volume in the first second was negatively correlated with the frequency of Th17 cells and plasma IL-17 levels, and positively correlated with the frequency of Treg cells. However, mean Asthma Control Questionnaire scores were positively correlated with the frequency of Th17 cells and plasma IL-17 levels, and negatively correlated with the frequency of Treg cells. Imbalances in Th1/Th2 and Th17/Treg were found in patients with allergic asthma. Furthermore, elevated Th17 cell responses, the absence of Tregs and an imbalance in Th17/Treg levels were associated with moderate to severe asthma.

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Itoi, Saori; Terao, Mika, E-mail: mterao@derma.med.osaka-u.ac.jp; Murota, Hiroyuki

Highlights: •We investigate the role of 11β-HSD1 in skin inflammation. •Various stimuli increase expression of 11β-HSD1 in keratinocytes. •11β-HSD1 knockdown by siRNA decreases cortisol levels in media. •11β-HSD1 knockdown abrogates the response to pro-inflammatory cytokines. •Low-dose versus high-dose cortisol has opposing effects on keratinocyte inflammation. -- Abstract: The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactivemore » cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10{sup −13} M cortisol, whereas 1 × 10{sup −5} M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic

Melatonin promotes diabetic wound healing in vitro by regulating keratinocyte activity

PubMed Central

Song, Ruipeng; Ren, Lijun; Ma, Haoli; Hu, Ruijing; Gao, Honghong; Wang, Li; Chen, Xuehui; Zhao, Zhigang; Liu, Jialin

2016-01-01

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Proper keratinocyte proliferation and migration are crucial steps during re-epithelialization. Melatonin (Mel) accelerates wound repair in full-thickness incisional wounds; however, its role in diabetic wound healing is unknown. This study explored the role of Mel in diabetic wound healing in vitro by using high glucose (HG)-cultured keratinocytes. Mel reduced the HG-induced mRNA expression and release of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8, in keratinocytes. Mel inhibited oxidative stress, as evidenced by reduced production of reactive oxygen species and malondialdehyde and increased activity of superoxide dismutase in HG-stimulated keratinocytes. Mel also inhibited HG-induced nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome activation in keratinocytes. HG-induced reduced migration and proliferation and increased apoptosis of keratinocytes were counteracted by Mel treatment. The pro-proliferative, pro-migratory, and anti-apoptotic effects of Mel on HG-treated keratinocytes were mediated by extracellular signal-regulated kinase signaling pathway. Results collectively suggested that Mel is an alternative therapeutic strategy to ameliorate poor condition for diabetic wound healing by regulating keratinocyte activity. PMID:27904671

Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

PubMed

Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

2014-04-01

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo.

PubMed

Calapre, Leslie; Gray, Elin S; Kurdykowski, Sandrine; David, Anthony; Hart, Prue; Descargues, Pascal; Ziman, Mel

2016-05-26

UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells. In this study, we determined the effects of repeated UVB exposure 1 kJ/m(2) followed by heat (39 °C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2 days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes. Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells. Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis.

Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

PubMed

Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

2011-01-01

Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

Steroid synthesis by primary human keratinocytes; implications for skin disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk; Michael, Anthony E.; Jaulim, Adil

2011-01-07

Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary humanmore » keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively

Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

PubMed

Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

2012-10-01

Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

Down-regulation of Notch signaling pathway reverses the Th1/Th2 imbalance in tuberculosis patients.

PubMed

Li, Qifeng; Zhang, Hui; Yu, Liang; Wu, Chao; Luo, Xinhui; Sun, He; Ding, Jianbing

2018-01-01

Th1/Th2 imbalance to Th2 is of significance in the peripheral immune responses in Tuberculosis (TB) development. However, the mechanisms for Th1/Th2 imbalance are still not well determined. Notch signaling pathway is involved in the peripheral T cell activation and effector cell differentiation. However, whether it affects Th1/Th2 imbalance in TB patients is still not known. Here, we used γ-secretase inhibitor (DAPT) to treat the peripheral blood mononuclear cells (PBMCs) from healthy people or individuals with latent or active TB infection in vitro, respectively. Then, the Th1/Th2 ratios were determined by flow cytometry, and cytokines of IFN-γ, IL-4, IL-10 in the culture supernatant were measured by CBA method. The Notch signal pathway associated proteins Hes1, GATA3 and T-bet were quantitated by real-time PCR or immunoblotting. Our results showed that DAPT effectively inhibited the protein level of Hes1. In TB patients, the Th2 ratio increased in the PBMCs, alone with the high expression of GATA3 and IL-4, resulting in the high ratios of Th2/Th1 and GATA3/T-bet in TB patients. However, Th2 cells ratio decreased after blocking the Notch signaling pathway by DAPT and the Th2/Th1 ratio in TB patients were DAPT dose-dependent, accompanied by the decrease of IL-4 and GATA3. But, its influence on Th1 ratio and Th1 related T-bet and IFN-γ levels were not significant. In conclusion, our results suggest that blocking Notch signaling by DAPT could inhibit Th2 responses and restore Th1/Th2 imbalance in TB patients. Copyright © 2017. Published by Elsevier B.V.

Vitamin D signaling regulates oral keratinocyte proliferation in vitro and in vivo

PubMed Central

YUAN, FENG-NING F.; VALIYAPARAMBIL, JAYASANKER; WOODS, MICHAEL C.; TRAN, HUY; PANT, RIMA; ADAMS, JOHN S.; MALLYA, SANJAY M.

2014-01-01

The secosteroidal hormone 1,25-dihyroxyvitamin D [1,25(OH)2D3] and its receptor, the vitamin D receptor (VDR), are crucial regulators of epidermal proliferation and differentiation. However, the effects of 1,25(OH)2D3-directed signaling on oral keratinocyte pathophysiology have not been well studied. We examined the role of 1,25(OH)2D3 in regulating proliferation and differentiation in cultured oral keratinocytes and on the oral epithelium in vivo. Using lentiviral-mediated shRNA to silence VDR, we generated an oral keratinocyte cell line with stable knockdown of VDR expression. VDR knockdown significantly enhanced proliferation and disrupted calcium- and 1,25(OH)2D3-induced oral keratinocyte differentiation, emphasizing the anti-proliferative and pro-differentiation effects of 1,25(OH)2D3 in oral keratinocytes. Using vitamin D3-deficient diets, we induced chronic vitamin D deficiency in mice as evidenced by decreased serum 25-hydroxyvitamin D (25OHD) concentrations. The vitamin D-deficient mice manifested increased proliferation of the tongue epithelium, but did not develop any morphological or histological abnormalities in the oral epithelium, suggesting that vitamin D deficiency alone is insufficient to alter oral epithelial homeostasis and provoke carcinogenesis. Immunohistochemical analyses of human and murine oral squamous cell carcinomas showed increased VDR expression. Overall, our results provide strong support for a crucial role for vitamin D signaling in oral keratinocyte pathophysiology. PMID:24626468

Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

PubMed

Strittmatter, Gerhard E; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena; Beer, Hans-Dietmar

2016-01-01

Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1.

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis

PubMed Central

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-01-01

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways. PMID:28277539

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

PubMed

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-03-09

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

[Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

PubMed

Liao, W; Liu, Y; Ye, L

1999-09-01

To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

Noninvasive electromagnetic fields on keratinocyte growth and migration.

PubMed

Huo, Ran; Ma, Qianli; Wu, James J; Chin-Nuke, Kayla; Jing, Yuqi; Chen, Juan; Miyar, Maria E; Davis, Stephen C; Li, Jie

2010-08-01

Although evidence has shown that very small electrical currents produce a beneficial therapeutic result for wounds, noninvasive electromagnetic field (EMF) therapy has consisted mostly of anecdotal clinical reports, with very few well-controlled laboratory mechanistic studies. In this study, we evaluate the effects and potential mechanisms of a noninvasive EMF device on skin wound repair. The effects of noninvasive EMF on keratinocytes and fibroblasts were assessed via proliferation and incisional wound model migration assays. cDNA microarray and RT-PCR were utilized to assess genetic expression changes in keratinocytes after noninvasive EMF treatment. In vitro analyses with human skin keratinocyte cultures demonstrated that noninvasive EMFs have a strong effect on accelerating keratinocyte migration and a relatively weaker effect on promoting keratinocyte proliferation. The positive effects of noninvasive EMFs on cell migration and proliferation seem keratinocyte-specific without such effects seen on dermal fibroblasts. cDNA microarray and RT-PCR performed revealed increased expression of CRK7 and HOXC8 genes in treated keratinocytes. This study suggests that a noninvasive EMF accelerates wound re-epithelialization through a mechanism of promoting keratinocyte migration and proliferation, possibly due to upregulation of CRK7 and HOXC8 genes. Copyright 2010 Elsevier Inc. All rights reserved.

Enzyme-Treated Asparagus Extract (ETAS) Facilitates the Turnover of UV-B-Irradiated Keratinocytes.

PubMed

Koda, Tomoko; Shirato, Ken; Takanari, Jun; Imai, Hideki

2018-01-01

Enzyme-treated asparagus extract (ETAS) is prepared from the lower, residual parts of asparagus, and some functionalities, such as anti-oxidative and neuroprotective activities, have been suggested. The purpose of the present study was to investigate the effects of ETAS on photoaging in the epidermal layer of the skin using cultured keratinocytes. Normal human epidermal keratinocytes were irradiated or left unirradiated with UV-B (10 mJ/cm 2 ) and incubated with ETAS (0.5 or 2 mg/mL) or vehicle. After 3 or 13 h, molecular examinations were performed, and after 24 or 48 h, cell viabilities were determined by a CCK-8 assay. ETAS addition may induce keratinocyte migration and proliferation as well as apoptosis under molecular examination. These results suggest that ETAS might accelerate turnover of keratinocytes.

Keratinocytes in culture accumulate phagocytosed melanosomes in the perinuclear area.

PubMed

Ando, Hideya; Niki, Yoko; Yoshida, Masaki; Ito, Masaaki; Akiyama, Kaoru; Kim, Jin-Hwa; Yoon, Tae-Jin; Lee, Jeung-Hoon; Matsui, Mary S; Ichihashi, Masamitsu

2010-02-01

There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana-Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease-activated receptor-2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose-dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro.

Keratinocyte response to immobilized growth factors for enhanced dermal wound healing

NASA Astrophysics Data System (ADS)

Stefonek-Puccinelli, Tracy Jane

Chronic wounds cost billions of dollars per year to treat and wound care is limited to ineffective and/or expensive options. Chronic wounds are characterized by a failure to reepithelialize, as well as deficiencies in growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), normally present during wound healing. Our system described herein begins to tackle the problems associated with designing bioactive materials for chronic wound healing applications. We show that we can induce accelerated keratinocyte migration with photo-immobilized EGF and further control migration speed through the culture of cells on different types of gradient patterns of EGF. We also successfully immobilized IGF-1 while retaining its bioactivity, and further showed it induces directed keratinocyte migration, although not as potently as immobilized EGF. Potential synergy between co-immobilized IGF-1 and EGF was also investigated, although EGF continued to dominate the cellular response, and no significant increase in cell migration was achieved via the addition of IGF-1 to the system. To further understand cellular response to our immobilized growth factors, we investigated keratinocyte signaling and function in response to changes in EGF presentation. It was found that immobilized and soluble EGF can play different, yet complementary, roles in regulating keratinocyte function. Specifically, keratinocytes responded to immobilized EGF with high EGF receptor (EGFR) activation, accompanied by low proliferation and high migratory activity. In contrast, keratinocytes treated with soluble EGF displayed a highly proliferative, rather than migratory, phenotype. We then transitioned our photo-immobilization techniques to materials that may be more suitable as a wound dressing, such as silk fibroin films. Silk fibroin is a natural fiber with many desirable qualities for a biomaterial including high strength and elasticity, biocompatibility, a beta

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

PubMed Central

Tron, Victor A.; Trotter, Martin J.; Tang, Liren; Krajewska, Maryla; Reed, John C.; Ho, Vincent C.; Li, Gang

1998-01-01

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and

The incidence and multiplicity rates of keratinocyte cancers in Australia.

PubMed

Pandeya, Nirmala; Olsen, Catherine M; Whiteman, David C

2017-10-16

To assess the incidence and multiplicity of keratinocyte cancers (basal cell carcinoma [BCC] and squamous cell carcinoma [SCC]) excised in Australia, and to examine variations by age, sex, state, and prior skin cancer history. Analysis of individual-level Medicare data for keratinocyte cancer treatments (identified by eight specific MBS item codes) during 2011-2014. Histological data from the QSkin prospective cohort study were analysed to estimate BCC and SCC incidence. A 10% systematic random sample of all people registered with Medicare during 1997-2014. People aged at least 20 years in 2011 who made at least one claim for any MBS medical service during 2011-2014 (1 704 193 individuals). Age-standardised incidence rates (ASRs) and standardised incidence ratios (SIRs). The person-based incidence of keratinocyte cancer excisions in Australia was 1531 per 100 000 person-years; incidence increased with age, and was higher for men than women (SIR, 1.43; 95% CI, 1.42-1.45). Lesion-based incidence was 3154 per 100 000 person-years. The estimated ASRs for BCC and SCC were 770 per 100 000 and 270 per 100 000 person-years respectively. During 2011-2014, 3.9% of Australians had one keratinocyte cancer excised, 2.7% had more than one excised; 74% of skin cancers were excised from patients who had two or more lesions removed. Multiplicity was strongly correlated with age; most male patients over 70 were treated for multiple lesions. Keratinocyte cancer incidence was eight times as high among people with a prior history of excisions as among those without. The incidence and multiplicity of keratinocyte cancer in Australia are very high, causing a large disease burden that has not previously been quantified.

5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT Cell Line.

PubMed

Hartinger, Jan; Veselý, Pavel; Šíma, Martin; Netíková, Irena; Matoušková, Eva; Petruželka, Luboš

5-fluorouracil (5-FU) and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE) which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS) inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF) and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.

Thrombospondin-induced adhesion of human keratinocytes.

PubMed Central

Varani, J; Nickoloff, B J; Riser, B L; Mitra, R S; O'Rourke, K; Dixit, V M

1988-01-01

Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2+), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes. Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading. This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair. Images PMID:2452837

Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

PubMed Central

Parkinson, Eric Kenneth

2013-01-01

The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)—eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)—inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo. PMID:23892603

TRPV2 channel inhibitors attenuate fibroblast differentiation and contraction mediated by keratinocyte-derived TGF-β1 in an in vitro wound healing model of rats.

PubMed

Ishii, Taro; Uchida, Kunitoshi; Hata, Shozaburo; Hatta, Mitsutoki; Kita, Tomo; Miyake, Yuki; Okamura, Kazuhiko; Tamaoki, Sachio; Ishikawa, Hiroyuki; Yamazaki, Jun

2018-06-01

Keratinocytes release several factors that are involved in wound contracture and scar formation. We previously reported that a three-dimensional reconstruction model derived from rat skin represents a good wound healing model. We characterized the role of transient receptor potential (TRP) channels in the release of transforming growth factor (TGF)-β1 from keratinocytes and the differentiation of fibroblasts to identify possible promising pharmacological approaches to prevent scar formation and contractures. The three-dimensional culture model was made from rat keratinocytes seeded on a collagen gel in which dermal fibroblasts had been embedded. Among the TRP channel inhibitors tested, the TRPV2 inhibitors SKF96365 and tranilast attenuated most potently keratinocyte-dependent and - independent collagen gel contraction due to TGF-β signaling as well as TGF-β1 release from keratinocytes and α-smooth muscle actin production in myofibroblasts. Besides the low amounts detected in normal dermis, TRPV2 mRNA and protein levels were increased after fibroblasts were embedded in the gel. TRPV2 was also expressed in the epidermis and keratinocyte layers of the model. Both inhibitors and TRPV2 siRNA attenuated the intracellular increase of Ca 2+ induced by the TRPV agonist 2-aminoethoxydiphenyl borate in TGF-β1-pretreated fibroblasts. This is the first study to show that compounds targeting TRPV2 channels ameliorate wound contraction through the inhibition of TGF-β1 release and the differentiation of dermal fibroblasts in a culture model. Copyright © 2018. Published by Elsevier B.V.

The protective effects of piceatannol from passion fruit (Passiflora edulis) seeds in UVB-irradiated keratinocytes.

PubMed

Maruki-Uchida, Hiroko; Kurita, Ikuko; Sugiyama, Kenkichi; Sai, Masahiko; Maeda, Kazuhisa; Ito, Tatsuhiko

2013-01-01

The use of naturally occurring botanicals with substantial antioxidant activity to prevent photoageing is receiving increasing attention. We have previously identified piceatannol and scirpusin B, which is a dimer of piceatannol, as strong antioxidants that are present in passion fruit (Passiflora edulis) seeds. In the present study, the effects of passion fruit seed extract, piceatannol, and scirpusin B on human keratinocytes were investigated. The passion fruit seed extract and piceatannol upregulated the glutathione (GSH) levels in keratinocytes in a dose-dependent manner, indicating that piceatannol is an active component of the passion fruit seed extract in keratinocytes. The pretreatment with piceatannol also suppressed the UVB-induced generation of reactive oxygen species (ROS) in the keratinocytes. In addition, the transfer of the medium from the UVB-irradiated keratinocytes to non-irradiated fibroblasts enhanced matrix-metalloproteinase (MMP)-1 activity, and this MMP-1 induction was reduced when the keratinocytes were pretreated with piceatannol. These results suggest that piceatannol attenuates the UVB-induced activity of MMP-1 along with a reduction of ROS generation in keratinocytes. Thus, piceatannol and passion fruit seed extract containing high amounts of piceatannol are potential anti-photoageing cosmetic ingredients.

Wave Energy Prize - 1/20th Testing - Oscilla Power

DOE Data Explorer

Scharmen, Wesley

2016-09-16

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the Oscilla Power team, including the 1/20th Test Plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the WEPrize winners.

Upregulation of cathepsin S in psoriatic keratinocytes.

PubMed

Schönefuss, Alexander; Wendt, Wiebke; Schattling, Benjamin; Schulten, Roxane; Hoffmann, Klaus; Stuecker, Markus; Tigges, Christian; Lübbert, Hermann; Stichel, Christine

2010-08-01

Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II-mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS-immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T- and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS-immunostaining is also detectable in keratinocytes. We show that cocultivation with T-cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T-cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II-associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.

PubMed

Mohamadzadeh, M; Müller, M; Hultsch, T; Enk, A; Saloga, J; Knop, J

1994-12-01

To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

Rotation is the primary motion of paired human epidermal keratinocytes.

PubMed

Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

2015-09-01

Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Wave Energy Prize - 1/20th Testing - AquaHarmonics

DOE Data Explorer

Scharmen, Wesley

2016-09-02

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the AquaHarmonics team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Wave Energy Prize - 1/20th Testing - Waveswing America

DOE Data Explorer

Scharmen, Wesley

2016-08-19

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the Waveswing America team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Wave Energy Prize - 1/20th Testing - Sea Potential

DOE Data Explorer

Scharmen, Wesley

2016-09-23

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the Sea Potential team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses.

PubMed

Lambert, Sylviane; Swindell, William R; Tsoi, Lam C; Stoll, Stefan W; Elder, James T

2017-07-01

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The protease-activated receptor-2 upregulates keratinocyte phagocytosis.

PubMed

Sharlow, E R; Paine, C S; Babiarz, L; Eisinger, M; Shapiro, S; Seiberg, M

2000-09-01

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

AKT delays the early-activated apoptotic pathway in UVB-irradiated keratinocytes via BAD translocation.

PubMed

Claerhout, Sofie; Decraene, David; Van Laethem, An; Van Kelst, Sofie; Agostinis, Patrizia; Garmyn, Marjan

2007-02-01

Upon irradiation with a high dose of UVB, keratinocytes undergo apoptosis as a protective mechanism. In previous work, we demonstrated the existence of an early-activated UVB-induced apoptotic pathway in growth factor-depleted human keratinocytes, which can be substantially delayed by the exclusive supplementation of IGF-1. We now show that in human keratinocytes, IGF-1 inhibits the onset of UVB-triggered apoptosis through a transcriptional independent, AKT-mediated mechanism, involving BAD serine 136 phosphorylation. Our results show that the early UVB-induced apoptosis in growth factor-depleted human keratinocytes is exclusively triggered through the mitochondrial pathway. It is accompanied by BAX translocation, cytochrome c release, and procaspase-9 cleavage, but not by procaspase-8 or BID cleavage. In human keratinocytes, IGF-1 supplementation inhibits these events in a transcription-independent manner. Both IGF-1 supplementation and the transduction of a membrane-targeted form of AKT result in a shift of the BH3-only protein BAD from the mitochondria to the cytoplasm, paralleled by an increase of AKT-specific Ser136 phospho-BAD bound to 14-3-3zeta protein. These data indicate that AKT-induced BAD phosphorylation and its subsequent cytoplasmic sequestration by 14-3-3zeta is a major mechanism responsible for the postponement of UVB-induced apoptosis in human keratinocytes.

Vitamin D protects keratinocytes from deleterious effects of ionizing radiation.

PubMed

Langberg, M; Rotem, C; Fenig, E; Koren, R; Ravid, A

2009-01-01

Radiotherapy can induce severe skin responses that may limit the clinically acceptable radiation dose. The responses include erythema, dry and moist desquamation, erosions and dermal-epidermal blister formation. These effects reflect injury to, and reproductive failure of, epidermal cells and may also be due to dysregulation of the tissue remodelling process caused by excessive proteolytic activity. Calcitriol, the hormonally active vitamin D metabolite, protects keratinocytes from programmed cell death induced by various noxious stimuli. To examine whether calcitriol protects proliferating keratinocytes from the damage inflicted by ionizing radiation under conditions similar to those employed during radiotherapy. Autonomously proliferating HaCaT keratinocytes, used as a model for basal layer keratinocytes, were irradiated using a linear accelerator. Cell death was monitored by vital staining, executioner caspase activation, lactic dehydrogenase release and colony formation assay. Induction of matrix metalloproteinase-9 was assessed by gelatinase activity assay and mRNA determination. Levels of specific proteins were determined by immunoblotting. Treatment with calcitriol inhibited both caspase-dependent and -independent programmed cell death occurring within 48 h of irradiation and increased the colony formation capacity of irradiated cells. These effects may be attributable to inhibition of the c-Jun NH(2)-terminal kinase cascade and to upregulation of the truncated antiapoptotic isoform of p63. Treatment with the hormone also attenuated radiation-induced increase in matrix metalloproteinase-9 protein and mRNA levels. The results of this study suggest that active vitamin D derivatives may attenuate cell death and excessive proteolytic activity in the epidermis due to exposure to ionizing radiation in the course of radiotherapy.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells.

PubMed

Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan; Ahn, Kyu Joong

2016-08-01

We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

PubMed Central

Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

2016-01-01

Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

Autologous cultured keratinocytes on porcine gelatin microbeads effectively heal chronic venous leg ulcers.

PubMed

Liu, Jin Yu; Hafner, Jürg; Dragieva, Galya; Seifert, Burkhardt; Burg, Günter

2004-01-01

We have established a specific bioreactor microcarrier cell culture system using porcine gelatin microbeads as carriers to produce autologous keratinocytes on a large scale. Moreover, we have shown that autologous keratinocytes can be cultured on porcine collagen pads, thereby forming a single cell layer. The objective of this study was to compare efficacy and safety of autologous cultured keratinocytes on microbeads and collagen pads in the treatment of chronic wounds. Fifteen patients with recalcitrant venous leg ulcers were assigned to three groups in a single-center, prospective, uncontrolled study: five underwent a single treatment with keratinocyte monolayers on collagen pads (group 1); another five received a single grafting with keratinocyte-microbeads (group 2); and the last five received multiple, consecutive applications of keratinocyte-microbeads 3 days apart (group 3). All patients were followed for up to 12 weeks. By 12 weeks, there was a mean reduction in the initial wound area of 50, 83, and 97 percent in the three groups, respectively. The changes in wound size were statistically significant between the first and third groups (p= 0.0003). Keratinocyte-microbeads proved to be more effective than keratinocyte monolayers on collagen pads when the former were applied every 3 days. Rapid availability within 10-13 days after skin biopsy and easy handling represent particular advantages.

On the Mechanism Determining the Th1/Th2 Phenotype of an Immune Response, and its Pertinence to Strategies for the Prevention, and Treatment, of Certain Infectious Diseases

PubMed Central

Bretscher, P A

2014-01-01

It is well recognized that the physiological/pathological consequences of an immune response, against a foreign or a self-antigen, are often critically dependent on the class of immunity generated. Here we focus on how antigen interacts with the cells of the immune system to determine whether antigen predominantly generates Th1 or Th2 cells. We refer to this mechanism as the ‘decision criterion’ controlling the Th1/Th2 phenotype of the immune response. A plausible decision criterion should account for the variables of immunization known to affect the Th1/Th2 phenotype of the ensuing immune response. Documented variables include the nature of the antigen, in terms of its degree of foreignness, the dose of antigen and the time after immunization at which the Th1/Th2 phenotype of the immune response is assessed. These are quantitative variables made at the level of the system. In addition, the route of immunization is also critical. I describe a quantitative hypothesis as to the nature of the decision criterion, referred to as the Threshold Hypothesis. This hypothesis accounts for the quantitative variables of immunization known to affect the Th1/Th2 phenotype of the immune response generated. I suggest and illustrate how this is not true of competing, contemporary hypotheses. I outline studies testing predictions of the hypothesis and illustrate its potential utility in designing strategies to prevent or treat medical situations where a predominant Th1 response is required to contain an infection, such as those caused by HIV-1 and by Mycobacterium tuberculosis, or to contain cancers. PMID:24684592

Protective effects of amifostine and cyclooxygenase-1 inhibitor against normal human epidermal keratinocyte toxicity induced by methotrexate and 5-fluorouracil.

PubMed

Maiguma, Takayoshi; Kaji, Hiroaki; Makino, Kazutaka; Teshima, Daisuke

2009-07-01

Our study aimed to find more effective protective agents against mucosa toxicity induced by methotrexate and 5-fluorouracil. We focused on the relationship between oral mucositis and keratinocyte injury and examined methotrexate and 5-fluorouracil-induced cytotoxicity in normal human epidermal keratinocyte cell lines. Cell viability and superoxide radical activity were measured based on converting WST-1 (4-[3-(4-indophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzen disulfonate) to a water-soluble formazan dye. DNA synthesis by 5-bromo-2'-deoxyuridine incorporation was measured as an indirect parameter of cell proliferation. Allopurinol and amifostine were used as the radical scavengers. l-glutamine was used as a mucosa-protective agent. A cyclooxygenase inhibitor interrupting the production of hydroxyl radicals in the arachidonic acid cascade was also examined. 5-fluorouracil and methotrexate caused cytotoxicity due to the activation of intracellular superoxide radicals specifically on normal human epidermal keratinocytes. From the electron spin resonance study, it was found that allopurinol was a superoxide radical scavenger, while amifostine was hydroxyl radical scavenger. Allopurinol showed no effect on the cytotoxicity due to 5-fluorouracil and methotrexate. The cell injury induced by methotrexate was restored by amifostine. However, the cell injury induced by 5-fluorouracil was markedly recovered by a selective cyclooxygenase-1 inhibitor compared to amifostine. It was suggested that amifostine and cyclooxygenase-1 inhibitor could be useful protective agents against methotrexate and 5-fluorouracil chemotherapeutic toxicity. Additionally, this in vitro cell injury model using normal human epidermal keratinocytes may be useful for understanding the pathophysiology of oral mucositis induced by chemotherapeutic agents.

A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

PubMed Central

Hammiller, Brianna O.; El-Abaseri, Taghrid Bahig; Dlugosz, Andrzej A.; Hansen, Laura A.

2015-01-01

Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation. PMID:26284198

Stimulatory effect of fibroblast-derived prostaglandin E₂ on keratinocyte stratification in the skin equivalent.

PubMed

Arai, Koji Y; Fujioka, Atsuko; Okamura, Ryoko; Nishiyama, Toshio

2014-01-01

Epidermal-dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin-1 (IL-1) and an IL-1-induced inflammatory mediator prostaglandin E₂ (PGE₂) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL-1α mRNA (IL1A mRNA) and relatively low levels of IL-1β mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin-endoperoxide synthase 2 mRNA (PTGS2 mRNA) and production of PGE₂ in three-dimensionally cultured fibroblasts were noticeably stimulated by co-culture with keratinocytes, whereas PTGS2 mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15-(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL-1β stimulated PTGS2 mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67-positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE₂ was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL-1 and PGE₂. © 2014 by the Wound Healing Society.

In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold.

PubMed

Pajoum Shariati, Seyed Ramin; Shokrgozar, Mohammad Ali; Vossoughi, Manouchehr; Eslamifar, Ali

2009-07-01

Extensive full-thickness burns require replacement of both epidermis and dermis. In designing skin replacements, the goal has been to re-create this model and make a product which has both essential components. In the present study, we developed procedures for establishing confluent, stratified layers of cultured human keratinocytes on the surface of modified collagen-chitosan scaffold that contains fibroblasts. The culture methods for propagation of keratinocytes and fibroblasts isolated from human neonatal foreskin were developed. The growth and proliferation of normal human keratinocytes were evaluated in serum-free (keratinocyte growth medium) and our modified medium. Characterization of human keratinocytes was determined by using pan-keratin and anti-involucrin monoclonal antibodies. For fabrication of relevant biodegradable and biocompatible collagen-chitosan porous scaffold with improved biostability, modified method of freeze-gelation was used. In generating organotypic co-cultures, epidermal keratinocytes were plated onto the upper surface of scaffold containing embedded fibroblasts. The results showed that the growth of isolated human skin fibroblasts and keratinocytes in our modified medium was more than that in the serum-free medium. The different evaluations of collagen-chitosan scaffold showed that it is relevant to growth of cells (fibroblast and keratinocyte) and has a good flexibility in manipulation of the living skin equivalents. These findings indicate that the integration of collagen-chitosan scaffold with co-cultured keratinocyte and fibroblast in vitro provides a potential source of living skin for grafting in vivo.

Candida albicans-induced inflammatory response in human keratinocytes.

PubMed

Wollina, U; Künkel, W; Bulling, L; Fünfstück, C; Knöll, B; Vennewald, I; Hipler, U-C

2004-06-01

Candida albicans strains 3153a, ATCC 48867, CBS 2730, DSM 70014, and Vir 13 were cultivated and sterile C. albicans filtrates were produced. The interaction of soluble Candida factors of these infiltrates with human HaCaT keratinocytes was assayed in vitro. The following parameters were analyzed: cell proliferation, protein synthesis, nuclear matrix protein (NMP) 41 release, cytokine release (IL-1beta, soluble IL-2 receptor, IL-6, and IL-8), and reactive oxygen species (ROS). Cell counts at 1, 12, and 24 h were significantly lower for C. albicans strains CBS 2730 and VIR 13 (P < 0.05). There was no significant change for the remaining strains. Neither the protein synthesis nor the NMP-41 release was significantly affected. IL-6 and IL-8 were stimulated by C. albicans filtrates to different amounts with higher levels in strains of low virulence. There was no effect on the other cytokines. The production of ROS by HaCaT keratinocytes was suppressed. The induction of an inflammatory keratinocyte response by soluble C. albicans factors may play a role among the host-yeast interactions.

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

PubMed Central

Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels.

PubMed

Barr, Travis P; Albrecht, Phillip J; Hou, Quanzhi; Mongin, Alexander A; Strichartz, Gary R; Rice, Frank L

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.

IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

PubMed Central

Yoon, Juhan; Oyoshi, Michiko K.; Hoff, Sabine; Chervonsky, Alexander; Oppenheim, Joost J.; Rosenstiel, Philip

2016-01-01

Atopic dermatitis (AD) is a Th2-dominated inflammatory skin disease characterized by epidermal thickening. Serum levels of IL-22, a cytokine known to induce keratinocyte proliferation, are elevated in AD, and Th22 cells infiltrate AD skin lesions. We show that application of antigen to mouse skin subjected to tape stripping, a surrogate for scratching, induces an IL-22 response that drives epidermal hyperplasia and keratinocyte proliferation in a mouse model of skin inflammation that shares many features of AD. DC-derived IL-23 is known to act on CD4+ T cells to induce IL-22 production. However, the mechanisms that drive IL-23 production by skin DCs in response to cutaneous sensitization are not well understood. We demonstrate that IL-23 released by keratinocytes in response to endogenous TLR4 ligands causes skin DCs, which selectively express IL-23R, to up-regulate their endogenous IL-23 production and drive an IL-22 response in naive CD4+ T cells that mediates epidermal thickening. We also show that IL-23 is released in human skin after scratching and polarizes human skin DCs to drive an IL-22 response, supporting the utility of IL-23 and IL-22 blockade in AD. PMID:27551155

Effects of 25-hydroxyvitamin D3 on cathelicidin production and antibacterial function of human oral keratinocytes.

PubMed

Wang, Qi; Zhang, Wu; Li, Hao; Aprecio, Raydolf; Wu, Wan; Lin, Yiqiao; Li, Yiming

2013-01-01

Vitamin D and its metabolites have been recognized as key determinants in innate immune modulation. In this study, we investigated the regulation of antibacterial functions of oral keratinocyte cells by 25-hydroxyvitamin D3 (25VD3). OKF6/TERT2 cells, an immortalized human oral keratinocyte cell line, were transfected with or without 24-hydroxylase small interfering RNA (siRNA) and incubated with different amounts of 25VD3. These epithelial cells expressed high levels of inactivating 24-hydroxylase (CYP24A1) and relatively low levels of activating 1α-hydroxylase (CYP27B1) in the presence of 25VD3. 25VD3 influenced the expression of vitamin D-driven genes and cathelicidin in a dose-related manner. SiRNA specific to 24-hydroxylase augmented the cathelicidin production and subseqently influenced the antibacterial activity on multispecies of oral pathogens. These observations suggest that 25VD3 is capable of stimulating cathelicidin production and modulating antibacterial function upon CYP24A1 knochdown in oral epithelial cells, and indicate novel mechanisms that 25VD3 may enhance antibacterial ability in oral keratinocytes. Copyright © 2013 Elsevier Inc. All rights reserved.

Wave Energy Prize - 1/20th Testing - SEWEC

DOE Data Explorer

Wesley Scharmen

2016-10-07

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the SEWEC team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners. * Note: During the TG4 judging meeting, the Wave Energy Prize judges reviewed the data collected during the testing of SEWEC's device at Carderock and determined that the data were inconclusive and did not allow an ACE value to be calculated for the device. Consequently, the SEWEC device was deemed ineligible to be considered for the Wave Energy Prize.

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

PubMed Central

He, Yong; Fisher, Robert; Chowdhury, Soma; Sultana, Ishrat; Pereira, Claudia P.; Bray, Mike; Reed, Jennifer L.

2014-01-01

Rationale Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. Methods To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. Results Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. Conclusions Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus. PMID:25419841

Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

PubMed Central

Guha, Gunjan; Li, Shan; Kyrylkova, Kateryna; Kioussi, Chrissa; Leid, Mark; Ganguli-Indra, Gitali; Indra, Arup K.

2012-01-01

Background Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice. Methodology/Principal Findings To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes. Conclusions/Significance Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in

Studies on acute toxic effects to keratinocytes induced by hematoporphyrin derivatives and laser light.

PubMed

Artuc, M; Ramshad, M; Kappus, H

1989-01-01

Human epidermal keratinocytes were grown in culture and the uptake of hematoporphyrin derivatives (HPDs) used in photodynamic therapy was estimated. Keratinocytes loaded with HPDs were irradiated with laser light of 632 nm generated by a helium-neon laser and cell toxicity was determined by the trypan blue exclusion test and the measurement of enzyme release. With increasing intracellular concentration of HPDs and with increasing intensity of the laser light, an increasing number of cells took up trypan blue and released the cytosolic enzyme lactate dehydrogenase and the lysosomal enzyme acid phosphatase after 1 h incubation of the irradiated cells at 37 degrees C. Cytotoxicity was less pronounced when the irradiated cells were incubated at 0 degree C indicating the involvement of enzyme reactions in cell death. No lipid peroxidation as measured by malondialdehyde and ethane formation was detectable. Our results suggest that during photodynamic therapy with HPDs and laser light epidermal keratinocytes may be seriously damaged. The data indicate that not lipid peroxidation but rather the activation of lysosomal enzymes is responsible for the cytotoxicity observed.

Lyophilized keratinocyte cell lysates contain multiple mitogenic activities and stimulate closure of meshed skin autograft-covered burn wounds with efficiency similar to that of fresh allogeneic keratinocyte cultures.

PubMed

Duinslaeger, L; Verbeken, G; Reper, P; Delaey, B; Vanhalle, S; Vanderkelen, A

1996-07-01

For several years, grafting with allogeneic keratinocyte cultures has been used successfully as a wound-healing therapy both by us and by many other groups. Since their postgrafting survival time is limited, the effect of these cultures is generally explained by the production of wound repair-stimulating factors that promote proliferation and migration of resident cells. In this study we show that lysates of cultured keratinocytes contain mitogenic activity for keratinocytes, endothelial cells, and fibroblasts. In addition, the lysates inhibit the contraction of collagen gels by human skin fibroblasts. On the basis of these observations and of in vivo data obtained by ourselves and others, we have evaluated the effect of total keratinocyte lysates on the healing of meshed skin autograft-covered burn wounds. Twenty burn wounds were tangentially excised and autografted with one to three meshed conventional skin transplants. An area treated with a gel containing lysated keratinocyte cultures was compared with an area treated with placebo-gel in terms of epithelialization on day 5. In six patients an additional fresh keratinocyte alloculture was applied as a positive control. Results indicate that the newly formed epithelium (difference between percentage of epithelialization on day 5 and on day 0) was 31.1 percent in the treated area compared with 16.5 percent in the placebo area. This result is comparable with the value obtained by treatment with fresh keratinocyte allocultures, namely, 33.8 percent. These figures show a twofold stimulation of epithelialization.

Pimecrolimus enhances TLR2/6-induced expression of antimicrobial peptides in keratinocytes.

PubMed

Büchau, Amanda S; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Gallo, Richard L

2008-11-01

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

The herpes simplex virus-induced demise of keratinocytes is associated with a dysregulated pattern of p63 expression.

PubMed

Megyeri, Klára; Orosz, László; Kormos, Bernadett; Pásztor, Katalin; Seprényi, György; Ocsovszki, Imre; Mándi, Yvette; Bata-Csörgo, Zsuzsanna; Kemény, Lajos

2009-01-01

p63 plays a pivotal role in the development and maintenance of stratified epithelial tissues. In an effort to gain insight into the pathogenic mechanisms of skin infections caused by HSV-1 and HSV-2, we determined the patterns of p63 expression in primary keratinocytes and in the HaCaT cell line. The levels of DeltaNp63alpha and a 50kDa p73 isoform were decreased, Bax-alpha remained unaffected, while the expressions of the Bax-beta, TAp63gamma and a 44.5kDa p73 isoform were highly increased in both HSV-1-infected HaCaT cells and primary keratinocytes. In contrast, in response to HSV-2 infection the levels of DeltaNp63alpha, a 50kDa p73 isoform and a 44.5kDa p73 protein were decreased, Bax-alpha and TAp63gamma remained unaffected, while the expression of Bax-beta was slightly increased. The knockdown of TAp63 expression enhanced the viability of HSV-1-infected cells. Thus, HSV-1 and HSV-2 modulate the patterns of p63 and Bax expression in a serotype-specific manner. The dysregulated pattern of p63 expression observed in HSV-infected keratinocytes may comprise part of a mechanism by which these viruses perturb the functions of keratinocytes and lead to their demise.

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection

PubMed Central

Bourke, Claire D.; Prendergast, Catriona T.; Sanin, David E.; Oulton, Tate E.; Hall, Rebecca J.; Mountford, Adrian P.

2015-01-01

Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation*

PubMed Central

Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

2016-01-01

Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. PMID:26627828

Wave Energy Prize - 1/20th Testing - RTI Wave Power

DOE Data Explorer

Scharmen, Wesley

2016-09-30

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the RTI Wave Power team, including the 1/20th Test Plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Effects of lunar and mars dust on HaCaT keratinocytes and CHO-K1 fibroblasts

NASA Astrophysics Data System (ADS)

Brix, Klaudia; Slenzka, Klaus; Rehders, Maren; Sadhukhan, Annapurna; Mistry, Rima; Duenne, Matthias; Kempf, Juergen

Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respira-tory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of lunar dust on human health is required to best support future missions to moon. In this study, we used different methods to assess the specific effects of lunar dust onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and since a well orchestrated program ensures proper repair in cases of wounding. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology, metabolic state, survival and proliferation of the cells were determined. Cytotoxi-city and proliferation were measured using the MTT assay, metabolic activity was analyzed by vital staining of mitochondria, and phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells. It was found that the effects of the two types of soils on the different features of both cell lines varied to considerable extent, and that lunar and mars dust were specific in their effects. The obtained results will facilitate detailed inves-tigations of dust exposure during wound healing and will ease risk assessment studies for e.g. lunar lander approaches. The investigations will help to assess the risks and to determine safety measures to be taken during extraterrestrial expeditions in order to minimize risks to human health associated with exposure of human skin to dust contaminants.

The effect of growth hormone on fibroblast proliferation and keratinocyte migration.

PubMed

Lee, Sang Woo; Kim, Suk Hwa; Kim, Ji Youn; Lee, Yoonho

2010-04-01

The beneficial effects of growth hormones (GHs) on wound healing have been reported. Although the mechanism of how GH promotes wound healing is unclear, there are reports showing that the principal factor lies in the GH-stimulated production of IGF-1 in topical wounds. In this study, a human primary cell model was devised to examine how the topical application of GHs affects fibroblast proliferation and keratinocyte migration, which play fundamental roles in wound healing. The fibroblasts were cultured in media with different concentrations of GH. The amount of fibroblast proliferation was assessed using a tetrazolium-based colourimetric assay (MTT assay). The amount of newly formed IGF-I mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR). Keratinocyte migration was compared using a migration assay. Fibroblast proliferation was significantly higher in the experimental group than in the control group (the absorbance of 2.5IU L(-1) GH applied group: 0.3954+/-0.056, control group: 0.2943+/-0.0554, P<0.05), and the promotion of IGF-I formation by fibroblasts was observed. There was more keratinocyte migration in the experimental group than in the control group (the remaining gap in the 2.5IU L(-1) GH applied group after keratinocyte migration: 46.57+/-2.22% of the primary gap, control group: 75.14+/-3.44%, P<0.05). GH enhances the local formation of IGF-1, which activates fibroblast proliferation and keratinocyte migration. These results highlight the potential of the topical application of GHs in the treatment of wounds. Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. All rights reserved.

The Th1/Th2/Th17/Treg paradigm induced by stachydrine hydrochloride reduces uterine bleeding in RU486-induced abortion mice.

PubMed

Li, Xia; Wang, Bin; Li, Yuzhu; Wang, Li; Zhao, Xiangzhong; Zhou, Xianbin; Guo, Yuqi; Jiang, Guosheng; Yao, Chengfang

2013-01-09

The Th1/Th2/Th17/Treg paradigm plays an important role in achieving maternal-fetal immunotolerance and participates in RU486-induced abortion. Excessive uterine bleeding is the most common side effect of RU486-induced abortion; however, its etiopathogenesis has not been fully understood. Therefore, elucidating the correlation between the Th1/Th2/Th17/Treg paradigm and the volume of uterine bleeding may offer novel therapeutic target for reducing uterine bleeding in RU486-induced abortion. Leonurus sibiricus has been used in clinics to reduce postpartum hemorrhage with low toxicity and high efficiency; however, the effective constituents and therapeutic mechanism have not been described. Stachydrine hydrochloride is the main constituent of L. sibiricus, therefore L. sibiricus is regarded as a candidate for reducing uterine bleeding in RU486-induced abortion mice by regulating the Th1/Th2/Th17/Treg paradigm. The purpose of this study was to determine the Th1/Th2/Th17/Treg paradigm in uterine bleeding of RU486-induced abortion mice and to elucidate the immunopharmacologic effects of stachydrine hydrochloride on inducing the Th1/Th2/Th17/Treg paradigm in reducing the uterine bleeding volume in RU486-induced abortion mice. To investigate the Th1/Th2/Th17/Treg paradigm in uterine bleeding during RU486-induced abortion mice, pregnant BALB/c mice were treated with high- and low-dose RU486 (1.5mg/kg and 0.9 mg/kg, respectively), and the serum progesterone (P(4)) protein level, uterine bleeding volume, and proportions of Th1/Th2/Th17/Treg cells in mice at the maternal-fetal interface were detected by ELISA assay, alkaline hematin photometric assay, and flow cytometry, respectively. To determine the regulatory effect of stachydrine hydrochloride on the Th1/Th2/Th17/Treg paradigm in vitro, splenocytes of non-pregnant mice were separated and treated with P(4,) RU486, and/or stachydrine hydrochloride (10(-5)M, 10(-4)M, and 10(-3)M, respectively). The proportions of Th1/Th2/Th17

Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: Implications for oral carcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, San San; Crabb, Simon J.; Janghra, Nari

2007-09-10

In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1{alpha},25-dihydroxyvitamin D{sub 3.} BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified themore » nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1{alpha},25-dihydroxyvitamin D{sub 3}. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.« less

Wave Energy Prize - 1/20th Testing - M3 Wave

DOE Data Explorer

Wesley Scharmen

2016-08-12

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the M3 Wave team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Wave Energy Prize - 1/20th Testing - Harvest Wave Energy

DOE Data Explorer

Wesley Scharmen

2016-08-26

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the Harvest Wave Energy team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Determination of neutron capture cross sections of 232Th at 14.1 MeV and 14.8 MeV using the neutron activation method

NASA Astrophysics Data System (ADS)

Lan, Chang-Lin; Zhang, Yi; Lv, Tao; Xie, Bao-Lin; Peng, Meng; Yao, Ze-En; Chen, Jin-Gen; Kong, Xiang-Zhong

2017-04-01

The 232Th(n, γ)233Th neutron capture reaction cross sections were measured at average neutron energies of 14.1 MeV and 14.8 MeV using the activation method. The neutron flux was determined using the monitor reaction 27Al(n,α)24Na. The induced gamma-ray activities were measured using a low background gamma ray spectrometer equipped with a high resolution HPGe detector. The experimentally determined cross sections were compared with the data in the literature, and the evaluated data of ENDF/B-VII.1, JENDL-4.0u+, and CENDL-3.1. The excitation functions of the 232Th(n,γ)233Th reaction were also calculated theoretically using the TALYS1.6 computer code. Supported by Chinese TMSR Strategic Pioneer Science and Technology Project-The Th-U Fuel Physics Term (XDA02010100) and National Natural Science Foundation of China (11205076, 21327801)

Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We foundmore » that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.« less

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

PubMed Central

Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-01-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

PubMed

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic atopic dermatitis (AD) shows similar Th2 and higher Th17 immune activation compared to extrinsic AD

PubMed Central

Suárez-Fariñas, M; Dhingra, N; Gittler, J; Shemer, A; Cardinale, I; de Guzman Strong, C; Krueger, JG; Guttman-Yassky, E

2013-01-01

Background Atopic dermatitis (AD) is classified as extrinsic (ADe) and intrinsic (ADi), representing approximately 80% and 20% of patients, respectively. While sharing a similar clinical phenotype, only ADe is characterized by high serum IgE. Since most AD patients exhibit high IgE, an “allergic”/IgE-mediated disease pathogenesis was hypothesized. However, current models associate AD with T-cell activation, particularly Th2/Th22 polarization, and epidermal barrier defects. Objective To define if both variants share a common pathogenesis. Methods We stratified 51 severe AD patients as ADe (42) and ADi (9) (with similar mean disease activity/SCORAD), and analyzed the molecular and cellular skin pathology of lesional and non-lesional ADi and ADe using gene-expression (RT-PCR) and immunohistochemistry. Results A significant correlation between IgE levels and SCORAD (r=0.76, p<10−5) was found only in ADe. Marked infiltrates of T-cells and dendritic cells and corresponding epidermal alterations (K16, Mki67, S100A7/A8/A9) defined lesional skin of both variants. However, higher activation of all inflammatory axes (including Th2) was detected in ADi, particularly Th17 and Th22-cytokines. Positive correlations between Th17-related molecules and SCORAD were only found in ADi, while only ADe showed positive correlations between SCORAD and Th2-cytokines (IL-4, IL-5), and negative correlations with differentiation products (loricrin, periplakin). Conclusions Although differences in Th17 and Th22 activation exist between ADi and ADe, we identified common disease-defining features of T-cell activation, production of polarized cytokines, and keratinocyte responses to immune products. Our data indicate that a Th2 bias is not the sole cause of high IgE in ADe, with important implications for similar therapeutic interventions. Clinical Implications Both extrinsic and intrinsic AD variants might be treated with T-cell targeted therapeutics or agents that modify keratinocyte

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell

Pleiotropic Effects of White Willow Bark and 1,2-Decanediol on Human Adult Keratinocytes.

PubMed

Bassino, Eleonora; Gasparri, Franco; Munaron, Luca

2018-01-01

Acne vulgaris is a common skin defect, usually occurring during adolescence, but often it can persist in adults leaving permanent face scarring. Acne is usually treated with topical drugs, oral antibiotics, retinoids, and hormonal therapies, but medicinal plants are increasingly employed. To investigate the protective role of white willow bark (WWB) and 1,2-decanediol (DD) on the damage caused by lipopolysaccharides (LPS) on human adult keratinocytes (HaCaT). HaCaT were exposed to LPS alone or in association with WWB and DD. Epidermal viability, metabolic modulation, inflammatory activity, and cell migration were assessed with both common standardized protocols or high-throughput screening systems. The preincubation of HaCaT with WWB and DD (used separately or in combination) differently prevented the alterations induced by LPS on HaCaT in terms of growth factor release (IGF, EGF, VEGF), cytokine production (IL-1α, IL-6, IL-8), or expression of the transcription factor FOXO-I. Moreover, they partially restore wound repair lowered by LPS. These results suggest that both natural compounds were able to differently affect several functions of LPS-stressed keratinocytes suggesting their potential role for the prevention of acne vulgaris, without adverse effects. © 2017 S. Karger AG, Basel.

Optimized delivery of skin keratinocytes by aerosolization and suspension in fibrin tissue adhesive.

PubMed

Harkin, Damien G; Dawson, Rebecca A; Upton, Zee

2006-01-01

Aerosolized suspensions of keratinocytes provide a potential therapy for wounds, but the effects of aerosolization on cell viability remain unclear. Likewise, little is known of the resulting cell distribution pattern and how this compares to the density required for epithelialization. The potential benefits of cospraying cells in the presence of fibrin adhesive are equally uncertain. Thus, in the present study we have optimized conditions for the aerosolization of cultured keratinocytes using a device (Tissomat) that supports the option for coapplication with fibrin (Tisseel). Cell viability was unaffected when sprayed at 10 psi, but a significant reduction in metabolic activity, as determined by the methylthiazoyldiphenol-tetrazolium assay, was observed at higher pressure. Bursts of 0.2 mL cell suspension (1.5x10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm2 within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm2 at 24 hours. Optimal cell growth in Tisseel was achieved through dilution of fibrinogen (1-3 mg/mL) and thrombin (2-5 IU/mL). This optimized formulation eliminated fluid run-off postspraying and stimulated a twofold increase in cellular response. Therefore, our in vitro data supports the theory that aerosolized suspensions of keratinocytes in fibrin will benefit healing.

Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component.

PubMed

Lin, Yin-Ku; Leu, Yann-Lii; Yang, Sien-Hung; Chen, Hsiao-Wen; Wang, Chin-Ting; Pang, Jong-Hwei Su

2009-06-01

Indigo naturalis has shown efficacy in treating psoriasis in our previous clinical studies. To investigate the potential effect of indigo naturalis on regulating keratinocyte proliferation and differentiation. Skin samples from six patients were analyzed for proliferating cell nuclear antigen (PCNA) and involucrin expression by immunohistochemical staining. In addition, indigo naturalis extracts from 10 to 500 microg/ml were added to cultured keratinocytes and cell viability determined. Real-time RT-PCR, Western blotting analysis and indirect immunofluorescent labeling were used to investigate the messenger (m)RNA and protein expressions of PCNA and involucrin. Finally, high-performance liquid chromatography (HPLC) was used to identify major components of indigo naturalis and their in vitro effects compared. Immunohistochemical results demonstrated decreased PCNA and increased involucrin in psoriatic lesions after indigo naturalis treatment. Cultured keratinocytes decreased after indigo naturalis treatment, while G(0)/G(1) arrest was observed to dose-dependently increase. Staining revealed decreased PCNA-stained nuclei and increased cytosolic involucrin in treated keratinocytes. Decreased PCNA and increased involucrin at both the mRNA and protein levels were confirmed. Both major components, indirubin and indigo, could cause G(0)/G(1) phase arrest; however, only indirubin modulated the expressions of PCNA and involucrin similar to indigo naturalis. Together, these findings indicate that the anti-psoriatic effects of indigo naturalis are mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes, with indirubin as the major active component.

Exosomes released by keratinocytes modulate melanocyte pigmentation

PubMed Central

Cicero, Alessandra Lo; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

2015-01-01

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

Pimecrolimus Enhances TLR2/6-Induced Expression of Antimicrobial Peptides in Keratinocytes

PubMed Central

Büchau, Amanda S.; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Gallo, Richard L.

2009-01-01

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human β-defensin-2 and β-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureusand decreased TLR2/6-induced expression of IL-10 and IL-1β. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-κB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense. PMID:18496569

Improvement of Human Keratinocyte Migration by a Redox Active Bioelectric Dressing

PubMed Central

Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K.; Bellman, Karen; Dickinson, Bryan C.; Suri, Prerna; Subramaniam, Vish V.; Chang, Christopher J.; Sen, Chandan K.

2014-01-01

Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization. PMID:24595050

The effects of phototherapy and melanocytes on keratinocytes

PubMed Central

Tang, Luyan; Wu, Wenyu; Fu, Wenwen; Hu, Yao

2018-01-01

Phototherapy is widely used in the treatment of vitiligo. Previous studies have focused on the effects of ultraviolet (UV) radiation on melanocytes; however, the biological effects of phototherapy and melanocytes on keratinocytes remain to be elucidated. To investigate and assess the effects of clinically doses of broad band (BB)-UVA, narrow band (NB)-UVB and melanocytes on human keratinocytes in vitro, clinical doses of BB-UVA or NB-UVB radiation and human melanoma cell A375 co-culture were performed as stress divisors to HaCaT cells. Cell proliferation, expression of protease-activated receptor-2 (PAR-2) and nuclear factor E2-related factor 2 mRNA, lipid peroxidation and intracellular antioxidant level of keratinocytes were analyzed. It was demonstrated that UV radiation inhibited the proliferation of cells apart from following exposure to low dose (1 J/cm2) UVA. Medium dose (5 J/cm2) UVA radiation had no adverse effects on lipid peroxidation and increased antioxidant levels in HaCaT cells. Medium (200 mJ/cm2) and high (400 mJ/cm2) doses of UVB radiation induced cellular damage due to increased lipid peroxidation as indicated by levels of malondialdehyde. Furthermore, A375 co-culture treatment induced a similar effect on the lipid peroxidation of HaCaT as with low dose UVB radiation. Therefore, the results of the present study determined that clinical doses of BB-UVA and NB-UVB radiation had varying effects on proliferation and related protein levels in HaCaT cells. Co-culture with A375 had similar effects as those of low dose UVA and UVB radiation, in which the PAR-2 expression was significantly upregulated. PMID:29545869

The Benefit of Microskin in Combination With Autologous Keratinocyte Suspension to Treat Full Skin Loss In Vivo.

PubMed

Yuru, Shang; Dawei, Li; Chuanan, Shen; Kai, Yin; Li, Ma; Longzhu, Li; Dongxu, Zhao; Wenfeng, Cheng

Patients with extensive deep burns often lack enough autologous skin to cover the wounds. This study explores a new method using microskin in combination with autologous keratinocytes in the treatment of extensive deep burn. Wounds in the combination group were treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area) and autologous keratinocyte suspension, which were compared with the following treatments: no autotransplant, only allografts (control group); autologous keratinocyte suspension only (keratinocyte only group); automicroskin at an area expansion ratio of 20:1 (20:1 group); and automicroskin at an area expansion ratio of 10:1 (10:1 group, positive control). The authors used epithelialization rate (epithelialized area on day 21 divided by original wound area), hematoxylin and eosin staining, laminin, and type IV collagen immunohistochemistry to assess wound healing. The epithelialization rate of combination group (74.2% ± 8.0%) was similar to that of 10: 1 group (84.3% ± 11.9%, P = .085) and significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). The hematoxylin and eosin staining and immunohistochemistry showed the epithelialization in the combination group was better than that in the keratinocyte only group and control group. Microskin in combination with autologous keratinocyte suspension can promote the reepithelialization of full-thickness wounds and reduce the requirements for automircoskin, and it is a useful option in the treatment of extensive deep burns.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

PubMed

Pomari, Elena; Dalla Valle, Luisa; Pertile, Paolo; Colombo, Lorenzo; Thornton, M Julie

2015-02-01

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin. © FASEB.

NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis.

PubMed

Raad, Houssam; Serrano-Sanchez, Martin; Harfouche, Ghida; Mahfouf, Walid; Bortolotto, Doriane; Bergeron, Vanessa; Kasraian, Zeinab; Dousset, Lea; Hosseini, Mohsen; Taieb, Alain; Rezvani, Hamid Reza

2017-06-01

The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Considerations in the improvement of human epidermal keratinocyte culture in vitro.

PubMed

Kaviani, Maryam; Geramizadeh, Bita; Rahsaz, Marjan; Marzban, Saeed

2015-04-01

Large-scale expansion of epidermal keratinocytes is essential in the application of these cells for severe burn treatment in patients. Therefore, this study was designed to evaluate various conditions in the expansion of human epidermal keratinocytes. The epidermis was separated from the dermis of skin samples using dispase. The epidermis was trypsinized for keratinocyte isolation. Keratinocytes were cultured in various conditions, with or without a human dermal fibroblast feeder layer, mitomycin C treatment, and different culture media. Our results suggest that keratinocytes cultured on a human dermal fibroblast feeder layer were grown for several passages. Extensive deformation and rapid deterioration were observed in the cultured cells without a feeder layer and in serum-free medium. Human dermal fibroblasts treated with mitomycin C can provide optimal conditions for proliferation of keratinocytes.

Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes.

PubMed

Avezov, Katia; Reznick, Abraham Z; Aizenbud, Dror

2014-06-01

Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers. The objectives of the current study were to examine the effect of aldehydes originating from CS on intracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells. Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaT keratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possible intracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase (LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein) assay and GSH (glutathione) oxidation. Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to 1 μmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CS and acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels compared to controls. Activity of intracellular LDH was preserved. α,β-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role in intracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ligand activation of peroxisome proliferator-activated receptor-beta/delta inhibits cell proliferation in human HaCaT keratinocytes.

PubMed

Borland, Michael G; Foreman, Jennifer E; Girroir, Elizabeth E; Zolfaghari, Reza; Sharma, Arun K; Amin, Shantu; Gonzalez, Frank J; Ross, A Catharine; Peters, Jeffrey M

2008-11-01

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-beta/delta induces terminal differentiation and attenuates cell growth, some studies suggest that PPARbeta/delta actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARbeta/delta and potentiates cell proliferation by activating PPARbeta/delta. The present study examined the effect of ligand activation of PPARbeta/delta on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARbeta/delta ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARbeta/delta ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARbeta/delta target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARbeta/delta-null primary mouse keratinocytes to determine the specific role of PPARbeta/delta in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARbeta/delta-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARbeta/delta inhibits keratinocyte proliferation through PPARbeta/delta-dependent mechanisms. In contrast, the observed inhibition of

Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch*

PubMed Central

Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y.; MacLeod, Amanda S.; Hall, Russell P.; Liedtke, Wolfgang B.

2016-01-01

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance. PMID:26961876

Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

PubMed

Banovic, F; Dunston, S; Linder, K E; Rakich, P; Olivry, T

2017-03-01

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

A Model to Predict the Risk of Keratinocyte Carcinomas.

PubMed

Whiteman, David C; Thompson, Bridie S; Thrift, Aaron P; Hughes, Maria-Celia; Muranushi, Chiho; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

2016-06-01

Basal cell and squamous cell carcinomas of the skin are the commonest cancers in humans, yet no validated tools exist to estimate future risks of developing keratinocyte carcinomas. To develop a prediction tool, we used baseline data from a prospective cohort study (n = 38,726) in Queensland, Australia, and used data linkage to capture all surgically excised keratinocyte carcinomas arising within the cohort. Predictive factors were identified through stepwise logistic regression models. In secondary analyses, we derived separate models within strata of prior skin cancer history, age, and sex. The primary model included terms for 10 items. Factors with the strongest effects were >20 prior skin cancers excised (odds ratio 8.57, 95% confidence interval [95% CI] 6.73-10.91), >50 skin lesions destroyed (odds ratio 3.37, 95% CI 2.85-3.99), age ≥ 70 years (odds ratio 3.47, 95% CI 2.53-4.77), and fair skin color (odds ratio 1.75, 95% CI 1.42-2.15). Discrimination in the validation dataset was high (area under the receiver operator characteristic curve 0.80, 95% CI 0.79-0.81) and the model appeared well calibrated. Among those reporting no prior history of skin cancer, a similar model with 10 factors predicted keratinocyte carcinoma events with reasonable discrimination (area under the receiver operator characteristic curve 0.72, 95% CI 0.70-0.75). Algorithms using self-reported patient data have high accuracy for predicting risks of keratinocyte carcinomas. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Foreskin-isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts.

PubMed

Mcheik, Jiad N; Barrault, Christine; Pedretti, Nathalie; Garnier, Julien; Juchaux, Franck; Levard, Guillaume; Morel, Franck; Lecron, Jean-Claude; Bernard, François-Xavier

2016-03-01

Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls. Copyright © 2013 John Wiley & Sons, Ltd.

Transcription factor-dependent chromatin remodeling of Il18r1 during Th1 and Th2 differentiation 1

PubMed Central

Yu, Qing; Chang, Hua-Chen; Ahyi, Ayele-Nati N.; Kaplan, Mark H.

2008-01-01

The IL-18Rα chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Rα) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report we demonstrate that the establishment of DNase hypersensitivity patterns that are distinct among undifferentiated CD4 T cells, Th1 and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Rα. These data provide further mechanistic insight into transcription factor dependent establishment of Th subset-specific patterns of gene expression. PMID:18714006

Elevated YAP and its downstream targets CCN1 and CCN2 in basal cell carcinoma: impact on keratinocyte proliferation and stromal cell activation.

PubMed

Quan, Taihao; Xu, Yiru; Qin, Zhaoping; Robichaud, Patrick; Betcher, Stephanie; Calderone, Ken; He, Tianyuan; Johnson, Timothy M; Voorhees, John J; Fisher, Gary J

2014-04-01

Yes-associated protein (YAP) is a transcriptional co-activator of hippo signaling pathway, which plays an important role in organ size control and tumorigenesis. Here we report that YAP and its downstream transcriptional targets CCN1 and CCN2 are markedly elevated in keratinocytes in human skin basal cell carcinoma tumor islands. In human keratinocytes, knockdown of YAP significantly reduced expression of CCN1 and CCN2, and repressed proliferation and survival. This inhibition of proliferation and survival was rescued by restoration of CCN1 expression, but not by CCN2 expression. In basal cell carcinoma stroma, CCN2-regulated genes type I collagen, fibronectin, and α-smooth muscle actin were highly expressed. Furthermore, atomic force microscopy revealed increased tissue stiffness in basal cell carcinoma stroma compared to normal dermis. These data provide evidence that up-regulation of YAP in basal cell carcinoma impacts both aberrant keratinocyte proliferation, via CCN1, and tumor stroma cell activation and stroma remodeling, via CCN2. Targeting YAP and/or CCN1 and CCN2 may provide clinical benefit in basal cell carcinoma. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Wave Energy Prize - 1/20th Testing - CalWave Power Technologies

DOE Data Explorer

Scharmen, Wesley

2016-09-09

Data from the 1/20th scale testing data completed on the Wave Energy Prize for the CalWave Power Technologies team, including the 1/20th scale test plan, raw test data, video, photos, and data analysis results. The top level objective of the 1/20th scale device testing is to obtain the necessary measurements required for determining Average Climate Capture Width per Characteristic Capital Expenditure (ACE) and the Hydrodynamic Performance Quality (HPQ), key metrics for determining the Wave Energy Prize (WEP) winners.

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.

PubMed

Niwano, Takao; Terazawa, Shuko; Nakajima, Hiroaki; Wakabayashi, Yuki; Imokawa, Genji

2015-06-01

Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity

Protective properties of ginsenoside Rb1 against UV-B radiation-induced oxidative stress in human dermal keratinocytes.

PubMed

Oh, Sun-Joo; Kim, Kyunghoon; Lim, Chang-Jin

2015-06-01

Ginsenosides, also known as ginseng saponins, are responsible for most pharmacological effect of ginseng. Ginsenoside Rb1 (Rb1) exerts a variety of pharmacological properties, including anti-inflammatory, antistress, anti-aging and anti-neurodegenerative activities. The aim of the present work was to assess the skin anti-photoaging properties of Rb1 in human dermal keratinocyte HaCaT cells. The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) as well as cell viability for HaCaT cells under UV-B irradiation. Rb1 was able to suppress the ROS levels which were elevated under UV-B irradiation, and unable to influence cellular survival in UV-B-irradiated HaCaT cells. Rb1 diminished the enhancement of MMP-2 gelatinolytic activity in conditioned medium, which corresponded with the decreased MMP-2 protein levels in both conditioned medium and cellular lysate prepared from UV-B-irradiated HaCaT cultures. Rb1 could restore the total glutathione (GSH) and superoxide dismutase (SOD) activity diminished in UV-B-irradiated HaCaT cells. Ginsenoside Rb1 possesses skin anti-photoaging properties through scavenging ROS and decreasing MMP-2 levels possibly by enhancing antioxidant activity in keratinocytes under UV-B irradiation.

Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models.

PubMed

Smits, Jos P H; Niehues, Hanna; Rikken, Gijs; van Vlijmen-Willems, Ivonne M J J; van de Zande, Guillaume W H J F; Zeeuwen, Patrick L J M; Schalkwijk, Joost; van den Bogaard, Ellen H

2017-09-19

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Sustainability of keratinocyte gene transfer and cell survival in vivo.

PubMed

Choate, K A; Khavari, P A

1997-05-20

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

[Current Topics on Vitamin D. The vitamin D functions in keratinocytes and its therapeutic applications].

PubMed

Sawatsubashi, Shun

2015-03-01

1,25 (OH) 2D and calcium have been shown to promote epidermal keratinocyte differentiation and prevent proliferation. These prodifferentiation and antiproliferative effects of 1,25 (OH) 2D have led to its clinical use in the treatment of psoriasis. However, the mechanism of vitamin D action on keratinocytes remains largely unknown. While the actions of calcium and the vitamin D receptor signaling pathways on epidermal keratinocyte differentiation are redundant, their effects on the hair follicle are not. In this review, we discuss how the vitamin D and its receptor contribute to skin and hair follicle homeostasis.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

PubMed

Mehalick, Leslie A; Poulsen, Christopher; Fischer, Carol L; Lanzel, Emily A; Bates, Amber M; Walters, Katherine S; Cavanaugh, Joseph E; Guthmiller, Janet M; Johnson, Georgia K; Wertz, Philip W; Brogden, Kim A

2015-12-01

Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Ligand Activation of Peroxisome Proliferator-Activated Receptor-β/δ Inhibits Cell Proliferation in Human HaCaT KeratinocytesS

PubMed Central

Borland, Michael G.; Foreman, Jennifer E.; Girroir, Elizabeth E.; Zolfaghari, Reza; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Ross, A. Catharine; Peters, Jeffrey M.

2009-01-01

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-β/δ induces terminal differentiation and attenuates cell growth, some studies suggest that PPARβ/δ actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARβ/δ and potentiates cell proliferation by activating PPARβ/δ. The present study examined the effect of ligand activation of PPARβ/δ on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARβ/δ ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARβ/δ ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARβ/δ target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARβ/δ-null primary mouse keratinocytes to determine the specific role of PPARβ/δ in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARβ/δ-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARβ/δ inhibits keratinocyte proliferation through PPARβ/δ-dependent mechanisms. In contrast, the observed inhibition of cell proliferation in mouse and human keratinocytes by RA is

Overexpressed TRPV3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

PubMed Central

Huang, Susan M.; Lee, Hyosang; Chung, Man-Kyo; Park, Una; Yu, Yin Yin; Bradshaw, Heather B.; Coulombe, Pierre A.; Walker, J. Michael; Caterina, Michael J.

2009-01-01

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive TRPV ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared to wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naïve mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Co-administration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. PMID:19091963

Sulfation degree not origin of chondroitin sulfate derivatives modulates keratinocyte response.

PubMed

Corsuto, Luisana; Rother, Sandra; Koehler, Linda; Bedini, Emiliano; Moeller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Schiraldi, Chiara; Scharnweber, Dieter

2018-07-01

Chondroitin sulfate (CS) sulfation-dependently binds transforming growth factor-β1 (TGF-β1) and chronic wounds often accompany with epidermal hyperproliferation due to downregulated TGF-β signaling. However, the impact of CS on keratinocytes is unknown. Especially biotechnological-chemical strategies are promising to replace animal-derived CS. Thus, this study aims to evaluate the effects of CS derivatives on the interaction with vascular endothelial growth factor-A (VEGF-A) and on keratinocyte response. Over-sulfated CS (sCS3) interacts stronger with VEGF-A than CS. Furthermore, collagen coatings with CS variants are prepared by in vitro fibrillogenesis. Stability analyses demonstrate that collagen is firmly integrated, while the fibril diameters decrease with increasing sulfation degree. CS variants sulfation-dependently decelerate keratinocyte (HaCaT) migration and proliferation in a scratch assay. HaCaT cultured on sCS3-containing coatings produced increased amounts of solute active TGF-β1 which could be translated into biomaterials able to decrease epidermal hyperproliferation in chronic wounds. Overall, semi-synthetic and natural CS yield to comparable responses. Copyright © 2018 Elsevier Ltd. All rights reserved.

Apigenin Attenuates Experimental Autoimmune Myocarditis by Modulating Th1/Th2 Cytokine Balance in Mice.

PubMed

Zhang, Shouxin; Liu, Xiaoyan; Sun, Chengming; Yang, Jun; Wang, Lihong; Liu, Jie; Gong, Lei; Jing, Yanyan

2016-04-01

This study aims to investigate the protective effect of apigenin on the development of experimental autoimmune myocarditis (EAM) and the underlying mechanisms. An EAM model was induced in BALB/c mice by the injection of porcine cardiac myosin. Apigenin was orally administered from day 1 to 21. The severity of myocarditis was assessed by determination of heart weight/body weight ratio (HW/BW) and histopathological evaluation. Echocardiography was conducted to evaluate the cardiac function and heart structure. Antigen-specific T cell proliferation responses to cardiac myosin were evaluated by the lymphocyte proliferation assay. ELISA was used to determine serum levels of type 1 helper (Th1) and Th2 cytokines. Apigenin treatment significantly decreased HW/BW. Histopathologic analysis showed that the infiltration of inflammatory cells was reduced significantly by apigenin treatment. Meanwhile, apigenin administration effectively ameliorated autoimmune myocarditis-induced cardiac hypertrophy and cardiac dysfunction as well as inhibited lymphocyte proliferation in mice immunized with myosin. Furthermore, Th1 cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-2 (IL-2) were significantly downregulated, while Th2 cytokines IL-4 and IL-10 were markedly upregulated. The results indicated that apigenin can alleviate EAM due to its immunomodulatory reactions in modification of helper T cell balance.

Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

PubMed Central

Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

2013-01-01

Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

MicroRNA-17-92 cluster promotes the proliferation and the chemokine production of keratinocytes: implication for the pathogenesis of psoriasis.

PubMed

Zhang, Weigang; Yi, Xiuli; An, Yawen; Guo, Sen; Li, Shuli; Song, Pu; Chang, Yuqian; Zhang, Shaolong; Gao, Tianwen; Wang, Gang; Li, Chunying

2018-05-11

Keratinocytes are the main epidermal cell type that constitutes the skin barrier against environmental damages, which emphasizes the balance between the growth and the death of keratinocytes in maintaining skin homeostasis. Aberrant proliferation of keratinocytes and the secretion of inflammatory factors from keratinocytes are related to the formation of chronic inflammatory skin diseases like psoriasis. MicroRNA-17-92 (miRNA-17-92 or miR-17-92) is a miRNA cluster that regulates cell growth and immunity, but the role of miR-17-92 cluster in keratinocytes and its relation to skin diseases have not been well investigated. In the present study, we initially found that miR-17-92 cluster promoted the proliferation and the cell-cycle progression of keratinocytes via suppressing cyclin-dependent kinase inhibitor 2B (CDKN2B). Furthermore, miR-17-92 cluster facilitated the secretion of C-X-C motif chemokine ligand 9 (CXCL9) and C-X-C motif chemokine ligand 10 (CXCL10) from keratinocytes by inhibiting suppressor of cytokine signaling 1 (SOCS1), which enhanced the chemotaxis for T lymphocytes formed by keratinocytes. In addition, we detected increased expression of miR-17-92 cluster in psoriatic lesions and the level of lesional miR-17-92 cluster was positively correlated with the disease severity in psoriasis patients. At last, miR-17-92 cluster was increased in keratinocytes by cytokines through the activation of signal transducers and activators of transcription 1 (STAT1) signaling pathway. Our findings demonstrate that cytokine-induced overexpression of miR-17-92 cluster can promote the proliferation and the immune function of keratinocytes, and thus may contribute to the development of inflammatory skin diseases like psoriasis, which implicates miR-17-92 cluster as a potential therapeutic target for psoriasis and other skin diseases with similar inflammatory pathogenesis.

Vitamin D receptor-mediated control of Soggy, Wise, and Hairless gene expression in keratinocytes.

PubMed

Hsieh, Jui-Cheng; Estess, Rudolf C; Kaneko, Ichiro; Whitfield, G Kerr; Jurutka, Peter W; Haussler, Mark R

2014-02-01

The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is required for the progression of the mammalian hair cycle. We studied three genes relevant to hair cycle signaling, DKKL1 (Soggy), SOSTDC1 (Wise), and HR (Hairless), to determine whether their expression is regulated by VDR and/or its 1,25D ligand. DKKL1 mRNA was repressed 49-72% by 1,25D in primary human and CCD-1106 KERTr keratinocytes; a functional vitamin D responsive element (VDRE) was identified at -9590 bp in murine Soggy. Similarly, SOSTDC1 mRNA was repressed 41-59% by 1,25D in KERTr and primary human keratinocytes; a functional VDRE was located at -6215 bp in human Wise. In contrast, HR mRNA was upregulated 1.56- to 2.77-fold by 1,25D in primary human and KERTr keratinocytes; a VDRE (TGGTGAgtgAGGACA) consisting of an imperfect direct repeat separated by three nucleotides (DR3) was identified at -7269 bp in the human Hairless gene that mediated dramatic induction, even in the absence of 1,25D ligand. In parallel, a DR4 thyroid hormone responsive element, TGGTGAggccAGGACA, was identified at +1304 bp in the human HR gene that conferred tri-iodothyronine (T3)-independent transcriptional activation. Because the thyroid hormone receptor controls HR expression in the CNS, whereas VDR functions in concert with the HR corepressor specifically in skin, a model is proposed wherein unliganded VDR upregulates the expression of HR, the gene product of which acts as a downstream comodulator to feedback-repress DKKL1 and SOSTDC1, resulting in integration of bone morphogenic protein and Wnt signaling to drive the mammalian hair cycle and/or influencing epidermal function.

A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration.

PubMed

Peplow, Philip V; Chatterjee, Marissa P

2013-04-01

Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cutaneous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play an important role, and several in vitro assays have been performed to elucidate this. The purposes of this study were to review in vitro human studies on keratinocyte migration to identify those growth factors or cytokines that stimulate keratinocyte migration and whether these assays might serve as a screening procedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo. Research papers investigating effect of growth factors and cytokines on human keratinocyte migration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, and searches of relevant journals. Fourteen different growth factors and cytokines enhanced migration in scratch wound assay and HGF together with TGF-β, and IGF-1 with EGF, were more stimulatory than either growth factor alone. HGF with TGF-β1 had a greater chemokinetic effect than either growth factor alone in transmigration assay. TGF-β1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-α, IL-1α, IGF and MGSA enhanced cell migration on ECM proteins. Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certain combinations of growth factors were more stimulatory than either alone. These and other combinations that stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repair in vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening method for testing individual or combinations of growth factors or cytokines, or growth factors combined with other modalities such as laser irradiation, prior to performing wound healing studies with laboratory animals. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chemosensory Information Processing between Keratinocytes and Trigeminal Neurons

PubMed Central

Sondersorg, Anna Christina; Busse, Daniela; Kyereme, Jessica; Rothermel, Markus; Neufang, Gitta; Gisselmann, Günter; Hatt, Hanns; Conrad, Heike

2014-01-01

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2′,4′-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling. PMID:24790106

Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

PubMed Central

Cerimele, Francesca; Curreli, Francesca; Ely, Scott; Friedman-Kien, Alvin E.; Cesarman, Ethel; Flore, Ornella

2001-01-01

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission. PMID:11160746

Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway.

PubMed

McMullan, Rachel; Lax, Siân; Robertson, Vicki H; Radford, David J; Broad, Simon; Watt, Fiona M; Rowles, Alison; Croft, Daniel R; Olson, Michael F; Hotchin, Neil A

2003-12-16

The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. We have analyzed ROCK's role in the regulation of epidermal keratinocyte function by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. We report that blocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes.

No evidence for induction of key components of the Notch signaling pathway (Notch-1, Jagged-1) by treatment with UV-B, 1,25(OH)(2)D(3), and/or epigenetic drugs (TSA, 5-Aza) in human keratinocytes in vitro.

PubMed

Reichrath, Sandra; Reichrath, Jörg

2012-01-01

Notch signaling is of high importance for growth and survival of various cell types. We now analyzed the protein expression of two key components of the Notch signaling pathway (Notch-1, Jagged-1) in spontaneously immortalized (HaCaT) and in malignant (SCL-1) human keratinocytes, using western analysis. We found that Notch-1 and its corresponding ligand Jagged-1 are expressed in both cell lines, with no marked change following UV-B treatment. Moreover, treatment of both cell lines before or after UV-B irradiation with 1,25-dihydroxyvitamin D(3), the biologically active form of vitamin D, and/or epigenetic modulating drugs (TSA; 5-Aza) did not result in a marked modulation of the protein expression of Notch-1 or Jagged-1. Under the experimental conditions of this study, treatment with 1,25(OH)(2)D(3) protected human keratinocytes in part against the antiproliferative effects of UV-B-radiation. In conclusion, our findings do not point at a differential expression of these two key components of Notch signaling in non-malignant as compared to malignant human keratinocytes, indicating that alterations in their expression are not of importance for the photocarcinogenesis of human squamous cell carcinomas. Moreover, our findings do not support the hypothesis that modulation of Notch signaling may be involved in the photoprotective effect of 1,25-dihydroxyvitamin D(3), that we and others reported previously. Additionally, we demonstrate that epigenetic modulating drugs (TSA, 5-Aza) do not markedly modulate the expression Notch-1 or Jagged-1 in UV-B-treated human keratinocytes in vitro.

Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes

PubMed Central

Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

2017-01-01

Background: Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. Objective: To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. Materials and Methods: We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Results: Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. Conclusion: These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. SUMMARY Hisbiscus syriacus L. flower absolute increases HaCaT cell migration and proliferation.Hisbiscus syriacus L. flower absolute regulates phosphorylation of ERK 1/2 and Akt in HaCaT cell.Treatment with Hisbiscus syriacus L. flower induced sprout outgrowth.The wound in the tail-skin of rat was reduced by Hisbiscus syriacus

Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes.

PubMed

Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

2017-01-01

Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. Hisbiscus syriacus L. flower absolute increases HaCaT cell migration and proliferation. Hisbiscus syriacus L. flower absolute regulates phosphorylation of ERK 1/2 and Akt in HaCaT cell.Treatment with Hisbiscus syriacus L. flower induced sprout outgrowth.The wound in the tail-skin of rat was reduced by Hisbiscus syriacus L. flower absolute Abbreviations used: HSF: Hibiscus syriacus L. flower

Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

EPA Science Inventory

Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes Nanoparticle uptake in cells may be an important determinant of their potential cytotoxic and inflammatory effects. Six commercial TiO2 NP (A=Alfa Aesar,10nm, A*=Alfa Aesar 32nm, B=P25 27...

Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes

PubMed Central

Köllisch, Gabriele; Kalali, Behnam Naderi; Voelcker, Verena; Wallich, Reinhard; Behrendt, Heidrun; Ring, Johannes; Bauer, Stefan; Jakob, Thilo; Mempel, Martin; Ollert, Markus

2005-01-01

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-κB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1–10 expression profile. First, reverse transcription–polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1–10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-κB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-κB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin. PMID:15804290

UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense

PubMed Central

Nechifor, Marina T.; Niculiţe, Cristina M.; Urs, Andreea O.; Regalia, Teodor; Mocanu, Mihaela; Popescu, Alexandra; Manda, Gina; Dinu, Diana; Leabu, Mircea

2012-01-01

UVA affects epidermal cell physiology in a complex manner, but the harmful effects have been studied mainly in terms of DNA damage, mutagenesis and carcinogenesis. We investigated UVA effects on membrane integrity and antioxidant defense of dysplastic keratinocytes after one and two hours of irradiation, both immediately after exposure, and 24 h post-irradiation. To determine the UVA oxidative stress on cell membrane, lipid peroxidation was correlated with changes in fatty acid levels. Membrane permeability and integrity were assessed by propidium iodide staining and lactate dehydrogenase release. The effects on keratinocyte antioxidant protection were investigated in terms of catalase activity and expression. Lipid peroxidation increased in an exposure time-dependent manner. UVA exposure decreased the level of polyunsaturated fatty acids, which gradually returned to its initial value. Lactate dehydrogenase release showed a dramatic loss in membrane integrity after 2 h minimum of exposure. The cell ability to restore membrane permeability was noted at 24 h post-irradiation (for one hour exposure). Catalase activity decreased in an exposure time-dependent manner. UVA-irradiated dysplastic keratinocytes developed mechanisms leading to cell protection and survival, following a non-lethal exposure. The surviving cells gained an increased resistance to apoptosis, suggesting that their pre-malignant status harbors an abnormal ability to control their fate. PMID:23222638

Keratinocyte-driven contraction of reconstructed human skin.

PubMed

Chakrabarty, K H; Heaton, M; Dalley, A J; Dawson, R A; Freedlander, E; Khaw, P T; Mac Neil, S

2001-01-01

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes.

PubMed

Yu, Ran; Ding, Yumei; Zhu, Lijuan; Qu, Yinying; Zhang, Chenguang; Liu, Lin; Chen, Lili

2016-12-01

Wounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity. The function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22, collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes. IL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1, which may contribute to scarless healing. Our study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

PubMed

Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro toxicity testing for antibacterials against human keratinocytes.

PubMed

Smoot, E C; Kucan, J O; Roth, A; Mody, N; Debs, N

1991-05-01

The use of cultured human keratinocytes in an in vitro comparison of topical antibacterial toxicity for epithelial cells was examined. The complement of three assessments allows testing of epithelial migration, growth, and survival. The three assessments included (1) flow cytometry for determination of cell survival, (2) a comparison of confluent cell culture growth after antibacterial exposures, and (3) an evaluation of cell migration using a technique of dermal explants to study radial migration. A comparative ranking of the toxicities of the various topical antibacterials was determined with the three assessments. This has confirmed anecdotal reports that many of the topical antibacterials are cell-toxic and may inhibit wound healing. This information can be directly extrapolated to the clinical setting, unlike many of the animal data for wound healing that currently exist.

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

PubMed

Sun, Baihui; Ding, Ruoting; Yu, Wenlin; Wu, Yanhong; Wang, Bulin; Li, Qin

2016-07-01

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalizationmore » analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.« less

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.

PubMed

Seiberg, M; Paine, C; Sharlow, E; Andrade-Gordon, P; Costanzo, M; Eisinger, M; Shapiro, S S

2000-01-10

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2. Copyright 2000 Academic Press.

Arsenite suppression of BMP signaling in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, Marjorie A.; Qin, Qin; Hu, Qin

2013-06-15

Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction,more » BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of

The Galvanotactic Migration of Keratinocytes is Enhanced by Hypoxic Preconditioning

PubMed Central

Guo, Xiaowei; Jiang, Xupin; Ren, Xi; Sun, Huanbo; Zhang, Dongxia; Zhang, Qiong; Zhang, Jiaping; Huang, Yuesheng

2015-01-01

The endogenous electric field (EF)-directed migration of keratinocytes (galvanotaxis) into wounds is an essential step in wound re-epithelialization. Hypoxia, which occurs immediately after injury, acts as an early stimulus to initiate the healing process; however, the mechanisms for this effect, remain elusive. We show here that the galvanotactic migration of keratinocytes was enhanced by hypoxia preconditioning as a result of the increased directionality rather than the increased motility of keratinocytes. This enhancement was both oxygen tension- and preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours. Hypoxic preconditioning (2% O2, 6 hours) decreased the threshold voltage of galvanotaxis to < 25 mV/mm, whereas this value was between 25 and 50 mV/mm in the normal culture control. In a scratch-wound monolayer assay in which the applied EF was in the default healing direction, hypoxic preconditioning accelerated healing by 1.38-fold compared with the control conditions. Scavenging of the induced ROS by N-acetylcysteine (NAC) abolished the enhanced galvanotaxis and the accelerated healing by hypoxic preconditioning. Our data demonstrate a novel and unsuspected role of hypoxia in supporting keratinocyte galvanotaxis. Enhancing the galvanotactic response of cells might therefore be a clinically attractive approach to induce improved wound healing. PMID:25988491

The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin.

PubMed

Gutowska-Owsiak, D; Greenwald, L; Watson, C; Selvakumar, T A; Wang, X; Ogg, G S

2014-10-01

Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention. © 2014 British Association of Dermatologists.

Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

PubMed Central

2013-01-01

Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

Germacrane sesquiterpenes isolated from the rhizome of Curcuma xanthorrhiza Roxb. inhibit UVB-induced upregulation of MMP-1, -2, and -3 expression in human keratinocytes.

PubMed

Park, Ji-Hae; Mohamed, Mohamed Antar Aziz; Jung, Ye-Jin; Shrestha, Sabina; Lee, Tae Hoon; Lee, Chang-Ho; Han, Daeseok; Kim, Jiyoung; Baek, Nam-In

2015-10-01

Four sesquiterpenes were isolated from the rhizome of Curcuma xanthorrhiza Roxb.: furanodiene (1), germacrone (2), furanodienone (3), and 13-hydroxygermacrone (4). Importantly, this was the first time compounds 1 and 4 were isolated from this plant. The chemical structures of these compounds were determined using 1D- and 2D-nuclear magnetic resonance, infrared spectroscopy, and electron ionization mass spectrometry analyses. Among the isolated compounds, compounds 2 and 4 inhibited UVB-induced upregulation of the mRNA and protein expression levels of MMP-1, MMP-2, and MMP-3 in human keratinocytes (HaCaT). Moreover, this upregulation occurred in a dose-dependent manner over the range of 1-10 μM for each compound.

Barrier Function of the Repaired Skin Is Disrupted Following Arrest of Dicer in Keratinocytes

PubMed Central

Ghatak, Subhadip; Chan, Yuk Cheung; Khanna, Savita; Banerjee, Jaideep; Weist, Jessica; Roy, Sashwati; Sen, Chandan K

2015-01-01

Tissue injury transiently silences miRNA-dependent posttranscriptional gene silencing in its effort to unleash adult tissue repair. Once the wound is closed, miRNA biogenesis is induced averting neoplasia. In this work, we report that Dicer plays an important role in reestablishing the barrier function of the skin post-wounding via a miRNA-dependent mechanism. MicroRNA expression profiling of skin and wound-edge tissue revealed global upregulation of miRNAs following wound closure at day 14 post-wounding with significant induction of Dicer expression. Barrier function of the skin, as measured by trans-epidermal water loss, was compromised in keratinocyte-specific conditional (K14/Lox-Cre) Dicer-ablated mice because of malformed cornified epithelium lacking loricrin expression. Studies on human keratinocytes recognized that loricrin expression was inversely related to the expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Compared to healthy epidermis, wound-edge keratinocytes from Dicer-ablated skin epidermis revealed elevated p21Waf1/Cip1 expression. Adenoviral and pharmacological suppression of p21Waf1/Cip1 in keratinocyte-specific conditional Dicer-ablated mice improved wound healing indicating a role of Dicer in the suppression of p21Waf1/Cip1. This work upholds p21Waf1/Cip1 as a druggable target to restore barrier function of skin suffering from loss of Dicer function as would be expected in diabetes and other forms of oxidant insult. PMID:25896246

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

PubMed Central

Mehalick, Leslie A.; Poulsen, Christopher; Fischer, Carol L.; Lanzel, Emily A.; Bates, Amber M.; Walters, Katherine S.; Cavanaugh, Joseph E.; Guthmiller, Janet M.; Johnson, Georgia K.; Wertz, Philip W.; Brogden, Kim A.

2015-01-01

Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2–10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0–80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26550599

Platelet-released growth factors inhibit proliferation of primary keratinocytes in vitro.

PubMed

Bayer, Andreas; Tohidnezhad, Mersedeh; Berndt, Rouven; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Simanski, Maren; Gläser, Regine; Harder, Jürgen

2018-01-01

Autologous thrombocyte concentrate lysates as platelet-released growth factors (PRGF) or Vivostat Platelet Rich Fibrin (PRF ® ) represent important tools in modern wound therapy, especially in the treatment of chronic, hard-to-heal or infected wounds. Nevertheless, underlying cellular and molecular mechanisms of the beneficial clinical effects of a local wound therapy with autologous thrombocyte concentrate lysates are poorly understood. Recently, we have demonstrated that PRGF induces antimicrobial peptides in primary keratinocytes and accelerates keratinocytes' differentiation. In the present study we analyzed the influence of PRGF on primary human keratinocytes' proliferation. Using the molecular proliferation marker Ki-67 we observed a concentration- and time dependent inhibition of Ki-67 gene expression in PRGF treated primary keratinocytes. These effects were independent from the EGFR- and the IL-6-R pathway. Inhibition of primary keratinocytes' proliferation by PRGF treatment was confirmed in colorimetric cell proliferation assays. Together, these data indicate that the clinically observed positive effects of autologous thrombocytes concentrates in the treatment of chronic, hard-to-heal wounds are not based on an increased keratinocytes proliferation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Staphlyococcus aureus phenol-soluble modulins stimulate the release of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation.

PubMed

Syed, Adnan K; Reed, Tamra J; Clark, Kaitlyn L; Boles, Blaise R; Kahlenberg, J Michelle

2015-09-01

Staphylococcus aureus is a human commensal that colonizes the skin. While it is normally innocuous, it has strong associations with atopic dermatitis pathogenesis and has become the leading cause of skin and soft tissue infections in the United States. The factors that dictate the role of S. aureus in disease are still being determined. In this work, we utilized primary keratinocyte culture and an epidermal murine colonization model to investigate the role of S. aureus phenol-soluble modulins (PSMs) in proinflammatory cytokine release and inflammation induction. We demonstrated that many species of Staphylococcus are capable of causing release of interleukin 18 (IL-18) from keratinocytes and that S. aureus PSMs are necessary and sufficient to stimulate IL-18 release from keratinocytes independently of caspase 1. Further, after 7 days of epicutaneous exposure to wild-type S. aureus, but not S. aureus Δpsm, we saw dramatic changes in gross pathology, as well as systemic release of proinflammatory cytokines. This work demonstrates the importance of PSM peptides in S. aureus-mediated inflammatory cytokine release from keratinocytes in vitro and in vivo and further implicates PSMs as important contributors to pathogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Protective effect of silk lutein on ultraviolet B-irradiated human keratinocytes.

PubMed

Pongcharoen, Sutatip; Warnnissorn, Prateep; Leŗtkajornsin, Ongart; Limpeanchob, Nanteetip; Sutheerawattananonda, Manote

2013-01-01

Carotenoids are efficient antioxidants that are of great importance for human health. Lutein and zeaxanthin are carotinoids present in high concentrations in the human retina which are involved in the photoprotection of the human eye. Lutein may also protect the skin from ultraviolet (UV)-induced damage. The present study investigated the protective effect of lutein extracted from yellow silk cocoons of Bombyx mori on human keratinocytes against UVB irradiation. A human keratinocyte cell line and primary human keratinocytes were used to investigate the UVB protection effects of silk lutein and plant lutein. Silk lutein showed no cytotoxicity to keratinocytes. Treatment with silk lutein prior to UVB irradiation enhanced cell viability and cell proliferation, and reduced cell apoptosis. The protective effects of silk lutein may be superior to those of plant lutein. Silk lutein may have a benefit for protection of keratinocytes against UVB-irradiation.

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

PubMed

Ghaffari, Abdi; Li, Yunyaun; Karami, Ali; Ghaffari, Mazyar; Tredget, Edward E; Ghahary, Aziz

2006-05-15

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for

Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

PubMed

Srivastava, Ankit; Ståhle, Mona; Pivarcsi, Andor; Sonkoly, Enikö

2018-05-08

Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

The effect of different biologic and biosynthetic wound covers on keratinocyte growth, stratification and differentiation in vitro

PubMed Central

Mestak, Ondrej

2014-01-01

The purpose of this study was to compare, by means of in vitro cultivation technique, five marketed brands of wound covers used in the treatment of burns and other skin defects (Biobrane®, Suprathel®, Veloderm®, Xe-Derma®, and Xenoderm®) for their ability to stimulate the keratinocyte growth, stratification, and differentiation. In three independent experiments, human keratinocytes were grown on the tested covers in organotypic cultures by the 3T3 feeder layer technique. Vertical paraffin sections of the wound covers with keratinocytes were processed using hematoxylin–eosin staining and immunostaining for involucrin. Keratinocyte populations on the dressings were assessed for (1) number of keratinocyte strata (primary variable), (2) quantitative growth, (3) thickness of the keratinocyte layer, and (4) cell differentiation. The Xe-Derma wound cover provided the best support to keratinocyte proliferation and stratification, with the number of keratinocyte strata significantly (p < 0.05) higher in comparison to all products studied, except Xenoderm. However, in contrast to Xe-Derma, Xenoderm did not significantly differ from the other dressings. The results of this in vitro study show that the brands based on porcine dermal matrix possess the strongest effect on keratinocyte proliferation and stratification. The distinctive position of Xe-Derma may be related to its composition, where natural dermal fibers form a smooth surface, similar to the basement membrane. Furthermore, the results indicate that in vitro evaluation of effects on epithelial growth may accelerate the development of new bio-engineering-based wound covers. PMID:25383177

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

PubMed

Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

2018-06-14

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

MicroRNA-146a alleviates chronic skin inflammation in atopic dermatitis through suppression of innate immune responses in keratinocytes.

PubMed

Rebane, Ana; Runnel, Toomas; Aab, Alar; Maslovskaja, Julia; Rückert, Beate; Zimmermann, Maya; Plaas, Mario; Kärner, Jaanika; Treis, Angela; Pihlap, Maire; Haljasorg, Uku; Hermann, Helen; Nagy, Nikoletta; Kemeny, Lajos; Erm, Triin; Kingo, Külli; Li, Mei; Boldin, Mark P; Akdis, Cezmi A

2014-10-01

Chronic skin inflammation in atopic dermatitis (AD) is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. microRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation. The aim of this study was to investigate the role of miR-146a in skin inflammation in AD. RNA and protein expression was analyzed using miRNA and mRNA arrays, RT-quantitative PCR, Western blotting, and immunonohistochemistry. Transfection of miR-146a precursors and inhibitors into human primary keratinocytes, luciferase assays, and MC903-dependent mouse model of AD were used to study miR-146a function. We show that miR-146a expression is increased in keratinocytes and chronic lesional skin of patients with AD. miR-146a inhibited the expression of numerous proinflammatory factors, including IFN-γ-inducible and AD-associated genes CCL5, CCL8, and ubiquitin D (UBD) in human primary keratinocytes stimulated with IFN-γ, TNF-α, or IL-1β. In a mouse model of AD, miR-146a-deficient mice developed stronger inflammation characterized by increased accumulation of infiltrating cells in the dermis, elevated expression of IFN-γ, CCL5, CCL8, and UBD in the skin, and IFN-γ, IL-1β, and UBD in draining lymph nodes. Both tissue culture and in vivo experiments in mice demonstrated that miR-146a-mediated suppression in allergic skin inflammation partially occurs through direct targeting of upstream nuclear factor kappa B signal transducers caspase recruitment domain-containing protein 10 and IL-1 receptor-associated kinase 1. In addition, human CCL5 was determined as a novel, direct target of miR-146a. Our data demonstrate that miR-146a controls nuclear factor kappa B-dependent inflammatory responses in keratinocytes and chronic skin inflammation in AD. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

PubMed Central

Nam, Ju-Suk; Chakraborty, Chiranjib; Sharma, Ashish Ranjan; Her, Young; Bae, Kee-Jeong; Sharma, Garima; Doss, George Priya; Lee, Sang-Soo; Hong, Myung-Sun; Song, Dong-Keun

2014-01-01

Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers. PMID:24686518

Tualang honey protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damage.

PubMed

Ahmad, Israr; Jimenez, Hugo; Yaacob, Nik Soriani; Yusuf, Nabiha

2012-01-01

Malaysian tualang honey possesses strong antioxidant and anti-inflammatory properties. Here, we evaluated the effect of tualang honey on early biomarkers of photocarcinogenesis employing PAM212 mouse keratinocyte cell line. Keratinocytes were treated with tualang honey (1.0%, v/v) before a single UVB (150 mJ cm(-2) ) irradiation. We found that the treatment of tualang honey inhibited UVB-induced DNA damage, and enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers and 8-oxo-7,8-dihydro-2'-deoxyguanosine. Treatment of tualang honey inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in murine keratinocyte cell line. The treatment of tualang honey also inhibited UVB-induced inflammatory cytokines and inducible nitric oxide synthase protein expression. Furthermore, the treatment of tualang honey inhibited UVB-induced COX-2 expression and PGE2 production. Taken together, we provide evidence that the treatment of tualang honey to keratinocytes affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Simultaneous analysis of multiple T helper subsets in leprosy reveals distinct patterns of Th1, Th2, Th17 and Tregs markers expression in clinical forms and reactional events.

PubMed

Azevedo, Michelle de Campos Soriani; Marques, Heloisa; Binelli, Larissa Sarri; Malange, Mariana Silva Vieira; Devides, Amanda Carreira; Silva, Eliane Aparecida; Fachin, Luciana Raquel Vincenzi; Ghidella, Cassio Cesar; Soares, Cleverson Teixeira; Garlet, Gustavo Pompermaier; Rosa, Patrícia Sammarco; Belone, Andrea de Farias Fernandes; Trombone, Ana Paula Favaro

2017-12-01

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Previous studies have demonstrated that the difference among clinical forms of leprosy can be associated with the immune response of patients, mainly by T helper (Th) and regulatory T cells (Tregs). Then, aiming at clarifying the immune response, the expression of cytokines related to Th1, Th2, Th17 and Tregs profiles were evaluated by qPCR in 87 skin biopsies from leprosy patients. Additionally, cytokines and anti-PGL-1 antibodies were determined in serum by ELISA. The results showed that the expression of various targets (mRNA) related to Th1, Th2, Th17 and Tregs were significantly modulated in leprosy when compared with healthy individuals, suggesting the presence of a mixed profile. In addition, the targets related to Th1 predominated in the tuberculoid pole and side and Th2 and Tregs predominated in the lepromatous pole and side; however, Th17 targets showed a mixed profile. Concerning reactional events, Tregs markers were decreased and IL-15 was increased in reversal reaction and IL-17F, CCL20 and IL-8 in erythema nodosum leprosum, when compared with the respective non-reactional leprosy patients. Additionally, ELISA analysis demonstrated that IL-22, IL-6, IL-10 and anti-PGL-1 antibody levels were significantly higher in the serum of patients when compared with healthy individuals, and IL-10 and anti-PGL-1 antibodies were also increased in the lepromatous pole and side. Together, these results indicate that Th1, Th2 and Th17 are involved in the determination of clinical forms of leprosy and suggest that decreased Tregs activity may be involved in the pathogenesis of reactional events.

TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.

2014-05-01

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highlymore » induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic

Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice.

PubMed

Elhaik Goldman, Shirin; Dotan, Shahar; Talias, Amir; Lilo, Amit; Azriel, Shalhevet; Malka, Itay; Portnoi, Maxim; Ohayon, Ariel; Kafka, Daniel; Ellis, Ronald; Elkabets, Moshe; Porgador, Angel; Levin, Ditza; Azhari, Rosa; Swiatlo, Edwin; Ling, Eduard; Feldman, Galia; Tal, Michael; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

2016-04-01

Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freund's adjuvant (CFA) in the initial immunization and with incomplete Freund's adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA‑pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA‑immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

PubMed

Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

2017-10-01

In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Induction of hyaluronan cables and monocyte adherence in epidermal keratinocytes.

PubMed

Jokela, Tiina A; Lindgren, Antti; Rilla, Kirsi; Maytin, Edward; Hascall, Vincent C; Tammi, Raija H; Tammi, Markku I

2008-01-01

Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.

α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes

PubMed Central

Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG

2009-01-01

Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their

Modulation of mycobacterial-specific Th1 and Th17 cells in latent tuberculosis by coincident hookworm infection

PubMed Central

George, Parakkal Jovvian; Anuradha, Rajamanickam; Kumaran, Paramasivam Paul; Chandrasekaran, Vedachalam; Nutman, Thomas B.; Babu, Subash

2013-01-01

Hookworm infections and tuberculosis are co-endemic in many parts of the world. It has been suggested that infection with helminth parasites could suppress the predominant Th1 (IFN-γ-mediated) response needed to control Mycobacterium tuberculosis (Mtb) infection and enhance susceptibility to infection and/or disease. To determine the role of coincident hookworm infection on responses at steady state and on Mtb – specific immune responses in latent tuberculosis (TB), we examined the cellular responses in individuals with latent TB with or without concomitant hookworm infection. By analyzing the expression of Th1, Th2 and Th17 subsets of CD4+ T cells, we were able to demonstrate that the presence of coincident hookworm infection significantly diminished both spontaneously expressed and Mtb – specific mono – and dual – functional Th1 and Th17 cells. Hookworm infection, in contrast, was associated with expanded frequencies of mono – and dual – functional Th2 cells at both steady state and upon antigen – stimulation. This differential induction of CD4+ T cell subsets was abrogated upon mitogen stimulation. In addition, coincident hookworm infection was associated with increased adaptive T regulatory (aTreg) cells but not natural regulatory T cells (nTregs) in latent TB. Finally, the CD4+ T cell cytokine expression pattern was also associated with alterations in the systemic levels of Th1 and Th2 cytokines. Thus, coincident hookworm infection exerts a profound inhibitory effect on protective Th1 and Th17 responses in latent tuberculosis and may predispose toward the development of active tuberculosis in humans. PMID:23576678

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes.

PubMed

Choi, Mi-Ra; Choi, Dae-Kyoung; Sohn, Kyung-Cheol; Lim, Seul Ki; Kim, Dong-Il; Lee, Young Ho; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok; Lee, Jeung-Hoon

2015-01-01

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1β and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.

Elevated Ratio of Th17 Cell-Derived Th1 Cells (CD161(+)Th1 Cells) to CD161(+)Th17 Cells in Peripheral Blood of Early-Onset Rheumatoid Arthritis Patients.

PubMed

Kotake, Shigeru; Nanke, Yuki; Yago, Toru; Kawamoto, Manabu; Kobashigawa, Tsuyoshi; Yamanaka, Hisashi

2016-01-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the destruction of articular cartilage and bone with elevated levels of proinflammatory cytokines. It has been reported that IL-17 and Th17 cells play important roles in the pathogenesis of RA. Recently, plasticity in helper T cells has been demonstrated; Th17 cells can convert to Th1 cells. It remains to be elucidated whether this conversion occurs in the early phase of RA. Here, we tried to identify Th17 cells, Th1 cells, and Th17 cell-derived Th1 cells (CD161(+)Th1 cells) in the peripheral blood of early-onset RA patients. We also evaluated the effect of methotrexate on the ratio of Th17 cells in early-onset RA patients. The ratio of Th17 cell-derived Th1 cells to CD161(+)Th17 cells was elevated in the peripheral blood of early-onset RA patients. In addition, MTX reduced the ratio of Th17 cells but not Th1 cells. These findings suggest that IL-17 and Th17 play important roles in the early phase of RA; thus, anti-IL-17 antibodies should be administered to patients with RA in the early phase.

Elevated Ratio of Th17 Cell-Derived Th1 Cells (CD161+Th1 Cells) to CD161+Th17 Cells in Peripheral Blood of Early-Onset Rheumatoid Arthritis Patients

PubMed Central

Kotake, Shigeru; Nanke, Yuki; Yago, Toru; Kawamoto, Manabu; Kobashigawa, Tsuyoshi; Yamanaka, Hisashi

2016-01-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the destruction of articular cartilage and bone with elevated levels of proinflammatory cytokines. It has been reported that IL-17 and Th17 cells play important roles in the pathogenesis of RA. Recently, plasticity in helper T cells has been demonstrated; Th17 cells can convert to Th1 cells. It remains to be elucidated whether this conversion occurs in the early phase of RA. Here, we tried to identify Th17 cells, Th1 cells, and Th17 cell-derived Th1 cells (CD161+Th1 cells) in the peripheral blood of early-onset RA patients. We also evaluated the effect of methotrexate on the ratio of Th17 cells in early-onset RA patients. The ratio of Th17 cell-derived Th1 cells to CD161+Th17 cells was elevated in the peripheral blood of early-onset RA patients. In addition, MTX reduced the ratio of Th17 cells but not Th1 cells. These findings suggest that IL-17 and Th17 play important roles in the early phase of RA; thus, anti-IL-17 antibodies should be administered to patients with RA in the early phase. PMID:27123445

Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

PubMed

Orlova-Fink, Nina; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-10-23

The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction.

PubMed

Danis, Judit; Janovák, Luca; Gubán, Barbara; Göblös, Anikó; Szabó, Kornélia; Kemény, Lajos; Bata-Csörgő, Zsuzsanna; Széll, Márta

2018-03-08

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction

PubMed Central

Danis, Judit; Janovák, Luca; Gubán, Barbara; Göblös, Anikó; Szabó, Kornélia; Bata-Csörgő, Zsuzsanna; Széll, Márta

2018-01-01

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types—normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line—upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT). PMID:29518010

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

2013-08-01

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Epidermal cornification is preceded by the expression of a keratinocyte-specific set of pyroptosis-related genes.

PubMed

Lachner, Julia; Mlitz, Veronika; Tschachler, Erwin; Eckhart, Leopold

2017-12-12

The homeostasis of the epidermis depends on keratinocyte differentiation and cornification, a mode of programmed cell death that does not elicit inflammation. Here, we report that cornification is associated with the expression of specific genes that control multiple steps of pyroptosis, another form of cell death that involves the processing and release of interleukin-1 family (IL1F) cytokines. Expression levels of pro-inflammatory IL1A and IL1B and of the pyroptotic pore-forming gasdermin (GSDM) D were downregulated during terminal differentiation of human keratinocytes in vitro. By contrast, negative regulators of IL-1 processing, including NLR family pyrin domain containing 10 (NLRP10) and pyrin domain-containing 1 (PYDC1), the anti-inflammatory IL1F members IL-37 (IL1F7) and IL-38 (IL1F10), and GSDMA, were strongly induced in differentiated keratinocytes. In human tissues, these keratinocyte differentiation-associated genes are expressed in the skin at higher levels than in any other organ, and mammalian species, that have lost the epidermal cornification program during evolution, i.e. whales and dolphins, lack homologs of these genes. Together, our results suggest that human epidermal cornification is accompanied by a tight control of pyroptosis and warrant further studies of potential defects in the balance between cornification and pyroptosis in skin pathologies.

Expression of MIF and TNFA in psoriatic arthritis: relationship with Th1/Th2/Th17 cytokine profiles and clinical variables.

PubMed

Bautista-Herrera, L A; De la Cruz-Mosso, U; Morales-Zambrano, R; Villanueva-Quintero, G D; Hernández-Bello, J; Ramírez-Dueñas, M G; Martínez-López, E; Brennan-Bourdon, L M; Baños-Hernández, C J; Muñoz-Valle, J F

2018-05-01

Psoriatic arthritis (PsA) is an autoimmune inflammatory disease associated with psoriasis. The cause of this pathology is still unknown, but research suggests the diseases are caused by a deregulated cytokine production. MIF is a cytokine associated with immunomodulation of Th1, Th2, and Th17 cytokine profiles in inflammatory diseases. Based on this knowledge, the aim of this study was to determine the association of MIF and TNFA expression with Th1, Th2, and Th17 cytokine profiles in serum levels of PsA patients. A cross-sectional study was performed in 50 PsA patients and 30 control subjects (CS). The cytokine profiles were quantified by BioPlex MagPix system and the mRNA expression levels by real-time PCR. TNFA mRNA expression was 138.81-folds higher in PsA patients than CS (p < 0.001). Regarding MIF mRNA expression, no significant differences were observed; however, a positive correlation was identified between MIF mRNA expression and PsA time of evolution (r = - 0.53, p = 0.009). An increase of Th1 (IFNγ: PsA = 37.1 pg/mL vs. CS = 17 pg/mL, p < 0.05; TNFα: PsA = 24.6 pg/mL vs. CS = 9.8 pg/mL, p < 0.0001) and Th17 cytokine profiles (IL-17: PsA = 6.4 pg/mL vs. CS = 2.7 pg/mL, p < 0.05; IL-22: PsA = 8.4 pg/mL vs. CS = 1.8 pg/mL, p < 0.001), were found in PsA patients. Th2 cytokines were not significantly different in both groups. In conclusion, a high expression of TNFA mRNA, as well as an increase of Th1 and Th17 cytokine profiles evaluated by IFNγ, TNFα, IL-17, and IL-22 cytokines, was observed in PsA patients.

Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

PubMed

Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

2016-10-01

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. Copyright © 2016 Elsevier B.V. All rights reserved.

Loss of Robustness and Addiction to IGF1 during Early Keratinocyte Transformation by Human Papilloma Virus 16

PubMed Central

Geiger, Tamar; Levitzki, Alexander

2007-01-01

Infection of keratinocytes with high risk human Papilloma virus causes immortalization, and when followed by further mutations, leads to cervical cancer and other anogenital tumors. Here we monitor the progressive loss of robustness in an in vitro model of the early stages of transformation that comprises normal keratinocytes and progressive passages of HPV16 immortalized cells. As transformation progresses, the cells acquire higher proliferation rates and gain the ability to grow in soft agar. Concurrently, the cells lose robustness, becoming more sensitive to serum starvation and DNA damage by Cisplatin. Loss of robustness in the course of transformation correlates with significant reductions in the activities of the anti-apoptotic proteins PKB/Akt, Erk, Jnk and p38 both under normal growth conditions and upon stress. In parallel, loss of robustness is manifested by the shrinkage of the number of growth factors that can rescue starving cells from apoptosis, with the emergence of dependence solely on IGF1. Treatment with IGF1 activates PKB/Akt and Jnk and through them inhibits p53, rescuing the cells from starvation. We conclude that transformation in this model induces higher susceptibility of cells to stress due to reduced anti-apoptotic signaling and hyper-activation of p53 upon stress. PMID:17622350

Photodynamic toxicity of hematoporphyrin derivatives to human keratinocytes in culture.

PubMed

Kappus, H; Reinhold, C; Artuc, M

Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

PubMed Central

Chapman, Sandra; Liu, Xuefeng; Meyers, Craig; Schlegel, Richard; McBride, Alison A.

2010-01-01

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. PMID:20516646

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Sun Hee; Choi, Dalwoong; Chun, Young-Jin

Keratinocytes are the major cellular components of human epidermis and play a key role in the modulating cutaneous inflammation and toxic responses. In human chronic skin diseases, the common skin inflammatory phenotypes like skin barrier disruption and epidermal hyperplasia are manifested in epidermal keratinocytes by interactions with T helper (Th) cells. To find a common gene expression signature of human keratinocytes in chronic skin diseases, we performed a whole genome microarray analysis on normal human epidermal keratinocytes (NHKs) treated with IFNγ, IL-4, IL-17A or IL-22, major cytokines from Th1, Th2, Th17 or Th22 cells, respectively. The microarray results showed thatmore » the four genes, IL-24, PDZK1IP1, H19 and filaggrin, had common expression profiles in NHKs exposed to Th cell cytokines. In addition, the acute phase pro-inflammatory cytokines, IL-1β, IL-6 and TNFα, also change the gene transcriptional profile of IL-24, PDZK1IP1, H19, and filaggrin in NHKs as those of Th cytokines. Therefore, the signature gene set, consisting of IL-24, PDZK1IP1, H19, and filaggrin, provides essential insights for understanding the process of cutaneous inflammation and toxic responses. We demonstrate that environmental toxic stressors, such as chemical irritants and ultraviolet irradiation stimulate the production of IL-24 in NHKs. IL-24 stimulates the JAK1-STAT3 and MAPK pathways in NHKs, and promotes the secretion of pro-inflammatory mediators IL-8, PGE2, and MMP-1. These results suggest that keratinocyte-derived IL-24 participates in the positive feedback regulation of epidermal inflammation in response to both endogenous and environmental toxic stressors. - Highlights: • Cutaneous inflammatory gene signature consists of PDZK1IP1, IL-24, H19 and filaggrin. • Pro-inflammatory cytokines increase IL-24 production in human keratinocytes. • Environmental toxic stressors increase IL-24 production in human keratinocytes. • IL-24 stimulates human

Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β–Slug signaling

PubMed Central

Cheng, Fang; Shen, Yue; Mohanasundaram, Ponnuswamy; Lindström, Michelle; Ivaska, Johanna; Ny, Tor; Eriksson, John E.

2016-01-01

Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial–mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM−/−) wounds. Correspondingly, VIM−/− wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM−/− wounds. Vimentin reconstitution in VIM−/− fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β–Slug–EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1–Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation. PMID:27466403

Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNFalpha in human epidermal keratinocytes (HaCaT).

PubMed

Onda, Kenji; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

2006-12-08

Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) alpha reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNFalpha was not accompanied by changes in mRNA expressions of GR isoforms (alpha or beta). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNFalpha. Additionally, we observed that TNFalpha reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNFalpha-mediated GC insensitivity. Our data suggest that overexpression of TNFalpha leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

Cytotoxicity measurement of Bisphenol A (BPA) and its substitutes using human keratinocytes.

PubMed

Son, Seogho; Nam, KeeSoo; Kim, Hyungjoo; Gye, Myung Chan; Shin, Incheol

2018-07-01

Bisphenol-A (BPA) was first synthesized in the 1890s and has been used in many plastic products. However, BPA is known to act as an endocrine disruptor and has been found to be toxic to human health. Many alternative substances have been developed to replace BPA, but it is still widely used worldwide. In this study, we identified the potential cytotoxicity of BPA by evaluating toxicity using human keratinocytes. Also, we evaluated cytotoxicity of BPA substitutes to determine their suitability as an alternative to BPA. The proliferation assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and western blot analysis showed that BPA significantly affect cell viability, induction of apoptotic fraction and increased activation of DNA-damage marker protein. In addition, through the same experiments, the substitutes of BPA were shown to be significantly less toxic than BPA, and the least toxicity was observed with 1,4-cyclohexanedimethanol (CHDM) and terephthalic acid (TPA). In conclusion, this study suggests that cytotoxicity of BPA induces apoptosis of human keratinocytes, and that CHDM and TPA are the most suitable substitutes for BPA. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of Th1 and Th17 cells in glomerulonephritis.

PubMed

Azadegan-Dehkordi, Fatemeh; Bagheri, Nader; Shirzad, Hedayatollah; Rafieian-Kopaei, Mahmoud

2015-04-01

T helper (Th) cells as an important part of the immune is responsible for elimination of invading pathogens. But, if Th cell responses are not regulated effectively, the autoimmune diseases might develop. The Th17 subset usually produces interleukin-17A which in experimental models of organ-specific autoimmune inflammation is very important. Directory of open access journals (DOAJ), Google Scholar, Embase, Scopus, PubMed and Web of Science have been searched. Fifty-six articles were found and searched. In the present review article, we tried to summarize the recently published data about characteristics and role of Th1 and Th17 cells and discuss in detail, the potential role of these T helpers immune responses in renal inflammation and renal injury, focusing on glomerulonephritis. Published papers in animal and human studies indicated that autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, classically believed to be Th1-mediated, are mainly derived from a Th17 immune response. Identification of the Th17 subgroup has explained seemingly paradoxical observations and improved our understanding of immune-mediated inflammatory responses. Secretion of IL-17A, as well as IL-17F, IL-21, IL-22, suggests that Th17 subset may play a crucial role as a pleiotropic pro-inflammatory Th subset. There is experimental evidence to support the notion that Th1 and Th17 cells contribute to kidney injury in renal inflammatory diseases like glomerulonephritis.

Inflammasome and Fas-Mediated IL-1β Contributes to Th17/Th1 Cell Induction in Pathogenic Bacterial Infection In Vivo.

PubMed

Uchiyama, Ryosuke; Yonehara, Shin; Taniguchi, Shun'ichiro; Ishido, Satoshi; Ishii, Ken J; Tsutsui, Hiroko

2017-08-01

CD4 + Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4 + Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium Mycobacterium tuberculosis in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1β induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium Listeria monocytogenes In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In Pycard -/- mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1β production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in Fas -/- mice, whereas supplementation with recombinant IL-1β restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of Fas -/- mice infected with Listeria IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1β stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1β, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo. Copyright © 2017 by The American Association of Immunologists

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes.

PubMed

Baek, Jin Young; Park, Sujin; Park, Jiyoung; Jang, Ji Yong; Wang, Su Bin; Kim, Sin Ri; Woo, Hyun Ae; Lim, Kyung Min; Chang, Tong-Shin

2017-06-01

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent H 2 O 2 -removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial H 2 O 2 levels were differentiated from other types of ROS using mitochondria-specific fluorescent H 2 O 2 indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII -/- ) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial H 2 O 2 compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII -/- mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial H 2 O 2 is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial H 2 O 2 . These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Human Papillomavirus Type 8 E6 Protein Interferes with NOTCH Activation during Keratinocyte Differentiation

PubMed Central

Meyers, Jordan M.; Spangle, Jennifer M.

2013-01-01

Cutaneous β-human papillomavirus (β-HPV) E6 proteins inhibit NOTCH signaling by associating with the transcriptional coactivator MAML1. NOTCH has tumor suppressor activities in epithelial cells and is activated during keratinocyte differentiation. Here we report that HPV type 8 (HPV8) E6 subverts NOTCH activation during keratinocyte differentiation by inhibiting RBPJ/MAML1 transcriptional activator complexes at NOTCH target DNA. NOTCH inhibition impairs epithelial differentiation and may thus contribute to β-HPV replication and viral oncogenesis. PMID:23365452

Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

PubMed Central

Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

2009-01-01

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

Keratinocytes at the uppermost layer of epidermis might act as sensors of atmospheric pressure change.

PubMed

Denda, Mitsuhiro

2016-01-01

It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.

Fibroblast Growth Factor-Peptide Improves Barrier Function and Proliferation in Human Keratinocytes After Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Kunzhong; Tian Yeping; Yin Liangjie

2011-09-01

Purpose: Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. Methods and Materials: Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed bymore » using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([{sup 3}H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin {beta} burns created with a strontium applicator. Results: We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [{sup 3}H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin {beta} burns. Conclusions: FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates

Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes

PubMed Central

Niki, Yoko; Yoshida, Masaki; Ito, Masaaki; Akiyama, Kaoru; Kim, Jin-Hwa; Yoon, Tae-Jin; Matsui, Mary S; Yarosh, Daniel B; Ichihashi, Masamitsu

2011-01-01

The mechanism of melanosome transfer from melanocytes to keratinocytes has not been fully clarified. We now show a route of melanosome transfer using co-cultures of normal human melanocytes and keratinocytes. Substantial levels of melanosome transfer were elicited in co-cultures of melanocytes and keratinocytes separated by a microporous membrane filter. The melanocyte dendrites penetrated into the keratinocyte layer through the filter and many pigment globules were observed in keratinocytes. Electron microscopic observations revealed that melanosomes incorporated in keratinocytes were packed in clusters enclosed by a double membrane. Numerous pigment globules budded off from melanocyte dendrites and were released into the culture medium. Those pigment globules were filled with multiple melanosomes and a few mitochondria but no nuclei. When those globules were added to the culture medium of keratinocytes, they were incorporated and showed double membrane-enclosed melano-phagolysosomes consistent with the structures obtained from the co-culture system. In contrast, when individual naked melanosomes isolated from melanocytes were added to keratinocytes, they were also phagocytosed by keratinocytes but were enclosed by a single membrane in a manner distinct from the co-culture system. These results suggest a novel mechanism of melanosome transfer, wherein melanosomes are packed in membrane globules that bud off from melanocyte dendrites, where they are released into the extracellular space and then phagocytosed by keratinocytes. PMID:21686100

Effect of isosecotanapartholide isolated from Artemisia princeps Pampanini on IL‑33 production and STAT‑1 activation in HaCaT keratinocytes.

PubMed

Oh, Chang Taek; Jang, Yu-Jin; Kwon, Tae-Rin; Im, Songi; Kim, Soon Re; Seok, Joon; Kim, Gun-Yong; Kim, Young-Heui; Mun, Seog Kyun; Kim, Beom Joon

2017-05-01

The present study aimed to investigate the anti‑inflammatory effect and mechanism of action of isosecotanapartholide (ISTP), isolated from Artemisia princeps Pampanini extract (APE). The effects of ISTP and APE on the proliferation of human keratinocytes following stimulation by tumor necrosis factor‑α/interferon‑γ were assessed. ISTP and APE downregulated the expression levels of signal transducer and activator of transcription‑1 (STAT‑1), and reduced interleukin‑33 (IL‑33) production. ISTP and APE inhibited the mRNA expression levels of thymus and activation‑regulated chemokine (TARC/CCL17) in a dose‑dependent manner. Western blot analysis demonstrated that ISTP and APE dose‑dependently inhibited protein expression levels of intercellular adhesion molecule‑1 and phosphorylation of STAT‑1. The results of the present study indicate that ISTP may inhibit TARC/CCL17 production in human epidermal keratinocytes via the STAT‑1 signaling pathway and may be associated with the inhibition of IL‑33 production. The current study indicated that ISTP is an active component in APE and may be a potential therapeutic agent for the treatment of inflammatory skin disorders.

Shining a Light on Black Holes in Keratinocytes.

PubMed

Bowman, Shanna L; Marks, Michael S

2018-03-01

The mechanisms by which melanins are transferred from melanocytes and stored within keratinocytes to generate skin pigmentation are hotly debated. Correia et al. and Hurbain et al. provide evidence that melanin cores of melanosomes are secreted from melanocytes and taken up and stored within non-degradative membranous organelles in keratinocytes in the basal epidermis of human skin. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onda, Kenji; Nagashima, Masahiro; Kawakubo, Yo

2006-12-08

Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} wasmore » not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.« less

Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease.

PubMed

Kidd, Parris

2003-08-01

One theory of immune regulation involves homeostasis between T-helper 1 (Th1) and T-helper 2 (Th2) activity. The Th1/Th2 hypothesis arose from 1986 research suggesting mouse T-helper cells expressed differing cytokine patterns. This hypothesis was adapted to human immunity, with Th1- and Th2-helper cells directing different immune response pathways. Th1 cells drive the type-1 pathway ("cellular immunity") to fight viruses and other intracellular pathogens, eliminate cancerous cells, and stimulate delayed-type hypersensitivity (DTH) skin reactions. Th2 cells drive the type-2 pathway ("humoral immunity") and up-regulate antibody production to fight extracellular organisms; type 2 dominance is credited with tolerance of xenografts and of the fetus during pregnancy. Overactivation of either pattern can cause disease, and either pathway can down-regulate the other. But the hypothesis has major inconsistencies; human cytokine activities rarely fall into exclusive pro-Th1 or -Th2 patterns. The non-helper regulatory T cells, or the antigen-presenting cells (APC), likely influence immunity in a manner comparable to Th1 and Th2 cells. Many diseases previously classified as Th1 or Th2 dominant fail to meet the set criteria. Experimentally, Th1 polarization is readily transformed to Th2 dominance through depletion of intracellular glutathione, and vice versa. Mercury depletes glutathione and polarizes toward Th2 dominance. Several nutrients and hormones measurably influence Th1/Th2 balance, including plant sterols/sterolins, melatonin, probiotics, progesterone, and the minerals selenium and zinc. The long-chain omega-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) significantly benefit diverse inflammatory and autoimmune conditions without any specific Th1/Th2 effect. Th1/Th2-based immunotherapies, e.g., T-cell receptor (TCR) peptides and interleukin-4 (IL-4) injections, have produced mixed results to date.

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

PubMed

Klymenko, T; Gu, Q; Herbert, I; Stevenson, A; Iliev, V; Watkins, G; Pollock, C; Bhatia, R; Cuschieri, K; Herzyk, P; Gatherer, D; Graham, S V

2017-10-11

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log 2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress. IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The

Attachment and growth of human keratinocytes in a serum-free environment.

PubMed

Gilchrest, B A; Calhoun, J K; Maciag, T

1982-08-01

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

PubMed

Kariya, Yoshinobu; Sato, Hiroki; Katou, Naoko; Kariya, Yukiko; Miyazaki, Kaoru

2012-01-01

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes.

PubMed

Uberti, Francesca; Bardelli, Claudio; Morsanuto, Vera; Ghirlanda, Sabrina; Cochis, Andrea; Molinari, Claudio

2017-01-01

The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe. © 2016 S. Karger AG, Basel.

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation.

PubMed

Kim, Mihwa; Morales, Liza D; Baek, Minwoo; Slaga, Thomas J; DiGiovanni, John; Kim, Dae Joon

2017-10-31

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

Dermoscopy of keratinocyte skin cancer.

PubMed

Kupsa, Romana; Deinlein, Teresa; Woltsche, Nora; Hofmann-Wellenhof, Rainer; Zalaudek, Iris

2016-12-01

Keratinocyte skin cancer (KSC) refers to a broad class of tumors with a regrettably rising incidence worldwide. The term KSC stands for different stages of skin cancer including actinic keratosis (AK), Bowen's Disease (BD) and invasive squamous cell carcinoma (SCC). These tumors tend to grow slow, are unlikely to result in distant metastatic disease and death but they frequently destroy underlying tissues and should therefore be removed at the earliest possible stage. The fact that the cure rate is very high when KSC is detected in early stages emphasizes once more the applicability of dermoscopy as an integrative part of the clinical examination of skin tumors. In the first part of this review article, we summarize key points of the dermoscopic diagnosis of KSC including different stages of AK, BD and SCC. In the second part we want to focus on the progression model of KSC and the role of dermoscopy in the management of keratinocyte skin cancer.

Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its regulation by Th2 cells.

PubMed

McQuillan, K; Lynch, Marina A; Mills, Kingston H G

2010-05-01

Microglia are innate immune cells of the CNS, that act as antigen-presenting cells (APC) for antigen-specific T cells and respond to inflammatory stimuli, such as amyloid-beta (Abeta), resulting in the release of neurotoxic factors and pro-inflammatory cytokines. Astrocytes can also act as APC and modulate the function of microglia. However, the role of distinct T cell subtypes, in particular Th17 cells, in glial activation and subsequent modulatory effects of Th2 cells are poorly understood. Here, we generated Abeta-specific Th1, Th2, and Th17 cells and examined their role in modulating Abeta-induced activation of microglia in a mixed glial culture, a preparation which mimics the complex APC types in the brain. We demonstrated that mixed glia acted as an effective APC for Abeta-specific Th1 and Th17 cells. Addition of Abeta-specific Th2 cells suppressed the Abeta-induced IFN-gamma production by Th1 cells and IL-17 production by Th17 cells with glia as the APC. Co-culture of Abeta-specific Th1 or Th17 cells with glia markedly enhanced Abeta-induced pro-inflammatory cytokine production and expression of MHC class II and co-stimulatory molecules on the microglia. Addition of Abeta-specific Th2 cells inhibited Th17 cell-induced IL-1beta and IL-6 production by mixed glia and attenuated Th1 cell-induced CD86 and CD40 expression on microglia. The modest enhancement of MHC class II and CD86 expression on astrocytes by Abeta-specific Th1 and Th17 was not attenuated by Th2 cells. These data indicate that Abeta-specific Th1 and Th17 cells induce inflammatory activation of glia, and that this is in part regulated by Th2 cells. Copyright 2010 Elsevier Inc. All rights reserved.

Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy

PubMed Central

Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

2013-01-01

Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors. PMID:23894651

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

PubMed

Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee

2015-03-01

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture.more » VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal

Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation*S⃞

PubMed Central

Müller, Margarethe; Essin, Kirill; Hill, Kerstin; Beschmann, Heike; Rubant, Simone; Schempp, Christoph M.; Gollasch, Maik; Boehncke, W. Henning; Harteneck, Christian; Müller, Walter E.; Leuner, Kristina

2008-01-01

The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation. PMID:18818211

Aged keratinocyte phenotyping: morphology, biochemical markers and effects of Dead Sea minerals.

PubMed

Soroka, Yoram; Ma'or, Zeev; Leshem, Yael; Verochovsky, Lilian; Neuman, Rami; Brégégère, François Menahem; Milner, Yoram

2008-10-01

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging. We showed that the expression of 16 biochemical markers was altered in aged cultured cells and in tissues, including caspases 1 and 3 and beta-galactosidase activities, immunoreactivities of p16, Ki67, 20S proteasome and effectors of the Fas-dependent apoptotic pathway. Aged cells diversity, and individual variability of aging markers, call for a multifunctional assessment of the aging phenomenon, and of its modulation by drugs. As a test case, we have measured the effects of Dead Sea minerals on keratinocyte cultures and human skin, and found that they stimulate proliferation and mitochondrial activity, decrease the expression of some aging markers, and limit apoptotic damage after UVB irradiation.

Changes in nuclear morphology and chromatin texture of basal keratinocytes in melasma.

PubMed

Brianezi, G; Handel, A C; Schmitt, J V; Miot, L D B; Miot, H A

2015-04-01

The pathogenesis of melasma and the role of keratinocytes in disease development and maintenance are not completely understood. Dermal abnormalities, the expression of inflammatory mediators, growth factors, epithelial expression of melanocortin and sexual hormones receptors suggest that not only melanocytes, but entire epidermal melanin unit is involved in melasma physiopathology. To compare nuclear morphological features and chromatin texture between basal keratinocytes in facial melasma and adjacent normal skin. We took facial skin biopsies (2 mm melasma and adjacent normal skin) from women processed for haematoxylin and eosin. Thirty non-overlapping basal keratinocyte nuclei were segmented and descriptors of area, highest diameter, perimeter, circularity, pixel intensity, profilometric index (Ra) and fractal dimension were extracted using ImageJ software. Basal keratinocyte nuclei from facial melasma epidermis displayed larger size, irregular shape, hyperpigmentation and chromatin heterogeneity by fractal dimension than perilesional skin. Basal keratinocytes from facial melasma display changes in nuclear form and chromatin texture, suggesting that the phenotype differences between melasma and adjacent facial skin can result from complete epidermal melanin unit alterations, not just hypertrophic melanocytes. © 2014 European Academy of Dermatology and Venereology.

[Cultivated keratinocytes on micro-carriers: in vitro studies of a new carrier system].

PubMed

Hecht, J; Hoefter, E A; Hecht, J; Haraida, S; Nerlich, A; Hartinger, A; Mühlbauer, W; Dimoudis, N

1997-03-01

Epidermal grafts from confluently cultivated keratinocytes have been used since the early eighties for the treatment of severe burns, where the shortage of donor sites for split-thickness skin grafts did not allow for adequate wound coverage. The difficult handling of these grafts as well as the advanced differentiation of their epithelial cells into a multilayer sheet poses a problem for their clinical application. The aim of the study was to characterize cultivated keratinocytes, as well as to observe their migration and proliferation from the MC onto a surface. Keratinocytes were isolated from human foreskin and cultivated in serum-free and serum-containing medium according to a modified method by Rheinwald and Green. Collagen-coated Dextran beads were used as MC. The MC were colonized with keratinocytes using the Spinner culture technique. After seeding the colonized MC into culture flasks, their migration and proliferation was monitored regularly through immunohistochemical studies and measurement of the metabolic cell activity. Immunohistological staining proved that the cells isolated from human foreskin represent keratinocytes of the basal type. Keratinocytes, cultivated with serum-containing and serum free medium, both adhered to the surface of the MC, then migrated onto the surface of the flasks and proliferated to form a multilayer of epithelial cells. In the long-term, a flexible epithelial graft consisting of poorly differentiated keratinocytes should be available, which is simple to produce and easy to handle. This would be an alternative method for treating wounds, where the conventional multilayer epithelial graft (ET) is insufficient.

BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes.

PubMed

Sun, Yang; Wang, Peiling; Li, Hongyu; Dai, Jun

2018-06-26

A diverse array of biological processes are under circadian controls. In mouse skin, ultraviolet ray (UVR)-induced apoptosis and DNA damage responses are time-of-day dependent, which are controlled by core clock proteins. This study investigates the roles of clock proteins in regulating UVB responses in human keratinocytes (HKCs). We found that the messenger RNA expression of brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) genes is altered by low doses (5 mJ/cm 2 ) of UVB in the immortalized HaCat HKCs cell line. Although depletion of BMAL1 or CLOCK has no effect on the activation of Rad3-related protein kinases-checkpoint kinase 1-p53 mediated DNA damage checkpoints, it leads to suppression of UVB-stimulated apoptotic responses, and downregulation of UVB-elevated expression of DNA damage marker γ-H2AX and cell cycle inhibitor p21. Diminished apoptotic responses are also observed in primary HKCs depleted of BMAL1 or CLOCK after UVB irradiation. While CLOCK depletion shows a suppressive effect on UVB-induced p53 protein accumulation, depletion of either clock gene triggers early keratinocyte differentiation of HKCs at their steady state. These results suggest that UVB-induced apoptosis and DNA damage responses are controlled by clock proteins, but via different mechanisms in the immortalized human adult low calcium temperature and primary HKCs. Given the implication of UVB in photoaging and photocarcinogenesis, mechanistic elucidation of circadian controls on UVB effects in human skin will be critical and beneficial for prevention and treatment of skin cancers and other skin-related diseases. © 2018 Wiley Periodicals, Inc.

Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

PubMed

Bigliardi-Qi, M; Sumanovski, L T; Büchner, S; Rufli, T; Bigliardi, P L

2004-01-01

We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

PubMed Central

Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

2009-01-01

EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

PubMed

Rasmussen, Cathy A; Allen-Hoffmann, B Lynn

2012-04-01

For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss. Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites. Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures. Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application. This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

PubMed Central

Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M.

2017-01-01

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema. PMID:28829355

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo.

PubMed

Wölfle, Ute; Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M

2017-08-22

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

PubMed

Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

2015-04-01

Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis

PubMed Central

Carvalho, Agostinho; Giovannini, Gloria; De Luca, Antonella; D'Angelo, Carmen; Casagrande, Andrea; Iannitti, Rossana G; Ricci, Giovanni; Cunha, Cristina; Romani, Luigina

2012-01-01

The recognition of β-glucans by dectin-1 has been shown to mediate cell activation, cytokine production and a variety of antifungal responses. Here, we report that the functional activity of dectin-1 in mucosal immunity to Candida albicans is influenced by the genetic background of the host. Dectin-1 was required for the proper control of gastrointestinal and vaginal candidiasis in C57BL/6, but not BALB/c mice; in fact, the latter showed increased resistance in the absence of dectin-1. The susceptibility of dectin-1-deficient C57BL/6 mice to infection was associated with defects in IL-17A and aryl hydrocarbon receptor-dependent IL-22 production and in adaptive Th1 responses. In contrast, the resistance of dectin-1-deficient BALB/c mice was associated with increased IL-17A and IL-22 production and the skewing towards Th1/Treg immune responses that provide immunological memory. Disparate canonical/noncanonical NF-κB signaling pathways downstream of dectin-1 were activated in the two different mouse strains. Thus, the net activity of dectin-1 in antifungal mucosal immunity is dependent on the host's genetic background, which affects both the innate cytokine production and the adaptive Th1/Th17 cell activation upon dectin-1 signaling. PMID:22543832

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production.

PubMed

Ham, Sun Ah; Hwang, Jung Seok; Yoo, Taesik; Lee, Hanna; Kang, Eun Sil; Park, Chankyu; Oh, Jae-Wook; Lee, Hoon Taek; Min, Gyesik; Kim, Jin-Hoi; Seo, Han Geuk

2012-05-15

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

Ecklonia cava Extract and Dieckol Attenuate Cellular Lipid Peroxidation in Keratinocytes Exposed to PM10.

PubMed

Lee, Jeong-Won; Seok, Jin Kyung; Boo, Yong Chool

2018-01-01

Airborne particulate matter can cause oxidative stress, inflammation, and premature skin aging. Marine plants such as Ecklonia cava Kjellman contain high amounts of polyphenolic antioxidants. The purpose of this study was to examine the antioxidative effects of E. cava extract in cultured keratinocytes exposed to airborne particulate matter with a diameter of <10  μ m (PM10). After the exposure of cultured HaCaT keratinocytes to PM10 in the absence and presence of E. cava extract and its constituents, cell viability and cellular lipid peroxidation were assessed. The effects of eckol and dieckol on cellular lipid peroxidation and cytokine expression were examined in human epidermal keratinocytes exposed to PM10. The total phenolic content of E. cava extract was the highest among the 50 marine plant extracts examined. The exposure of HaCaT cells to PM10 decreased cell viability and increased lipid peroxidation. The PM10-induced cellular lipid peroxidation was attenuated by E. cava extract and its ethyl acetate fraction. Dieckol more effectively attenuated cellular lipid peroxidation than eckol in both HaCaT cells and human epidermal keratinocytes. Dieckol and eckol attenuated the expression of inflammatory cytokines such as tumor necrosis factor- (TNF-) α , interleukin- (IL-) 1 β , IL-6, and IL-8 in human epidermal keratinocytes stimulated with PM10. This study suggested that the polyphenolic constituents of E. cava , such as dieckol, attenuated the oxidative and inflammatory reactions in skin cells exposed to airborne particulate matter.

Human eccrine sweat gland cells turn into melanin-uptaking keratinocytes in dermo-epidermal skin substitutes.

PubMed

Böttcher-Haberzeth, Sophie; Biedermann, Thomas; Pontiggia, Luca; Braziulis, Erik; Schiestl, Clemens; Hendriks, Bart; Eichhoff, Ossia M; Widmer, Daniel S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2013-02-01

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.

PubMed

Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J

2010-07-01

Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

PubMed

Scott, Glynis; Leopardi, Sonya; Parker, Lorelle; Babiarz, Laura; Seiberg, Miri; Han, Rujiing

2003-09-01

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.

Human antimicrobial peptides LL-37 and human β-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus.

PubMed

Crack, L R; Jones, L; Malavige, G N; Patel, V; Ogg, G S

2012-07-01

There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human β-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human β-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-incubation of VZV with LL-37, but not hBD-2, further reduced VZV load. Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE. © The Author(s). CED © 2012 British Association of Dermatologists.

Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients.

PubMed

Elango, Tamilselvi; Thirupathi, Anand; Subramanian, Swapna; Ethiraj, Purushoth; Dayalan, Haripriya; Gnanaraj, Pushpa

2017-08-01

Psoriasis is a chronic inflammatory skin disease characterized by hyper proliferation of keratinocytes. Recent data show that the epidermis thickening in psoriasis may be related to imbalance of homeostasis caused by abnormal apoptotic process. Maintenance of keratinocyte apoptotic process is very important in psoriasis. Methotrexate (MTX) has been used for many years to restore the normal skin in psoriasis condition. However, the exact mechanism of MTX in psoriasis condition is poorly understood. The aim of this study was to examine the role of MTX on keratinocyte apoptosis pathway in psoriasis patients. A total of 58 psoriasis vulgaris patients were recruited for this study. Nonlesional skin biopsies served as control. Skin biopsies of psoriatic patients were collected and analyzed for cytosolic, mitochondria and total cytochrome c by ELISA. Expression of caspase-9, NFκBp65, pAkt1 by western blot, real-time PCR and immunohistochemical analysis of c-FLIP protein was analyzed in nonlesional and lesional skin biopsies before (day 0) and after (at the end of 6 and 12 weeks) MTX treatment. After MTX treatment, a significant increase in cytochrome c was observed when compared with before MTX treatment in psoriasis patients (p < 0.001). Protein and gene expression of cleaved caspase-9 were significantly increased after MTX treatment, whereas the expression of Bcl-xL, c-FLIP, NFκBp65, pAkt1 significantly downregulated after MTX treatment. In conclusion, these results showed that intrinsic apoptotic pathway induced by MTX eventually adds the beneficial therapeutic role of MTX in psoriasis by controlling the acanthosis.

The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kadono, Nanako; Miyazaki, Takafumi; Okugawa, Yoji

2012-01-27

Highlights: Black-Right-Pointing-Pointer A subpopulation of syntaxin4 localizes extracellularly in the keratinocytes. Black-Right-Pointing-Pointer Epimorphin and syntaxin4 confer the resistance to the oxidative stress. Black-Right-Pointing-Pointer Epimorphin suppresses and syntaxin4 accelerates the CCE formation. Black-Right-Pointing-Pointer The antagonistic peptide to syntaxin4 blocks the syntaxin4-dependent CCE formation. -- Abstract: Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 viamore » a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103-108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.« less

Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway

PubMed Central

Lou, Hongfei; Lu, Jingning; Choi, Eun Byul; Oh, Min Hee; Jeong, Mingeum; Barmettler, Sara; Zhu, Zhou; Zheng, Tao

2017-01-01

Increased expression of Th22 cytokine IL-22 is a characteristic finding in atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of AD in vivo has yet to be elucidated. Consistent with observations in human AD, IL-22 was significantly increased in the AD skin of mice after epicutaneous sensitization to house dust mite allergen. Utilizing a skin-specific inducible transgenic system, we show here that expression of IL-22 in the skin of mice caused an AD-like phenotype characterized by chronic pruritic dermatitis associated with Th2-biased local and systemic immune responses, down-regulation of Epidermal Differentiation Complex genes and enhanced dermatitis upon epicutaneous allergen exposure. IL-22 potently induced the expression of gastrin-releasing peptide (GRP), a neuropeptide pruritogen, in dermal immune cells and sensory afferents and in their skin-innervating sensory neurons. IL-22 also differentially up-regulated the expression of GRP receptor (GRPR) on keratinocytes of AD skin. The number of GRP+ cells in the skin correlated with the AD severity and the intensity of pruritus. IL-22 directly upregulated the expression of epithelial-derived type 2 cytokines (TSLP and IL-33) and GRP in primary keratinocytes. Furthermore, GRP not only strongly induced TSLP but also increased the expression IL-33 and GRPR synergistically with IL-22. Importantly, we found that the expression of GRP was strikingly increased in the skin of patients with AD. These results indicate that IL-22 plays important pathogenic roles in the initiation and development of AD, in part through inducing keratinocyte production of type 2 cytokines and activation of the GRP/GRPR pathway. PMID:28228560

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

PubMed

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

Anti-apoptotic effects of suppressor of cytokine signaling 3 and 1 in psoriasis

PubMed Central

Madonna, S; Scarponi, C; Pallotta, S; Cavani, A; Albanesi, C

2012-01-01

Because of their genetically determined capacity to respond to pro-inflammatory stimuli, keratinocytes have a crucial role in the pathogenesis of psoriasis. Upon IFN-γ and TNF-α exposure, psoriatic keratinocytes express exaggerated levels of inflammatory mediators, and show aberrant hyperproliferation and terminal differentiation. The thickening of psoriasic skin also results from a peculiar resistance of keratinocytes to cytokine-induced apoptosis. In this study, we investigated on the molecular mechanisms concurring to the resistance of psoriatic keratinocytes to cell death, focusing on the role having suppressor of cytokine signaling (SOCS)1 and SOCS3, two molecules abundantly expressed in IFN-γ/TNF-α-activated psoriatic keratinocytes, in sustaining anti-apoptotic pathways. We found that SOCS1 and SOCS3 suppress cytokine-induced apoptosis by sustaining the activation of the PI3K/AKT pathway in keratinocytes. The latter determines the activation of the anti-apoptotic NF-κB cascade and, in parallel, the inhibition of the pro-apoptotic BAD function in keratinocytes. For the first time, we report that phosphorylated AKT and phosphorylated BAD are strongly expressed in lesional psoriatic skin, compared with healthy or not lesional skin, and they strictly correlate to the high expression of SOCS1 and SOCS3 molecules in the psoriatic epidermis. Finally, the depletion of SOCS1 and SOCS3, as well as the chemical inactivation of PI3K activity in psoriatic keratinocytes, definitively unveils the role of PI3K/AKT cascade on the resistance of diseased keratinocytes to apoptosis. PMID:22739986

SOCS3 inhibits the pathological effects of IL-22 in non-melanoma skin tumor-derived keratinocytes

PubMed Central

Madonna, Stefania; Scarponi, Claudia; Morelli, Martina; Sestito, Rosanna; Scognamiglio, Pasqualina Liana; Marasco, Daniela; Albanesi, Cristina

2017-01-01

Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes. In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo, in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts. PMID:28445952

SOCS3 inhibits the pathological effects of IL-22 in non-melanoma skin tumor-derived keratinocytes.

PubMed

Madonna, Stefania; Scarponi, Claudia; Morelli, Martina; Sestito, Rosanna; Scognamiglio, Pasqualina Liana; Marasco, Daniela; Albanesi, Cristina

2017-04-11

Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes.In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo, in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts.

1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

PubMed Central

Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A.; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU. PMID:25337708

The role of PRP and adipose tissue-derived keratinocytes on burn wound healing in diabetic rats

PubMed Central

Hosseini Mansoub, Navid; Gürdal, Mehmet; Karadadaş, Elif; Kabadayi, Hilal; Vatansever, Seda; Ercan, Gulinnaz

2018-01-01

Introduction: Diabetic burn wounds and ulcers are significant complications of diabetic patients. The aim of this study is to investigate the use of platelet rich-plasma (PRP) and/or keratinocyte-like cells (KLCs) in diabetic thermal wound rat model and to evaluate EGF, FGF-2, TGF-β1, COL1α2, MCP-1 and VEGF-α as wound healing markers at gene expression level. Method: In this study, we used adipose tissue as the source of mesenchymal stem cells (MSCs) and differentiated MSCs into KLCs. KLCs were characterized and transferred to the burn areas on the dorsum of streptozotocine (STZ)-induced diabetic rats. We prepared PRP from rat blood and evaluated its effect alone or in combination with KLCs. On 3rd, 7th, 10th and 14th days after treatment, wound areas were measured and biopsy samples were excised from the wound areas of the KLCs and/or PRP-treated and untreated diabetic rats to analyze gene expression levels of wound healing markers by qPCR. Results: We observed that, wound contraction started earlier in the PRP and/or KLCs-treated groups in comparison to the control group. However, PRP and KLCs when applied in combination showed additive affect in wound healing. In all groups treated with KLCs and/or PRP, the gene expression levels of evaluated growth factors and COL1α2 increased, while MCP-1 levels decreased when compared to the untreated diabetic rats. In addition, the most prominent difference in qPCR results belongs to combined PRP and KLCs-treated group. Conclusion: We demonstrated that applying PRP and KLCs in combination has a greater potential for treatment of diabetic burn wounds. PMID:29713597

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia

PubMed Central

Perez-Crespo, Ignacio; Chillón-Marinas, Carlos; Khoubbane, Messaoud; Quesada, Carla; Reguera-Gomez, Marta; Mas-Coma, Santiago; Fresno, Manuel; Gironès, Núria

2017-01-01

Background Fascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity. Methodology/Principal findings The experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

PubMed Central

Poulsen, Christopher; Mehalick, Leslie A.; Fischer, Carol L.; Lanzel, Emily A.; Bates, Amber M.; Walters, Katherine S.; Cavanaugh, Joseph E.; Guthmiller, Janet M.; Johnson, Georgia K.; Wertz, Philip W.; Brogden, Kim A.

2015-01-01

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propridium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2 to 10.0 µM long-chain bases and GML were not cytotoxic; 40.0 to 80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 minutes. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26005054

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

PubMed

Poulsen, Christopher; Mehalick, Leslie A; Fischer, Carol L; Lanzel, Emily A; Bates, Amber M; Walters, Katherine S; Cavanaugh, Joseph E; Guthmiller, Janet M; Johnson, Georgia K; Wertz, Philip W; Brogden, Kim A

2015-08-19

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 μM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propidium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2-10.0 μM long-chain bases and GML were not cytotoxic; 40.0-80.0 μM long-chain bases, but not GML, were cytotoxic; and 80.0 μM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Punicalagin and (-)-Epigallocatechin-3-Gallate Rescue Cell Viability and Attenuate Inflammatory Responses of Human Epidermal Keratinocytes Exposed to Airborne Particulate Matter PM10.

PubMed

Seok, Jin Kyung; Lee, Jeong-Won; Kim, Young Mi; Boo, Yong Chool

2018-01-01

Airborne particulate matter with a diameter of < 10 µm (PM10) causes oxidative damage, inflammation, and premature skin aging. In this study, we evaluated whether polyphenolic antioxidants attenuate the inflammatory responses of PM10-exposed keratinocytes. Primary human epidermal keratinocytes were exposed in vitro to PM10 in the absence or presence of punicalagin and (-)-epigallocatechin-3-gallate (EGCG), which are the major polyphenolic antioxidants found in pomegranate and green tea, respectively. Assays were performed to determine cell viability, production of reactive oxygen species (ROS), and expression of NADPH oxidases (NOX), proinflammatory cytokines, and matrix metalloproteinase (MMP)-1. PM10 decreased cell viability and increased ROS production in a dose-dependent manner. It also increased the expression levels of NOX-1, NOX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and MMP-1. Punicalagin was not cytotoxic up to 300 μM, and (-)-EGCG was cytotoxic above 30 μM, respectively. Further, punicalagin (3-30 μM) and EGCG (3-10 μM) rescued the viability of PM10-exposed cells. They also attenuated ROS production and the expression of NOX-1, NOX-2, TNF-α, IL-1β, IL-6, IL-8, and MMP-1 stimulated by PM10. This study demonstrates that polyphenolic antioxidants, such as punicalagin and (-)-EGCG, rescue keratinocyte viability and attenuate the inflammatory responses of these cells due to airborne particles. © 2018 S. Karger AG, Basel.

Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome.

PubMed

Borges, Alessandra Abel; Campos, Gelse Mazzoni; Moreli, Marcos Lázaro; Moro Souza, Ricardo Luiz; Saggioro, Fabiano Pinto; Figueiredo, Glauciane Garcia; Livonesi, Márcia Cristina; Moraes Figueiredo, Luiz Tadeu

2008-01-01

The hantavirus pulmonary syndrome (HPS) is an emerging syndrome in the Americas. The disease results from intense immune activation and changes in vascular permeability. The aim of this study was to determine the profile of serum cytokines in HPS patients looking for correlation with the clinical parameters, severity and outcome of illness. Studying 21 HPS patients, we found that IL-6 may have an important role in the pathogenesis of HPS, being associated with fatal outcome. Our results also support a mixed Th1/Th2 immune response during the course of HPS and that the magnitude of Th1 response effector cytokines is correlated to HPS severity. The decreased levels of TGF-beta observed in HPS patients suggest that immunoregulatory activity could be damaged in these patients.

The Th1-promoting effects of dehydroepiandrosterone can provide an explanation for the stronger Th1-immune response of women.

PubMed

Namazi, Mohammad Reza

2009-03-01

Estrogens foster immunological processes driven by CD4+ Th2 cells and B cells and androgens foster Th1 CD4+ and CD8+ cell activity. Higher levels of IFN-gamma and IL-2 and lower levels of IL-4 and IL-10 are detected in the phytohemagglutinin-stimulated lymphocyte culture supernatants of men compared with women. It is documented that the physiologic levels of estrogens produced during the luteal phase of the menstrual cycle shift the female immune system toward a Th2-type response and that the Th1 cytokines are increased in postmenopausal women. However, the Th1 immune response is also surprisingly stronger in women, hence affording them a better protection against infections. Nickel sensitivity, a Th1 immune reaction, seems to be more common in women even if men wear earrings. Further, not only the Th2 but also the Th1 autoimmune diseases are generally more common in women than men. How do women advance a stronger Th1 response than men? It is suggested that in contrast to the paradigm that estrogens lead to a Th2 bias, estrogens can enhance Th1 cytokine production also. However, the discrepant effects of estrogens are difficult to be reconciled from a molecular viewpoint and hence are not advocated by all authors. This paper provides an explanation: The effects of dehydroepiandrosterone on Th1/Th2 balance seem to be model-specific; in humans dehydroepiandrosterone, represents a pivotal up-regulator of Th1 immune response. Steroid sulphatase is a microsomal enzyme that cleaves the sulphate group of dehydroepiandrosterone sulphate. This enzyme is controlled by an X-linked gene that escapes the Lyon effect of X-inactivation; as a result, women usually have about twice steroid sulphatase in their cells, including macrophages, as have men. Putting all these facts together, it could be concluded that women's macrophages, which contain higher steroid sulphatase levels and enter peripheral lymphoid organs through afferent lymphatic drainage, produce higher levels of

Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA,more » which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.« less

Aqueous extract from Vitis vinifera tendrils is able to enrich keratinocyte antioxidant defences.

PubMed

Fraternale, Daniele; De Bellis, Roberta; Calcabrini, Cinzia; Potenza, Lucia; Cucchiarini, Luigi; Mancini, Umberto; Dachà, Marina; Ricci, Donata

2011-09-01

An aqueous extract of V. vinifera L. tendrils was evaluated for its ability to enrich the antioxidant capacity of cultured cells. The long-time antioxidant capability of the extract was measured by in vitro chemical methods, and its influence on reduced glutathione levels and plasma membrane oxido reductase activity was determined in cultured human keratinocytes (NCTC 2544). Keratinocytes are cells normally exposed to oxidative stress, and for this reason adequately equipped with antioxidant defences. However, it has long been suggested that exogenous antioxidants may play an important role in minimizing the adverse effects of oxidative stress on skin.We demonstrated that V. vinifera tendril aqueous extract was able to increase, in a time- and dose-dependent manner, the reduced glutathione concentration and activity of trans plasma membrane oxido reductase as an indirect evaluation of the intracellular redox status of the cells demonstrating a relevant antioxidant activity of this phytocomplex.

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, F.; Lyon, M.B.; Norris, D.A.

1989-09-01

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid ofmore » (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.« less

Modulation of LXR-α and the effector genes by Ascorbic acid and Statins in psoriatic keratinocytes.

PubMed

Soodgupta, Deepti; Kaul, Deepak; Kanwar, A J; Parsad, Davinder

2014-12-01

Recent studies have revealed critical roles that nuclear receptors like LXR-α (Liver X Receptor- alpha) plays as a class of post-transcriptional gene regulator in skin development and diseases. Keeping in view the fact that LXR-α plays crucial role in keratinocyte proliferation and differentiation, it becomes imperative to dissect the pathways and role of LXR-α genomics in the pathogenesis of psoriasis with ultimate aim to explore novel preventive/therapeutic strategies as treatment options. To explore the effects of agonists and activators of LXR-α on its own gene expression and the putative targets in psoriatic keratinocytes. Identification of promoter sequences for (vitamin D receptor) VDR and Catalase were done using in silico analysis followed by β-galactosidase (β-gal) reporter plasmid assay in keratinocytes from clinically heathy subjects. Determination of relative levels of LXR-α,VDR and catalase in control versus treated cells upon activation of LXR-α with Atorvastatin + 22R hydroxycholestrol and Ascorbic acid + 22R hydroxycholestrol was done by PCR and Cell Proliferation Assay. The cells transfected with the reporter plasmid element for VDR and catalase showed more than 5 and 4 fold increase respectively in the β-gal activity compared to the control. An increase of 55% in LXR-α gene expression at RNA level was observed in Atorvastatin + 22-R hydroxycholestrol compared to 24% in Ascorbic acid + 22-ROH cholesterol. The expression of the VDR and Catalase was significantly increased in both treated keratinocytes compared to its normal counterpart.

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation.

PubMed

Goto, Makiko; Ikeyama, Kazuyuki; Tsutsumi, Moe; Denda, Sumiko; Denda, Mitsuhiro

2010-07-01

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non-selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response. (c) 2010 Wiley-Liss, Inc.

Bullous pemphigoid followed by pustular psoriasis showing Th1, Th2, Treg and Th17 immunological changes.

PubMed

Yasukawa, Shinsuke; Dainichi, Teruki; Kokuba, Hisashi; Moroi, Yoichi; Urabe, Kazunori; Hashimoto, Takashi; Furue, Masutaka

2009-01-01

Psoriasis vulgaris is occasionally accompanied by autoimmune bullous diseases, but the opposite is very rare. We document here the first reported case of generalized pustular psoriasis that appeared during steroid therapy for bullous pemphigoid. The serum cytokine levels and the results of an immunohistochemical study over the disease course suggest that the immunological state was consistent with a shift from Th2-dominance to Th1-dominance. IL-17-producing cells appeared in the skin lesions when each disease was most exacerbated and disappeared after remission. Thus, the present case demonstrated a dynamic immunological state in which the appearances of Th1 and Th2 as well as Th17 varied during the course of the disease.

In vitro toxicity of photodynamic antimicrobial chemotherapy on human keratinocytes proliferation.

PubMed

Migliario, Mario; Rizzi, Manuela; Rocchetti, Vincenzo; Cannas, Mario; Renò, Filippo

2013-02-01

This in vitro experimental study has been designed to assess the effects of photodynamic antimicrobial chemotherapy (PACT) on human keratinocytes proliferation. Human keratinocytes (HaCaT) monolayers (∼0.5 cm(2)) have been irradiated with 635 nm red laser light with a fluence of 82.5 or 112.5 J/cm(2) in the absence or presence of toluidine (TB). Cell proliferation, monolayer area coverage, cytokeratin 5 (K5) and filaggrin (Fil) expression, and metalloproteinase (MMP)-2 and MMP-9 activity were measured after 72 h from laser irradiation. HaCaT proliferation was reduced by TB staining. Cell exposure to both low- and high-fluence laser irradiation in both presence and absence of TB staining reduced their proliferation and monolayer area extension. Moreover both laser treatments were able to reduce K5 and Fil expression and MMP-9 production in keratinocytes not treated with TB. These data indicate that PACT could exert toxic effects on normal proliferating keratinocytes present around parodontal pockets. The observed reduced cell proliferation along with a reduced production of enzymes involved in wound healing could alter the clinical outcome of the patients treated with PACT.

MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Haigang; Hou, Liyue; Liu, Jingjing

MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 bymore » luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.« less

Wave Energy Prize - 1/50th Testing - Iowec

DOE Data Explorer

Wesley Scharmen

2015-12-18

This submission of data includes all the 1/50th scale testing data completed on the Wave Energy Prize for the Iowec team, and includes: 1/50th test data (raw & processed) 1/50th test data video and pictures 1/50th Test plans and testing documents SSTF_Submission (summarized results)

Wave Energy Prize - 1/50th Testing - SEWEC

DOE Data Explorer

Wesley Scharmen

2015-12-11

This submission of data includes all the 1/50th scale testing data completed on the Wave Energy Prize for the SEWEC team, and includes: 1/50th test data (raw & processed) 1/50th test data video and pictures 1/50th Test plans and testing documents SSTF_Submission (summarized results)

Wave Energy Prize - 1/50th Testing - WECCA

DOE Data Explorer

Wesley Scharmen

2016-01-15

This submission of data includes all the 1/50th scale testing data completed on the Wave Energy Prize for the WECCA team, and includes: 1/50th test data (raw & processed) 1/50th test data video and pictures 1/50th Test plans and testing documents SSTF_Submission (summarized results)

Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

PubMed

Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

2015-08-02

Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence

miR-155 promotes cutaneous wound healing through enhanced keratinocytes migration by MMP-2.

PubMed

Yang, Longlong; Zheng, Zhao; Zhou, Qin; Bai, Xiaozhi; Fan, Lei; Yang, Chen; Su, Linlin; Hu, Dahai

2017-04-01

Inflammation, re-epithelization and tissue remodeling are three essential steps during wound healing. The re-epithelization process plays the most important role which mainly involves keratinocyte proliferation and migration. miR-155 has been reported to participate in cell migration and transformation, however, its function in skin wound healing is largely unknown. Here we hypothesize that overexpression of miR-155 at wound edges could accelerate wound healing mediated by enhanced keratinocyte migration. To test this hypothesis, direct local injection of miR-155 expression plasmid to wound edges was conducted to overexpress miR-155 in vivo. Results shown that miR-155 significantly promoted wound healing and re-epithelization compared to control, while did not affect wound contraction. Also, miR-155 overexpression accelerated primarily cultured keratinocyte migration in vitro, but had no effect on cell proliferation. Importantly, western blot analysis shown that MMP-2 was significantly upregulated whiles its inhibitor TIMP-1 downregulated after miR-155 treatment. Moreover, the use of ARP-101, an MMP-2 inhibitor, effectively attenuated the accelerative effects on cell migration induced by miR-155. Taken together, our results suggest that miR-155 has the promote effect on wound healing that is probably mediated by accelerating keratinocyte migration via upregulated MMP-2 level. This study provides a rationale for the therapeutic effect of miR-155 on wound healing.

Circulating endothelial progenitor cells, Th1/Th2/Th17-related cytokines, and endothelial dysfunction in resistant hypertension.

PubMed

Magen, Eli; Feldman, Arie; Cohen, Ziona; Alon, Dora Ben; Minz, Evegeny; Chernyavsky, Alexey; Linov, Lina; Mishal, Joseph; Schlezinger, Menacham; Sthoeger, Zev

2010-02-01

A possible link between chronic vascular inflammation and arterial hypertension is now an object of intensive studies. To compare Th1/Th2/Th17 cells-related cytokines, circulating endothelial progenitor cells (EPC), and endothelial function in subjects with resistant arterial hypertension (RAH) and controlled arterial hypertension (CAH). Blood pressure was measured by electronic sphygmomanometer. EPC were identified as CD34+/CD133+/kinase insert domain receptor (KDR)+ cells by flow cytometry. Th1/Th2/Th17 cells-related cytokines were identified using the Human Th1/Th2/Th17 Cytokines MultiAnalyte ELISArray Kit. Endothelium-dependent (FMD) vasodilatation of brachial artery was measured by Doppler ultrasound scanning. RAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, lower blood levels of EPC number (42.4 +/- 16.7 cells/mL) and EPC% (0.19 +/- 0.08%) were observed than in the CAH group (93.1 +/- 88.7 cells/mL; P = 0.017; 0.27 +/- 0.17; P = 0.036) and control group (68.5 +/- 63.6 cells/mL; P < 0.001; 0.28 +/- 0.17%; P = 0.003), respectively. Plasma transforming growth factor-beta1 levels were significantly higher in the RAH group (1767 +/- 364 pg/mL) than in the CAH group (1292 +/- 349; P < 0.001) and in control group (1203 +/- 419 pg/mL; P < 0.001). In the RAH group, statistically significant negative correlation was observed between systolic blood pressure and EPC% (r = -0.72, P < 0.01). FMD in the RAH group was significantly lower (5.5 +/- 0.8%) than in the CAH group (9.2 +/- 1.4; P < 0.001) and in healthy controls (10.1 +/- 1.1%; P < 0.001). RAH is characterized by reduced circulating EPC, substantial endothelial dysfunction, and increased plasma transforming growth factor-beta1 levels.

Epigenetic control of IL-23 expression in keratinocytes is important for chronic skin inflammation.

PubMed

Li, Hui; Yao, Qi; Mariscal, Alberto Garcia; Wu, Xudong; Hülse, Justus; Pedersen, Esben; Helin, Kristian; Waisman, Ari; Vinkel, Caroline; Thomsen, Simon Francis; Avgustinova, Alexandra; Benitah, Salvador Aznar; Lovato, Paola; Norsgaard, Hanne; Mortensen, Mette Sidsel; Veng, Lone; Rozell, Björn; Brakebusch, Cord

2018-04-12

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment.

PubMed

Nowak, Karolin; Linzner, Daniela; Thrasher, Adrian J; Lambert, Paul F; Di, Wei-Li; Burns, Siobhan O

2017-10-01

Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4 + T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Association Between Statin Use and Risk for Keratinocyte Carcinoma in the Veterans Affairs Topical Tretinoin Chemoprevention Trial

PubMed Central

Dore, David D.; Lapane, Kate L.; Trivedi, Amal N.; Mor, Vincent; Weinstock, Martin A.

2013-01-01

Background Recent evidence suggests that statins may prevent cancer. Objective To quantify the association between statin use and the occurrence of keratinocyte carcinoma in high-risk veterans. Design Cohort study. Setting 6 Veterans Affairs medical centers. Participants 1037 participants of the Veterans Affairs Topical Tretinoin Chemoprevention Trial, a randomized, multicenter, double-blind, vehicle-controlled trial of topical tretinoin, 0.1%, for prevention of keratinocyte carcinoma conducted from November 1998 to November 2004. Measurements Time to first occurrence of keratinocyte carcinoma on the face or ears. Participants using a statin at randomization, according to the Veterans Affairs Pharmacy Benefits Management database, were considered exposed. Study dermatologists conducted physical examinations at baseline and every 6 months during follow-up. The association between statin use at randomization and the outcome was evaluated by using propensity score matching (n = 608) and Cox proportional hazards regression (n = 1037). Results Among the 1037 participants, 37% used a statin at randomization (n = 397) for a median duration of at least 900 days over a median follow-up of 3.5 years. In the propensity score-matched analysis, statin use at randomization was not associated with keratinocyte carcinoma (rate ratio, 0.92 [95% CI, 0.73 to 1.16]), a finding that was consistent with the estimates derived from the Cox proportional hazards regression (rate ratio, 0.84 [CI, 0.70 to 1.02]). Limitations The extent of residual confounding is unknown, and the confidence bounds around the measures of association were wide. These data may not be generalizable to lower-risk populations. Conclusion These data show no conclusive or consistent relationship between long-term statin use and risk for keratinocyte carcinoma. Funding Department of Veterans Affairs, Agency for Healthcare Research and Quality, American Cancer Society, and National Institutes of Health. PMID:19124815

Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

PubMed

Ong, Huan Ting; Redmond, Sharon L; Marano, Robert J; Atlas, Marcus D; von Unge, Magnus; Aabel, Peder; Dilley, Rodney J

2017-03-15

Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CM ADSC ) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CM ADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CM ADSC . Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling

PubMed Central

Moehring, Francie; Cowie, Ashley M; Menzel, Anthony D; Weyer, Andy D; Grzybowski, Michael; Arzua, Thiago; Geurts, Aron M; Palygin, Oleg

2018-01-01

The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. PMID:29336303

Production and in vitro evaluation of soy protein-based biofilms as a support for human keratinocyte and fibroblast culture.

PubMed

Curt, Sèverine; Subirade, Muriel; Rouabhia, Mahmoud

2009-06-01

This study presents results on soy protein isolate (SPI) biofilm production and the corresponding effect on the stability and toxicity of the derived films. SPI biofilms were prepared from SPI chemically treated with formaldehyde at various concentrations (0%, 1%, 2%, and 3%) as cross-linking agents. In vitro SPI biofilm degradation was evaluated as a function of water absorption leading to weight and size modifications. SPI biofilm toxicity was determined as a function of human keratinocyte and fibroblast adhesion, viability, and proliferation. Cytokine gene expression supported this using reverse transcriptase polymerase chain reaction techniques. Our results confirm that SPI can be used to produce biofilms. The resulting SPI biofilms without formaldehyde swell significantly, which leads to their physical instability. Formaldehyde treatment enhanced the mechanical properties of these biofilms by covalently cross-linking polypeptide chains. The decreased water absorption was dependent on the amount of formaldehyde present. SPI biofilms with 2% and 3% formaldehyde were highly stable and easier to manipulate than those with 0% and 1% formaldehyde. Tissue culture analyses revealed that the SPI biofilms without formaldehyde were non-toxic to human cells (keratinocytes and fibroblasts). The presence of formaldehyde in biofilms did not have any effects on cell viability, adhesion, or proliferation. This was supported by the high level of messenger RNA expression of interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha by the keratinocytes and of IL-6 and IL-8 by the fibroblasts. Overall, we produced a stable, non-toxic soy protein support, which may be of potential interest in medical applications such as cell culture matrices and damaged tissue replacement.

Effects of Acute Low-Dose Exposure to the Chlorinated Flame Retardant Dechlorane 602 and Th1 and Th2 Immune Responses in Adult Male Mice

PubMed Central

Feng, Yu; Tian, Jijing; Xie, Heidi Qunhui; She, Jianwen; Xu, Sherry Li; Xu, Tuan; Tian, Wenjing; Fu, Hualing; Li, Shuaizhang; Tao, Wuqun; Wang, Lingyun; Chen, Yangsheng; Zhang, Songyan; Zhang, Wanglong; Guo, Tai L.; Zhao, Bin

2016-01-01

Background: Although the chlorinated flame retardant Dechlorane (Dec) 602 has been detected in food, human blood, and breast milk, there is limited information on potential health effects, including possible immunotoxicity. Objectives: We determined the immunotoxic potential of Dec 602 in mice by examining the expression of phenotypic markers on thymocyte and splenic lymphocyte subsets, Th1/Th2 transcription factors, and the production of cytokines and antibodies. Methods: Adult male C57BL/6 mice were orally exposed to environmentally relevant doses of Dec 602 (1 and 10 μg/kg body weight per day) for 7 consecutive days. Thymocyte and splenic CD4 and CD8 subsets and splenocyte apoptosis were examined by flow cytometric analysis. Cytokine expression was measured at both the mRNA and the protein levels. Levels of the transcription factors Th1 (T-bet and STAT1) and Th2 (GATA3) were determined using quantitative real-time polymerase chain reaction (qPCR). Serum levels of immunoglobulins IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA). Results: Splenic CD4+ and CD8+ T cell subsets were decreased compared with vehicle controls, and apoptosis was significantly increased in splenic CD4+ T cells. Expression (mRNA and protein) of Th2 cytokines [interleukin (IL)-4, IL-10, and IL-13] increased, and that of Th1 cytokines [IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] decreased. The Th2 transcriptional factor GATA3 increased, whereas the Th1 transcriptional factors T-bet and STAT1 decreased. As additional indicators of the Th2-Th1 imbalance, production of IgG1 was significantly increased, whereas IgG2a was reduced. Conclusions: To our knowledge, we are the first to report evidence of the effects of Dec 602 on immune function in mice, with findings indicating that Dec 602 exposure favored Th2 responses and reduced Th1 function. Citation: Feng Y, Tian J, Xie HQ, She J, Xu SL, Xu T, Tian W, Fu H, Li S, Tao W, Wang L, Chen Y

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging.

PubMed

Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

2017-05-01

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Proteome-wide changes in primary skin keratinocytes exposed to diesel particulate extract-A role for antioxidants in skin health.

PubMed

Rajagopalan, Pavithra; Jain, Ankit P; Nanjappa, Vishalakshi; Patel, Krishna; Mangalaparthi, Kiran K; Babu, Niraj; Cavusoglu, Nükhet; Roy, Nita; Soeur, Jeremie; Breton, Lionel; Pandey, Akhilesh; Gowda, Harsha; Chatterjee, Aditi; Misra, Namita

2018-05-21

Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress. To investigate global proteomic alterations in diesel particulate extract (DPE)/its vapor exposed skin keratinocytes. We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/DPE vapor on primary skin keratinocytes. We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p ≤ 0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/DPE vapor. Our study highlights distinct adverse effects of chronic exposure to DPE/DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis.

PubMed

Wu, Nan-Lin; Huang, Duen-Yi; Hsieh, Shie-Liang; Hsiao, Cheng-Hsiang; Lee, Te-An; Lin, Wan-Wan

2013-10-01

Decoy receptor 3 (DcR3) is a soluble receptor of Fas ligand (FasL), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A) and plays pleiotropic roles in many inflammatory and autoimmune disorders and malignant diseases. In cutaneous biology, DcR3 is expressed in primary human epidermal keratinocytes and is upregulated in skin lesions in psoriasis, which is characterized by chronic inflammation and angiogenesis. However, the regulatory mechanisms of DcR3 over-expression in skin lesions of psoriasis are unknown. Here, we demonstrate that DcR3 can be detected in both dermal blood vessels and epidermal layers of psoriatic skin lesions. Analysis of serum samples showed that DcR3 was elevated, but FasL was downregulated in psoriatic patients compared with normal individuals. Additional cell studies revealed a central role of epidermal growth factor receptor (EGFR) in controlling the basal expression of DcR3 in keratinocytes. Activation of EGFR by epidermal growth factor (EGF) and transforming growth factor (TGF)-α strikingly upregulated DcR3 production. TNF-αenhanced DcR3 expression in both keratinocytes and endothelial cells compared with various inflammatory cytokines involved in psoriasis. Additionally, TNF-α-enhanced DcR3 expression in keratinocytes was inhibited when EGFR was knocked down or EGFR inhibitor was used. The NF-κB pathway was critically involved in the molecular mechanisms underlying the action of EGFR and inflammatory cytokines. Collectively, the novel regulatory mechanisms of DcR3 expression in psoriasis, particularly in keratinocytes and endothelial cells, provides new insight into the pathogenesis of psoriasis and may also contribute to the understanding of other diseases that involve DcR3 overexpression. Copyright © 2013 Elsevier B.V. All rights reserved.

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation*

PubMed Central

Lee, Hyunseung; Morales, Liza D.; Slaga, Thomas J.; Kim, Dae Joon

2015-01-01

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer. PMID:25406309

Effects of Wannachawee Recipe with Antipsoriatic Activity on Suppressing Inflammatory Cytokine Production in HaCaT Human Keratinocytes

PubMed Central

Na Takuathung, Mingkwan; Wongnoppavich, Ariyaphong; Pitchakarn, Pornsiri; Panthong, Ampai; Khonsung, Parirat; Soonthornchareonnon, Noppamas

2017-01-01

Psoriasis is a chronic inflammatory and immune-mediated skin disease. The pathogenesis involves T cells activation via the IL-23/Th17 axis. Conventional treatments of psoriasis have adverse events influencing patients' adherence. Wannachawee Recipe (WCR) has been effectively used as Thai folk remedy for psoriasis patients; however, preclinical evidence defining how WCR works is still lacking. This study defined mechanisms for its antiproliferation and anti-inflammatory effects in HaCaT cells. The cytotoxicity and antiproliferation results from SRB and CCK-8 assays showed that WCR inhibited the growth and viability of HaCaT cells in a concentration-dependent manner. The distribution of cell cycle phases determined by flow cytometry showed that WCR did not interrupt cell cycle progression. Interestingly, RT-qPCR revealed that WCR significantly decreased the mRNA expression of IL-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF-α but induced IL-10 expression in TNF-α- and IFN-γ-induced HaCaT cells. At the protein level determined by ELISA, WCR significantly reduced the secretion of IL-17A, IL-22, and IL-23. The WCR at low concentrations was proved to possess anti-inflammatory effect without cytotoxicity and it did not interfere with cell cycle of keratinocytes. This is the first study to provide convincing evidence that WCR is a potential candidate for development of effective psoriasis therapies. PMID:28900461

Effects of Wannachawee Recipe with Antipsoriatic Activity on Suppressing Inflammatory Cytokine Production in HaCaT Human Keratinocytes.

PubMed

Na Takuathung, Mingkwan; Wongnoppavich, Ariyaphong; Pitchakarn, Pornsiri; Panthong, Ampai; Khonsung, Parirat; Chiranthanut, Natthakarn; Soonthornchareonnon, Noppamas; Sireeratawong, Seewaboon

2017-01-01

Psoriasis is a chronic inflammatory and immune-mediated skin disease. The pathogenesis involves T cells activation via the IL-23/Th17 axis. Conventional treatments of psoriasis have adverse events influencing patients' adherence. Wannachawee Recipe (WCR) has been effectively used as Thai folk remedy for psoriasis patients; however, preclinical evidence defining how WCR works is still lacking. This study defined mechanisms for its antiproliferation and anti-inflammatory effects in HaCaT cells. The cytotoxicity and antiproliferation results from SRB and CCK-8 assays showed that WCR inhibited the growth and viability of HaCaT cells in a concentration-dependent manner. The distribution of cell cycle phases determined by flow cytometry showed that WCR did not interrupt cell cycle progression. Interestingly, RT-qPCR revealed that WCR significantly decreased the mRNA expression of IL-1 β , IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF- α but induced IL-10 expression in TNF- α - and IFN- γ -induced HaCaT cells. At the protein level determined by ELISA, WCR significantly reduced the secretion of IL-17A, IL-22, and IL-23. The WCR at low concentrations was proved to possess anti-inflammatory effect without cytotoxicity and it did not interfere with cell cycle of keratinocytes. This is the first study to provide convincing evidence that WCR is a potential candidate for development of effective psoriasis therapies.

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes.

PubMed

Pène, Jérôme; Chevalier, Sylvie; Preisser, Laurence; Vénéreau, Emilie; Guilleux, Marie-Hélène; Ghannam, Soufiane; Molès, Jean-Pierre; Danger, Yannic; Ravon, Elisa; Lesaux, Sabine; Yssel, Hans; Gascan, Hugues

2008-06-01

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

PubMed

Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

2015-07-01

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Image enhancement of optical images for binary system of melanocytes and keratinocytes

NASA Astrophysics Data System (ADS)

Takanezawa, S.; Baba, A.; Sako, Y.; Ozaki, Y.; Date, A.; Toyama, K.; Morita, S.

2013-05-01

Automatic determination of the cell shapes of large numbers of melanocytes based on optical images of human skin models have been largely unsuccessful (the complexities introduced by dendrites and the melanin pigmentation over the keratinocytes to give unclear outlines). Here, we present an image enhancement procedure for enhancing the contrast of images with removing the non-uniformity of background. The brightness is normalized also for the non-uniform population density of melanocytes.

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kioka, Noriyuki, E-mail: nkioka@kais.kyoto-u.ac.jp; Ito, Takuya; Yamashita, Hiroshi

2010-06-10

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantiallymore » suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.« less

In vitro induction effect of 1,25(OH)2D3 on differentiation of hair follicle stem cell into keratinocyte.

PubMed

Joulai Veijouyeh, Sanaz; Mashayekhi, Farhad; Yari, Abazar; Heidari, Fatemeh; Sajedi, Nayereh; Moghani Ghoroghi, Fatemeh; Nobakht, Maliheh

2017-02-01

Stem cells are characterized by self-renewal and differentiation capabilities. The bulge hair follicle stem cells (HFSCs) are able to convert to epithelial components. The active metabolite of vitamin D, 1,25(OH) 2 D 3 , plays important roles in this differentiation process. In the present study has found that 1,25(OH) 2 D 3 induces the HFSCs differentiation into keratinocyte. HFSCs are isolated from rat whiskers and cultivated in DMEM medium. To isolate bulge stem cell population, flow cytometry and immunocytochemistry using K15, CD34 and nestin biomarkers were performed. In order to accelerate the HFSCs differentiation into eratinocyte, HFSCs were treated with 10 -12 M, 1,25(OH) 2 D 3 every 48 h for a week. Immunocytochemistry results showed that bulge stem cells are nestin and CD34 positive but K15 negative before differentiation. Subsequently flow cytometry results, showed that the expression of nestin, CD34 and K15 were 70.96%, 93.03% and 6.88% respectively. After differentiation, the immunocytochemical and flow cytometry results indicated that differentiated cells have positive reaction to K15 with 68.94% expression level. It was concluded that 10 -12 M, 1,25(OH) 2 D 3 could induce the HFSCs differentiation into keratinocytes. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation.

PubMed

Ma, Hak-Ling; Liang, Spencer; Li, Jing; Napierata, Lee; Brown, Tom; Benoit, Stephen; Senices, Mayra; Gill, Davinder; Dunussi-Joannopoulos, Kyriaki; Collins, Mary; Nickerson-Nutter, Cheryl; Fouser, Lynette A; Young, Deborah A

2008-02-01

Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocytes and infiltrating immune cells. We report on a psoriasis-like disease model, which is induced by the transfer of CD4(+)CD45RB(hi)CD25(-) cells to pathogen-free scid/scid mice. Psoriasis-like lesions had elevated levels of antimicrobial peptide and proinflammatory cytokine mRNA. Also, similar to psoriasis, disease progression in this model was dependent on the p40 common to IL-12 and IL-23. To investigate the role of IL-22, a Th17 cytokine, in disease progression, mice were treated with IL-22-neutralizing antibodies. Neutralization of IL-22 prevented the development of disease, reducing acanthosis (thickening of the skin), inflammatory infiltrates, and expression of Th17 cytokines. Direct administration of IL-22 into the skin of normal mice induced both antimicrobial peptide and proinflammatory cytokine gene expression. Our data suggest that IL-22, which acts on keratinocytes and other nonhematopoietic cells, is required for development of the autoreactive Th17 cell-dependent disease in this model of skin inflammation. We propose that IL-22 antagonism might be a promising therapy for the treatment of human psoriasis.

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.

PubMed

Naaldijk, Yahaira; Johnson, Adiv A; Friedrich-Stöckigt, Annett; Stolzing, Alexandra

2016-12-01

Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

PubMed

Harrison, Caroline A; Dalley, Andrew J; Mac Neil, Sheila

2005-01-01

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paes, Camila, E-mail: camilaquinetti@gmail.com; Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp; Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp

2012-10-19

Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalianmore » cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.« less

Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes

PubMed Central

Bernerd, Francoise; Sarasin, Alain; Magnaldo, Thierry

1999-01-01

Galectin-7 is a β-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300–305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis. PMID:10500176

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2}more » and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.« less

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

PubMed

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St Arnaud, René; Dedhar, Shoukat; D'Souza, Sudhir J A; Dagnino, Lina

2008-04-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

PubMed Central

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St. Arnaud, René; Dedhar, Shoukat

2008-01-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function. PMID:18234842

PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senthilkumar, P.K.; Robertson, L.W.; Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA

Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cellmore » cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length

Th1 differentiation drives the accumulation of intravascular, non-protective CD4 T cells during tuberculosis

PubMed Central

Sallin, Michelle A.; Sakai, Shunsuke; Kauffman, Keith D.; Young, Howard A.; Zhu, Jinfang; Barber, Daniel L.

2017-01-01

SUMMARY Recent data indicate that the differentiation state of Th1 cells determines their protective capacity against tuberculosis. Therefore, we examined the role of Th1 polarizing factors in the generation of protective and non-protective subsets of Mtb-specific Th1 cells. We find IL-12/23p40 promotes Th1 cell expansion and maturation beyond the CD73+CXCR3+T-betdim stage, and T-bet prevents deviation of Th1 cells into Th17 cells. Nevertheless, IL-12/23p40 and T-bet are also essential for the production of a prominent subset of intravascular CX3CR1+KLRG1+ Th1 cells that persists poorly and can neither migrate into the lung parenchyma nor control Mtb growth. Furthermore, T-bet suppresses development of CD69+CD103+ tissue resident phenotype effectors in lung. In contrast, Th1 cell-derived IFNγ inhibits the accumulation of intravascular CX3CR1+KLRG1+ Th1 cells. Thus, although IL-12 and T-bet are essential host survival factors, they simultaneously oppose lung CD4 T cell responses at several levels, demonstrating the dual nature of Th1 polarization in tuberculosis. PMID:28355562

Increased Th1 and Th2 allergen-induced cytokine responses in children with atopic disease.

PubMed

Smart, J M; Kemp, A S

2002-05-01

Polyclonal cytokine responses following stimulation of T cells with mitogens or superantigens provides information on cytokine production from a wide range of T cells. Alternatively allergen-induced T cell responses can provide information on cytokine production by allergen-reactive T cells. While there is evidence of increased Th2 and reduced Th1 cytokine production following T cell stimulation with non-specific mitogens and superantigens, the evidence that Th1 cytokine production to allergens is decreased in line with a postulated imbalance in Th1/Th2 responses is unclear, with studies finding decreased, no difference or increased IFN-gamma responses to allergens in atopic subjects. To examine childhood polyclonal and allergen-induced cytokine responses in parallel to evaluate cytokine imbalances in childhood atopic disease. PBMC cytokine responses were examined in response to a polyclonal stimulus, staphylococcal superantigen (SEB), in parallel with two inhalant allergens, house dust mite (HDM) and rye grass pollen (RYE), and an ingested allergen, ovalbumin (OVA), in (a) 35 healthy children (non-atopic) and (b) 36 children with atopic disease (asthma, eczema and/or rhinitis) (atopic). Atopic children had significantly reduced IFN-gamma and increased IL-4 and IL-5 but not IL13 production to SEB superantigen stimulation when compared with non-atopic children. HDM and RYE allergens stimulated significantly increased IFN-gamma, IL-5 and IL-13, while OVA stimulated significantly increased IFN-gamma production in atopic children. We show that a polyclonal stimulus induces a reduced Th1 (IFN-gamma) and increased Th2 (IL-4 and IL-5) cytokine pattern. In contrast, the allergen-induced cytokine responses in atopic children were associated with both increased Th1 (INF-gamma) and Th2 (IL-5 and IL-13) cytokine production. The increased Th1 response to allergen is likely to reflect prior sensitization and indicates that increases in both Th1 and Th2 cytokine production to

Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage.

PubMed

Sisto, Margherita; Lisi, Sabrina; D'Amore, Massimo; De Lucro, Raffaella; Carati, Davide; Castellana, Donatello; La Pesa, Velia; Zuccarello, Vincenzo; Lofrumento, Dario D

2012-12-05

Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Local expression of vaginal Th1 and Th2 cytokines in murine vaginal candidiasis under different immunity conditions.

PubMed

Chen, Shanjuan; Li, Shaohua; Wu, Yan; Liu, Zhixiang; Li, Jiawen

2008-08-01

To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 (IL-2)/Th2 (IL-4, IL-10, TGF-beta1) cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-beta1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-beta1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans (C. albicans), and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.

Enhancement of ovalbumin-specific Th1, Th2, and Th17 immune responses by amorphous silica nanoparticles

PubMed Central

Toda, Tsuguto; Yoshino, Shin

2016-01-01

Nanomaterials present in cosmetics and food additives are used for industrial applications. However, their safety profile is unclear. Amorphous silica nanoparticles (nSPs) are a widely used nanomaterial and have been shown to induce inflammatory cytokines following intratracheal administration in mice. The current study investigated the adjuvant effect of nSP30 (nSP with a diameter of 33 nm) on T helper (Th)1, Th2, and Th17 immune responses as well as immunoglobulin (Ig) levels in mice. BALB/c mice were intraperitoneally administered ovalbumin (OVA) with or without varying doses and varying sizes of nSPs. The adjuvant effect of nSPs was investigated by measuring OVA-specific IgG antibodies in sera, OVA-specific proliferative responses of splenocytes, and the production of Th1, Th2, and Th17 cytokines. Aluminum hydroxide was used as a positive adjuvant control. Anti-OVA IgG production, splenocyte proliferative responses, and secretion of IFN-γ, IL-2, IL-4, IL-5, and IL-17 were increased significantly in mice receiving a combined injection of nSP30 (30 or 300 µg) with OVA compared with OVA alone or a combined injection with nSP30 (3 µg). The responses were nSP30 dose-dependent. When different sized nSPs were used (with 30, 100, and 1000 nm diameters), the responses to OVA were enhanced and were size-dependent. The smaller sized nSP particles had a greater adjuvant effect. nSPs appear to exert a size-dependent adjuvant effect for Th1, Th2, and Th17 immune responses. Understanding the mechanisms of nSP adjuvanticity might lead to the development of novel vaccine adjuvants and therapies for allergic diseases caused by environmental factors. PMID:27343242