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Sample records for keratinocyte proliferative potential

  1. Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential

    SciTech Connect

    Patterson, Timothy J.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2007-05-15

    Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear {beta}-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative {beta}-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by {beta}-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.

  2. Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells.

    PubMed

    Chorachoo, Julalak; Saeloh, Dennapa; Srichana, Teerapol; Amnuaikit, Thanaporn; Musthafa, Khadar Syed; Sretrirutchai, Somporn; Voravuthikunchai, Supayang P

    2016-02-01

    Psoriasis is a skin disease associated with hyperproliferation and abnormal differentiation of keratinocytes. Available approaches using synthetic drugs for the treatment of severe psoriasis may cause side effects. Alternatively, plant-derived compounds are now receiving much attention as alternative candidates for the treatment of psoriasis. In this study, the effects of rhodomyrtone, a bioactive plant extract isolated from Rhodomyrtus tomentosa leaves on the proliferation, growth arrest, and apoptosis of HaCaT keratinocytes were investigated. Percentage anti-proliferative activity of rhodomyrtone on HaCaT cells at concentrations of 2-32µg/ml after 24, 48, and 72h ranged from 13.62-61.61%, 50.59-80.16%, and 61.82-85.34%, respectively. In a scratch assay, rhodomyrtone at 2 and 4µg/ml significantly delayed closure of a wound by up to 61.78%, and 71.65%, respectively, after 24h incubation. HaCaT keratinocytes treated with rhodomyrtone showed chromatin condensation and fragmentation of nuclei when stained with Hoechst 33342. This indicated that rhodomyrtone induced apoptosis in the keratinocytes. In addition, flow cytometric analysis demonstrated an increase in the percentage of apoptosis of keratinocytes after treatment with rhodomyrtone at 2-32µg/ml from 1.2-10%, 8.2-35.4%, and 21.0-77.8% after 24, 48, and 72h, respectively, compared with the control. To further develop the compound as a potential anti-psoriasis agent, a rhodomyrtone formulation was prepared and subjected to skin irritation tests in rabbits. The formulation caused no skin irritation including such as erythema and edema. The results indicated that rhodomyrtone had the potential as a promising candidate for further development as a natural anti-psoriasis agent. PMID:26687635

  3. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen

    PubMed Central

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  4. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen.

    PubMed

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  5. Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.

    PubMed

    Hegemann, L; Bonnekoh, B; van Rooijen, L A; Mahrle, G

    1992-07-01

    Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action. PMID:1390454

  6. Inhibition of Inflammatory and Proliferative Responses of Human Keratinocytes Exposed to the Sesquiterpene Lactones Dehydrocostuslactone and Costunolide

    PubMed Central

    Scarponi, Claudia; Butturini, Elena; Sestito, Rosanna; Madonna, Stefania; Cavani, Andrea; Mariotto, Sofia; Albanesi, Cristina

    2014-01-01

    The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH)/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE) and costunolide (CS), two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit hyperproliferation

  7. The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes.

    PubMed

    Nagy, Nikoletta; Bata-Csörgo, Zsuzsanna; Kopasz, Norbert; Szeg, Csilla; Pivarcsi, Andor; Koreck, Andrea; Dobozy, Attila; Kemény, Lajos; Széll, Márta

    2006-08-01

    Keratinocyte growth factor receptor (KGFR = FGFR2-IIIb) is a tyrosine kinase receptor expressed by keratinocytes, which mediates the effects of fibroblast growth factors (FGF). There are contradictory data in the literature regarding the role of FGFR2-IIIb during the proliferation/differentiation programme of keratinocytes. In this study, we aimed to investigate whether overexpression of FGFR2-IIIb may have a role in the regulation of keratinocyte proliferation. We analysed the expression of FGFR2-IIIb in an in vitro HaCaT model system representing different stages of proliferation and differentiation of keratinocytes. Real-time RT-PCR and Western blot analyses demonstrated a correlation between FGFR2-IIIb mRNA and protein expression and the proportion of cells in S/G2/M phase in synchronized HaCaT keratinocytes and thus with proliferation activity (r = 0.96). After treatment with the antipsoriatic drug, dithranol, FGFR2-IIIb is downregulated dose dependently both at mRNA and protein levels. Moreover, when the rate of proliferation is decreased by the lack of cell attachment to the culturing surface, FGFR2-IIIb mRNA (P = 0.0315) and protein expressions were also reduced (P = 0.0242), while a differentiation marker, keratin 10, mRNA (P = 0.0003) and protein levels (P = 0.001) were increased (r = -0.92). Based on our results we conclude that FGFR2-IIIb expression in HaCaT keratinocytes corresponds with the proliferative activation of the cells and is not related to the differentiation programme. PMID:16842598

  8. Abnormalities in the basement membrane structure promote basal keratinocytes in the epidermis of hypertrophic scars to adopt a proliferative phenotype

    PubMed Central

    YANG, SHAOWEI; SUN, YEXIAO; GENG, ZHIJUN; MA, KUI; SUN, XIAOYAN; FU, XIAOBING

    2016-01-01

    The majority of studies on scar formation have mainly focused on the dermis and little is known of the involvement of the epidermis. Previous research has demonstrated that the scar tissue-derived keratinocytes are different from normal cells at both the genetic and cell biological levels; however, the mechanisms responsible for the fundamental abnormalities in keratinocytes during scar development remain elusive. For this purpose, in this study, we used normal, wound edge and hypertrophic scar tissue to examine the morphological changes which occur during epidermal regeneration as part of the wound healing process and found that the histological structure of hypertrophic scar tissues differed from that of normal skin, with a significant increase in epidermal thickness. Notably, staining of the basement membrane (BM) appeared to be absent in the scar tissues. Moreover, immunofluorescence staining for cytokeratin (CK)10, CK14, CK5, CK19 and integrin-β1 indicated the differential expression of cell markers in the epidermal keratinocytes among the normal, wound edge and hypertrophic scar tissues, which corresponded with the altered BM structures. By using a panel of proteins associated with BM components, we validated our hypothesis that the BM plays a significant role in regulating the cell fate decision of epidermal keratinocytes during skin wound healing. Alterations in the structure of the BM promote basal keratinocytes to adopt a proliferative phenotype both in vivo and in vitro. PMID:26986690

  9. Effect of new curcumin-containing nanostructured lipid dispersions on human keratinocytes proliferative responses.

    PubMed

    Esposito, Elisabetta; Sticozzi, Claudia; Ravani, Laura; Drechsler, Markus; Muresan, Ximena M; Cervellati, Franco; Cortesi, Rita; Valacchi, Giuseppe

    2015-06-01

    This study describes the production and characterization of nanostructured lipid dispersions (NLDs) containing curcumin (CUR) as new tools for curcumin topical delivery. Four types of NLDs based on monoolein in association with different emulsifiers were produced: Na cholate and poloxamer 407 (NLD1), poloxamer alone (NLD2), the mixture of Na cholate and Na caseinate (NLD3) and Na cholate alone (NLD4). Morphology and dimensional distribution of lipid dispersions were investigated by cryo-TEM and photon correlation spectroscopy (PCS). In vitro studies based on Franz cell, membrane nylon and stratum corneum-epidermis (SCE) were carried out to compare the four NLDs in terms of cytotoxicity in human keratinocytes and CUR diffusion. Our PCS studies showed differences in particles diameter among the different NLDs. In addition, cytotoxicity results in HaCaT cells evidenced that NLD1 and NLD2 were toxic at doses over 1 μm. Therefore, cryo-TEM was determined only for NLD3 and NLD4 showing that CUR did not affect their structure. Diffusion measurement in SCE and nylon membrane evidenced that CUR had a time-delayed release for NLD4. The 'wound healing' effect of NLD3 and NLD4 with and without CUR analysed keratinocytes in vitro, and a clear inhibition of cell proliferation/migration by CUR was observed. This effect was mediated by the inhibition of cyclin D1 expression as a consequence of the impaired NFkB activation. This study confirms the antiproliferative properties of CUR and evidenced a new possible model of CUR topical delivery for hyperproliferative cutaneous diseases such as psoriasis. PMID:25808217

  10. Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

    SciTech Connect

    Ku, W.W.; Bernstein, I.A. )

    1988-04-01

    The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for {alpha}-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for {alpha}-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca{sup 2+} medium exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. Estimation of DNA synthesis showed (a) a higher ({sup 3}H)thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate ({sup 3}H)thymidine into their nuclei. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells.

  11. Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes

    PubMed Central

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A.; Breton, Lionel; Ruderman, Neil B.

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol’s effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated

  12. Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A; Breton, Lionel; Ruderman, Neil B

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by

  13. Transient receptor potential vanilloid 4 (TRPV4) is downregulated in keratinocytes in human non-melanoma skin cancer.

    PubMed

    Fusi, Camilla; Materazzi, Serena; Minocci, Daiana; Maio, Vincenza; Oranges, Teresa; Massi, Daniela; Nassini, Romina

    2014-09-01

    A subgroup of the transient receptor potential (TRP) channels, including vanilloid 1 (TRPV1), TRPV2, TRPV3, TRPV4, and TRP ankyrin 1 (TRPA1), is expressed in cutaneous peptidergic somatosensory neurons, and has been found in skin non-neuronal cells, such as keratinocytes. Different cancer cells express TRPs, where they may exert either pro- or antitumorigenic roles. Expression and function of TRPs in skin cancers have been, however, poorly investigated. Here, we have studied the distribution and expression of TRPs by immunohistochemistry and messenger RNA (mRNA) in human healthy skin and human keratinocytic tumors, including intraepidermal proliferative disorders (solar keratosis (SK) and Bowen's disease), and non-melanoma skin cancer (NMSC; basal cell and squamous cell carcinomas). Similar TRPV1, TRPV2, and TRPV3 staining was found in keratinocytes from healthy and tumor tissues. TRPA1 staining was increased solely in SK samples. However, the marked TRPV4 staining and TRPV4 mRNA expression, observed in healthy or inflamed skin, was abrogated both in premalignant lesions and NMSC. In a human keratinocyte cell line (HaCaT), TRPV4 stimulation released IL-8, which in turn downregulated TRPV4 expression. Selective reduction in TRPV4 expression could represent an early biomarker of skin carcinogenesis. Whether the cytokine-dependent, autocrine pathway that results in TRPV4 downregulation contributes to NMSC mechanism remains to be determined. PMID:24643128

  14. Potentiation of lymphocyte proliferative responses by nickel sulfide

    NASA Technical Reports Server (NTRS)

    Jaramillo, A.; Sonnenfeld, G.

    1992-01-01

    Crystalline nickel sulfide (NiS) induced a spleen cell proliferation that resembles a mixed lymphocyte reaction (MLR). It depended on cell-cell interaction, induced high levels of interleukin-1 (IL-1) and interleukin-2 (IL-2) and the responding cell subpopulation was composed of CD4+ T lymphocytes. Furthermore, the proliferation was inhibited in a dose-dependent manner by magnesium. Crystalline NiS also increased significantly the spleen cell proliferative response to concanavalin A (Con A) and lipopolysaccharide (LPS) with magnesium potentiating the combined effects of crystalline NiS and mitogens. Interestingly, crystalline NiS did not show any effect on the induction of IL-2 by Con A. The results described herein suggest that crystalline NiS can potentiate both antigenic (MLR) and mitogenic (Con A and LPS) proliferative responses in vitro. Crystalline NiS appears to potentiate these responses by acting in the form of ionic nickel on several intracellular targets for which magnesium ions have different noncompetitive interactions. The effects of magnesium on the potentiating action of crystalline NiS are different depending upon the type of primary stimulatory signal for proliferation (mitogenic or antigenic).

  15. Heterogeneity in proliferative potential of ovine mesenchymal stem cell colonies.

    PubMed

    Rhodes, N P; Srivastava, J K; Smith, R F; Longinotti, C

    2004-04-01

    Bone marrow biopsies were taken from the iliac crest of 28 individual sheep from three different breeds, ranging in age from 4 months to 8 years and mesenchymal stem cells (MSCs) isolated using selection due to plastic adherence. Cells were cultured in medium that had been selected for its effect on observed MSC proliferation, until populations of greater than 50 million had been obtained from each biopsy. The identity of the isolated cell populations as progenitors of the mesenchymal lineage was verified by deriving both osteoblastic and chondrocytic phenotypes when cultured in osteogenic and chondrogenic medium supplements, respectively. The rate of cell proliferation for each marrow biopsy was measured at each passage and the number of initial stem cells in each sample estimated. There was no statistically significant correlation between the age of the sheep and MSC proliferative potential, or age and estimated initial MSC number. There was no apparent significant difference between proliferation rate and sheep breed and colonies established from frozen cells grew at similar rates to pre-frozen cells. Counter intuitively, there appeared to be a negatively correlated trend between proliferation rate and MSC concentration in the samples. It is concluded that no initial descriptive statistics of the marrow biopsies can assist in estimating the proliferative potential, and therefore the timing of future surgeries, of MSCs sampled for the purposes of tissue engineering. PMID:15332606

  16. Potential applications of keratinocytes derived from human embryonic stem cells.

    PubMed

    Movahednia, Mohammad M; Kidwai, Fahad K; Jokhun, Doorgesh S; Squier, Christopher A; Toh, Wei Seong; Cao, Tong

    2016-01-01

    Although skin grafting is one of the most advanced cell therapy technique, wide application of skin substitutes is hampered by the difficulty in securing sufficient amount of epidermal substitute. Additionally, in understanding the progression of skin aging and disease, and in screening the cosmetic and pharmaceutical products, there is lack of a satisfactory human skin-specific in vitro model. Recently, human embryonic stem cells (hESCs) have been proposed as an unlimited and reliable cell source to obtain almost all cell types present in the human body. This review focuses on the potential off-the-shelf use of hESC-derived keratinocytes for future clinical applications as well as a powerful in vitro skin model to study skin function and integrity, host-pathogen interactions and disease pathogenesis. Furthermore, we discuss the industrial applications of hESC-derived keratinized multi-layer epithelium which provides a human-like test platform for understanding disease pathogenesis, evaluation of new therapeutic modalities and assessment of the safety and efficacy of skin cosmetics and therapeutics. Overall, we conclude that the hESC-derived keratinocytes have great potential for clinical, research and industrial applications. PMID:26663861

  17. Xenobiotics in vitro: the influence of L-cystine, pantothenat, and miliacin on metabolic and proliferative capacity of keratinocytes.

    PubMed

    Obrigkeit, D Hoeller; Oepen, T; Jugert, F K; Merk, H F; Kubicki, J

    2006-01-01

    To investigate the effect of cell growth-stimulating agents on human epidermal keratinocytes, we exposed monolayers of normal human keratinocytes derived from foreskin to different concentrations of the amino acid L-cystine, the member of the vitamin B family D-pantothenat, the phytosterol miliacin, and a combination thereof in keratinocyte growth medium. As a test system for the metabolic capacity, we used the activity of mitochondrial deyhdrogenases as measured by XTT, and for the cell proliferation, we determined the BrdU-uptake. The additives, active ingredients of the hair growth drug PRIORIN, were added in the presence of fully supplemented keratinocyte growth medium or a deficient medium without L-cystine, L-methionine, L-histidin, D-pantothenat, epidermal growth factor, and bovine pituary gland extract. Deficient medium itself reduced the metabolic capacity of keratinocytes to 35% compared with keratinocytes in fully supplemented growth medium. In deficient medium cell, proliferation was not measurable. Increasing doses of L-cystine restored the reduced metabolic capacity from 46% (0.009 mg/L) to 54% (0.09 mg/L) and 92% (0.45 mg/L) in deficient medium. Addition of D-pantothenat (0.43 mg/L) enhanced the metabolic capacity to 150% only in fully supplemented growth medium, compared with untreated controls with growth medium. Miliacin (6 mg/mL) increased not only the metabolic capacity (162%) but also stimulated cell proliferation (215%) as measured by BrdU-uptake in growth medium. The combination of all three additives increased the metabolic capacity (245%) synergistically in growth medium. We were able to show effects of D-panthenol, L-lysine, and miliacin on proliferation and metabolic capacity of keratinocyte monocell culture, which was further increased by combination of the three substances. These basic results suggest a beneficial effect on keratinocyte growth and stimulation by products combining these substances (e.g., Priorin). Furthermore, this work

  18. Proliferative Defects in Dyskeratosis Congenita Skin Keratinocytes are Corrected by Expression of the Telomerase Reverse Transcriptase, TERT, or by Activation of Endogenous Telomerase through Expression of Papillomavirus E6/E7 or the Telomerase RNA Component, TERC

    PubMed Central

    Gourronc, Francoise A.; Robertson, Mckaylee M.; Herrig, Annie K.; Lansdorp, Peter M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2010-01-01

    Dyskeratosis congenita (DC) is characterized by the triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Epidermal atrophy, hair growth defects, bone marrow failure, and increased risk of cancer are also common in DC patients. DC is caused by mutations in genes encoding for telomerase complex factors. Although there is an association of epidermal abnormalities with DC, epidermal cells from DC donors have not been previously characterized. We have isolated skin keratinocytes from affected members of a family with an autosomal dominant form of DC that is due to a mutation in the RNA component of telomerase, TERC. Here we demonstrate that, similar to DC fibroblasts from these donors, DC keratinocytes have short telomeres and a short lifespan. DC keratinocytes also exhibited impaired colony forming efficiency and migration capacity. Exogenous expression of the reverse transcriptase component of telomerase, TERT, activated telomerase levels to half that of TERT expressing normal cells and maintained telomeres at a short length with concomitant extension of lifespan. Unlike fibroblasts, transduction of human papillomavirus type 16 E6/E7 genes into DC keratinocytes activated telomerase to half that of E6/E7 expressing normal cells, and robust proliferation was observed. While expression of TERC has no measurable effect on telomerase in fibroblasts, expression of TERC in keratinocytes upregulated telomerase activity and, rarely, allowed rescue of proliferative defects. Our results point to important differences between DC fibroblasts and keratinocytes and show, for the first time, that expression of TERC can increase the lifespan of primary human epithelial cells. PMID:19558498

  19. Brm Inhibits the Proliferative Response of Keratinocytes and Corneal Epithelial Cells to Ultraviolet Radiation-Induced Damage

    PubMed Central

    Hassan, Nur Mohammad Monsur; Painter, Nicole; Howlett, C. Rolfe; Farrell, Andrew W.; Di Girolamo, Nick; Lyons, J. Guy; Halliday, Gary M.

    2014-01-01

    Ultraviolet radiation (UV) from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM) is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm−/− mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm−/− mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm−/−, Trp53+/−, and particularly the Brm−/− Trp53+/− mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm−/− Trp53+/+ and Brm−/− Trp53+/− mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm−/− mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis. PMID:25254962

  20. Evaluation of the skin sensitization potential of chemicals in THP-1/keratinocyte co-cultures.

    PubMed

    Cao, Yu-Ping; Ma, Peng-Cheng; Liu, Wei-Da; Zhou, Wu-Qing; Tao, Yue; Zhang, Meng-Li; Li, Ling-Jun; Chen, Zi-Yi

    2012-04-01

    Many attempts have been made to develop in vitro sensitization tests that employ dendritic cells (DCs), DC-like cell lines or keratinocytes. The aim of the present investigation was to establish a co-culture of THP-1 cells and keratinocytes for evaluation of skin sensitization potential of chemicals. Co-cultures were constructed by THP-1 cells cultured in lower compartments and keratinocytes cultured in upper compartments of cell culture inserts. After 24 h exposure to sensitizers (2, 4-dinitrochlorobenzene, p-phenylenediamine, formaldehyde, nickel sulfate, isoeugenol and eugenol) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride and lactic acid), the expression of CD86 and CD54 on THP-1 cells were evaluated by flow cytometry, and cell viabilities were determined. The sensitizers induced the augmentation of CD86 and CD54 expression, but the non-sensitizers had no significant effect. Compared with mono-cultures of THP-1 cells, the augmentation of CD86 and CD54 could be detected even at a non-toxic concentration of sensitizers in THP-1 cell/keratinocyte co-cultures. Moreover, isoeugenol was distinguished as a sensitizer in co-cultures, but failed to be identified in mono-cultures. These results revealed that the co-cultures of THP-1 cells and keratinocytes were successfully established and suitable for identifying sensitizers using CD86 and CD54 expression as identification markers. PMID:21721923

  1. Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

    PubMed Central

    Muiños-López, Emma; Rendal-Vázquez, Mª Esther; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

    2012-01-01

    Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes. PMID:22523526

  2. Antiproliferative potential of zidovudine in human keratinocyte cultures.

    PubMed

    Bonnekoh, B; Wevers, A; Geisel, J; Rasokat, H; Mahrle, G

    1991-09-01

    Because the beneficial effects of zidovudine in human immunodeficiency virus infection-associated psoriasis have recently been observed, this study focused on the drug's action on the rapidly proliferating human HaCaT keratinocyte line as an in vitro model for epidermal hyperproliferation. Cultures in log growth phase were exposed to zidovudine for 2 days. Zidovudine slowed proliferation in a dose-dependent fashion as evidenced by 50% inhibition concentrations of 33 mumol/L (cell number), 30 mumol/L (protein content), 0.9 mumol/L (protein synthesis), and 0.7 mumol/L (DNA synthesis). Significant (p less than 0.01) reduction of cell viability to 94.6% and 87.2%, as well as morphologic manifestations of cytotoxicity, were first evident after 2 days' exposure to maximal drug concentrations of 10 and 100 mumol/L, respectively. Control viability, assayed by trypan blue exclusion, was 98.0%. Direct cytotoxic plasma membrane injury could be ruled out by the absence of any increase in cytoplasmic lactate dehydrogenase release into supernatants at least during the 1 day of maximal dosage exposure. The drug-induced inhibition of proliferation was reversible within 7 days after a 2-day exposure to 100 mumol/L zidovudine. Two days of treatment with a 10 mumol/L dose did not alter the pattern and synthesis of keratins in vitro. Thus the known antipsoriatic efficacy of zidovudine might be explained, at least partly, by the drug's cytostatic potency. PMID:1918488

  3. Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro.

    PubMed

    Gönczi, Mónika; Szentandrássy, Norbert; Fülöp, László; Telek, Andrea; Szigeti, Gyula P; Magyar, János; Bíró, Tamás; Nánási, Péter P; Csernoch, László

    2007-04-01

    Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases. PMID:17359336

  4. [Importance of proliferative potential (as the ratio of a proliferative cells number and duration of mitosis) in diagnoses of malignant degree and prognosis of adrenocortical cancer].

    PubMed

    Raĭkhlin, N T; Bukaeva, I A; Filimoniuk, A V; Smirnova, E A; Probatova, N A; Pavlovskaia, A I; Shabanov, M A; Ponomareva, M V

    2011-01-01

    The aim of research has been the estimation of a proliferative potential as simultaneous detection of a proliferative cells number (Ki-67 index) and duration of mitosis (nucleolar argyrophilic protein expression--B23/nucleophosmin and C23/nucleolin) at patients with adrenocortical cancer. In according to lifetime of patients after operation 2 groups had been sorted out. The first one included patients surviving 56.12 months, the second one--9.25 months. We've found out that different aspects of tumor diagnosis as well distinction of benignant or malignant tumor growth, a malignant degree of tumors, a prognostic criteria of illness, survival of patients etc. must be characterized by total research both a proliferative cells fraction (Ki-67 index) and a rate of mitosis (expressions of B23/nucleophosmin and C23/nucleolin). PMID:22288173

  5. Cytotoxic T lymphocytes as a potential brake of keratinocyte proliferation in psoriasis.

    PubMed

    Vičić, Marijana; Peternel, Sandra; Simonić, Edita; Sotošek-Tokmadžić, Vlatka; Massari, Dražen; Brajac, Ines; Kaštelan, Marija; Prpić-Massari, Larisa

    2016-02-01

    Psoriasis is a chronic papulosquamous skin disease, histologically characterized by epidermal hyperproliferation and dermal infiltration of inflammatory cells. The majority of T lymphocytes infiltrating dermis are CD4+ T lymphocytes secreting type 1 and type 17 cytokines. These cytokines are responsible for triggering keratinocyte proliferation as well as chemokine secretion and subsequent migration of other inflammatory cells in the skin. Contrarily, lymphocytes that accumulate in epidermis are mainly CD8+ T lymphocytes. According to the recent findings, these cells can also secrete type 1 and type 17 cytokines. However, it is demonstrated so far that epidermal CD8+ T lymphocytes contain higher amounts of cytolytic molecules, such as perforin, granzyme B and granulysin whose role in psoriasis pathogenesis is still unknown. Therefore, in this article we hypothesize the active involvement of cell mediated cytotoxicity in killing the proliferating keratinocytes as a mechanism of potential self-defense and possible brake in psoriatic plaque formation, maintaining skin homeostasis. PMID:26826643

  6. Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage

    PubMed Central

    Fehrenbach, Sabrina; Novak, Daniel; Bernhardt, Mathias; Larribere, Lionel; Boukamp, Petra; Umansky, Viktor; Utikal, Jochen

    2016-01-01

    Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy. PMID:27387763

  7. Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.

    PubMed

    Fehrenbach, Sabrina; Novak, Daniel; Bernhardt, Mathias; Larribere, Lionel; Boukamp, Petra; Umansky, Viktor; Utikal, Jochen

    2016-01-01

    Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy. PMID:27387763

  8. 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment

    PubMed Central

    Mok, Chong-Fai; Xie, Chuan-Ming; Sham, Kathy Wai-Yan; Lin, Zhi-Xiu; Cheng, Christopher Hon-Ki

    2013-01-01

    Psoriasis, which affects approximately 1–3% of the population worldwide, is a chronic inflammatory skin disorder characterized by epidermal keratinocytes hyperproliferation, abnormal differentiation, and inflammatory infiltration. Decrease in keratinocyte apoptosis is a specific pathogenic phenomenon in psoriasis. Chinese herbs have been used for the treatment of psoriasis in China showing promising effect in clinical trials. A traditional Chinese medicine has relatively fewer side effects with longer remission time and lower recurrence rate. The extract of Rubia cordifolia L. (EA) was previously found by us to induce HaCaT keratinocytes apoptosis. In this study we identified one of the components in Rubia cordifolia L., the anthraquinone precursor 1,4-dihydroxy-2-naphthoic acid (DHNA), induces HaCaT keratinocytes apoptosis through G0/G1 cell cycle arrest. We have also demonstrated that DHNA acts through both caspase-dependent and caspase-independent pathways. Besides, cytotoxicity and IL-1α release assays indicate that DHNA causes less irritation problems than dithranol, which is commonly employed to treat psoriasis in many countries. Since DHNA possesses similar apoptotic effects on keratinocytes as dithranol but causes less irritation, DHNA therefore constitutes a promising alternative agent for treating psoriasis. Our studies also provide an insight on the potential of using EA and DHNA, alternatively, as a safe and effective treatment modality for psoriasis. PMID:23690852

  9. Transient receptor potential vanilloid 4 (TRPV4)-dependent calcium influx and ATP release in mouse oesophageal keratinocytes

    PubMed Central

    Mihara, Hiroshi; Boudaka, Ammar; Sugiyama, Toshiro; Moriyama, Yoshinori; Tominaga, Makoto

    2011-01-01

    Abstract Gastro-oesophageal reflux disease (GERD) is a multi-factorial disease that may involve oesophageal hypersensitivity to mechanical or heat stimulus as well as acids. Intraganglionic laminar endings (IGLEs) are the most prominent terminal structures of oesophageal vagal mechanosensitive afferents and may modulate mechanotransduction via purinergic receptors. Transient receptor potential channel vanilloid 4 (TRPV4) can detect various stimuli such as warm temperature, stretch and some chemicals, including 4α-phorbol 12,13-didecanoate (4α-PDD) and GSK1016790A. TRPV4 is expressed in many tissues, including renal epithelium, skin keratinocytes and urinary bladder epithelium, but its expression and function in the oesophagus is poorly understood. Here, we show anatomical and functional TRPV4 expression in mouse oesophagus and its involvement in ATP release. TRPV4 mRNA and protein were detected in oesophageal keratinocytes. Several known TRPV4 activators (chemicals, heat and stretch stimulus) increased cytosolic Ca2+ concentrations in cultured WT keratinocytes but not in TRPV4 knockout (KO) cells. Moreover, the TRPV4 agonist GSK1016790A and heat stimulus evoked TRPV4-like current responses in isolated WT keratinocytes, but not in TRPV4KO cells. GSK1016790A and heat stimulus also significantly increased ATP release from WT oesophageal keratinocytes compared to TRPV4KO cells. The vesicle-trafficking inhibitor brefeldin A (BFA) inhibited the ATP release. This ATP release could be mediated by the newly identified vesicle ATP transporter, VNUT, which is expressed by oesophageal keratinocytes at the mRNA and protein levels. In conclusion, in response to heat, chemical and possibly mechanical stimuli, TRPV4 contributes to ATP release in the oesophagus. Thus, TRPV4 could be involved in oesophageal mechano- and heat hypersensitivity. PMID:21540339

  10. Effects of Cryopreservation on the Cell Viability, Proliferative Capacity and Neuronal Differentiation Potential of Canine Bone Marrow Stromal Cells

    PubMed Central

    EDAMURA, Kazuya; NAKANO, Rei; FUJIMOTO, Kyohei; TESHIMA, Kenji; ASANO, Kazushi; TANAKA, Shigeo

    2013-01-01

    ABSTRACT We investigated the cell viability, proliferative capacity and neuronal differentiation potential of canine bone marrow stromal cells (BMSCs) after cryopreservation. BMSCs were cryopreserved using cryoprotectant solutions with 10% DMSO and 10% FBS (DF group) or without DMSO and FBS (DF-free group); fresh BMSCs were used as a control. The cell viability and proliferative capacity of BMSCs were similar in the DF-free and control groups, while those in the DF group were lower. In all groups, BMSCs differentiated into neuron-like cells that stained positive against neuron markers, and the mRNA expression levels of neuron markers increased after neuronal induction. In conclusion, cryopreservation with DF-free cryoprotectant solution did not diminish the cell viability, proliferative capacity or neuronal differentiation potential of canine BMSCs. PMID:24334862

  11. Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells

    PubMed Central

    Kokubu, Chikara; Yusa, Kosuke; Horie, Kyoji; Yoshimura, Yasuhide; Yamauchi, Kaori; Suemori, Hirofumi; Yokozeki, Hiroo; Toyoda, Masashi; Kiyokawa, Nobutaka; Okita, Hajime; Miyagawa, Yoshitaka; Akutsu, Hidenori; Umezawa, Akihiro; Katayama, Ichiro

    2014-01-01

    Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes. PMID:25024429

  12. Defective proliferative potential of MSCs from pediatric myelodysplastic syndrome patients is associated with cell senescence

    PubMed Central

    Liu, Qinghua; Zhu, Hongbo; Dong, Jing; Li, Helou; Zhang, Hong

    2015-01-01

    Objectives: Aberrant MSC function was shown to contribute to the pathophysiology of myelodysplastic syndromSe (MDS). In comparison to adult MDS, pediatric MDS displayed different features both in biologically and clinically. The mechanisms for adult MDS may not be applicable in pediatric MDS. However, understanding of the MSCs in pediatric MDS is lacking. In this study, we investigated the proliferation capacity of MSCs from pediatric MDS patients at clone cell level. Material and methods: Clone bone marrow MSCs were isolated from pediatric MDS patients and identified according to the criteria of the International Society for Cellular Therapy for MSCs. The proliferation capacity of pediatric MDS-derived MSCs was compared to healthy controls. Cell cycle was detected by flow cytometry following PI staining, as well as cell senescence was evaluated by β-galactosidase staining and telomere length. Results: Pediatric MDS-derived MSCs displayed similar basic biology characters as MSCs from healthy controls, including differentiation potential and surface markers. However, defective proliferative was displayed by pediatric MDS-derived MSCs. Pediatric MDS-derived MSCs were more prone to cellular senescence than healthy controls, and showed a decrease in the S phase. Conclusion: Pediatric MDS-derived MSCs possess the basic characteristics of normal MSCs, but display defective proliferation, which may be associated with cell senescence. PMID:26722501

  13. Toxicity of tannic acid-modified silver nanoparticles in keratinocytes: potential for immunomodulatory applications.

    PubMed

    Orlowski, Piotr; Soliwoda, Katarzyna; Tomaszewska, Emilia; Bien, Karolina; Fruba, Aleksandra; Gniadek, Marianna; Labedz, Olga; Nowak, Zuzanna; Celichowski, Grzegorz; Grobelny, Jarosław; Krzyzowska, Malgorzata

    2016-09-01

    Hydrolyzable tannins are known to exhibit anti-inflammatory activity, which can be used in combination with silver nanoparticles (AgNPs) for dermal uses. In this study, we investigated the effects of tannic acid-modified 13, 33, 46nm and unmodified 10-65nm AgNPs using the human-derived keratinocyte HaCaT and VK2-E6/E7 cell lines in the form of stationary and spheroids cultures. After exposition to tannic acid-modified AgNPs, VK2-E6/E7 cells showed higher toxicity, increased production of reactive oxygen species (ROS) and activity of JNK stress kinase, while HaCaT cell line demonstrated less ROS production and activation of ERK kinase. AgNPs internalization was detected both in the superficial and internal layers of spheroids prepared from both cell lines. Tannic acid modified AgNPs sized above 30nm did not induce DNA breaks in comet assay performed in both cell lines. Tannic acid-modified but not unmodified AgNPs down-regulated TNF-α and LPS-triggered production of IL-8 in VK2-E6/E7 but not in HaCaT cells. In summary, tannic acid-modified AgNPs sized above 30nm show good toxicological profile both in vitro and possess immunomodulatory properties useful for potential dermal applications in humans. PMID:27216470

  14. Transient Receptor Potential Vanilloid-1 in Epidermal Keratinocytes May Contribute to Acute Pain in Herpes Zoster.

    PubMed

    Han, Sang Bum; Kim, Hyeree; Cho, Sang Hyun; Lee, Jeong Deuk; Chung, Jin Ho; Kim, Hei Sung

    2016-03-01

    The role of transient receptor potential vanilloid-1 (TRPV1) in the initiation of neurogenic inflammation and transduction of pain is well established. In this study 33 patients with herpes zoster (HZ) were recruited from a single centre and underwent a questionnaire interview at their first visit. Punch biopsies from the HZ lesions and the contralateral unaffected skin were performed to localize and quantify the expression of TRPV1. Immunofluorescent staining for TRPV1 was most prominent in the epidermal keratinocytes. Both TRPV1 mRNA and protein levels were significantly higher in the HZ epidermis than in control epidermis (relative ratio 1.62 ± 0.27, p = 0.033 and 2.55 ± 0.51, p = 0.005, respectively). Protein TRPV1 ratio (HZ lesion/control) correlated with the degree of pain (measured on a visual analogue scale; VAS) (p = 0.017) and was significantly lower in patients who had taken either HZ medication or painkillers prior to their visit. These results suggest that non-neuronal TRPV1 may contribute to acute pain in herpes zoster. PMID:26390894

  15. Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential

    PubMed Central

    Huang, Enyi; Bi, Yang; Jiang, Wei; Luo, Xiaoji; Yang, Ke; Gao, Jian-Li; Gao, Yanhong; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Liu, Xing; Li, Mi; Hu, Ning; Liu, Hong; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Shen, Jikun; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Luo, Jinyong; He, Bai-Cheng; Wang, Huicong; Reid, Russell R.; Luu, Hue H.; Haydon, Rex C.; Yang, Li; He, Tong-Chuan

    2012-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications. PMID:22384246

  16. Human keratinocyte growth and differentiation on acellular porcine dermal matrix in relation to wound healing potential.

    PubMed

    Zajicek, Robert; Mandys, Vaclav; Mestak, Ondrej; Sevcik, Jan; Königova, Radana; Matouskova, Eva

    2012-01-01

    A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing. PMID:22629190

  17. MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases

    PubMed Central

    Heishima, Kazuki; Mori, Takashi; Ichikawa, Yukie; Sakai, Hiroki; Kuranaga, Yuki; Nakagawa, Takayuki; Tanaka, Yuiko; Okamura, Yasuhiko; Masuzawa, Mikio; Sugito, Nobuhiko; Murakami, Mami; Yamada, Nami; Akao, Yukihiro; Maruo, Kohji

    2015-01-01

    Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma. PMID:26512652

  18. Effects of progestins of human proliferative endometrium: an in vitro model of potential clinical relevance.

    PubMed

    Illouz, Séverine; Dales, Jean-Philippe; Sferlazzo, Karine; Garcia, Stéphane; Carpentier-Meunier, Séverine; Boubli, Léon; Lavaut, Marie-Noelle; Charpin, Colette

    2003-10-01

    Endometrium biopsy is a useful indicator of endometrium proliferation and is clinically relevant to diagnose cell proliferation and to evaluate response to progestin treatment and to monitor hormone replacement therapy. The aim of our study was to investigate the in vitro effects of progesterone and synthetic progestins on endometrium explants with a particular focus on estradiol receptor (ER) and progesterone receptor (PR) expression which reflects through cell secretion the hormone treatment efficiency. Most widely used progestagens belonging to three distinctive groups were investigated, i.e, medroxyprogesterone acetate (MPA), norethindrone acetate (NOR) and nomegestrol acetate (TX) which are respectively pregnane, 19-nortestosterone and norpregnane derivatives. We used organ culture from human proliferative endometrium, in which tissue integrity, particularly gland/stroma relationships are preserved. Progestins induce epithelial cell secretion and most effects were observed at the highest concentration tested (10(-7) M) and by TX and MPA on homogeneous and on heterogeneous (including also secretory glands) proliferative endometrium respectively. In these conditions, ER as well as PR expression were decreased on both glandular and stromal cells. In contrast, progesterone at 10(-7) M significantly decreased only PR, in glands and in stroma of homogeneous proliferative endometrium, and just in stroma of heterogeneous endometrium. NOR exhibited less effects. At lower concentrations (10(-8) M, 10(-9) M), significantly less effects were observed by synthetic progestins on proliferative endometrium. The experiments show that the different types of progestins do not exhibit in vitro similar effects. Since progestins variably act on proliferative endometrium, the exposure of endometrium explants to progestins may be a useful tool to predict clinical response to hormone therapy (individual "hormonogram") and to monitor endometrium proliferation. PMID:12964029

  19. Canine placenta: A promising potential source of highly proliferative and immunomodulatory mesenchymal stromal cells?

    PubMed

    Saulnier, Nathalie; Loriau, Julia; Febre, Marine; Robert, Clément; Rakic, Rodolphe; Bonte, Tancrède; Buff, Samuel; Maddens, Stéphane

    2016-03-01

    In veterinary medicine, therapeutic mesenchymal stromal cells (MSC) have been traditionally isolated from adult bone marrow or adipose tissue. Neonatal tissues, normally discarded at birth from all species have become an alternative source of cells for regenerative medicine in the human clinic. These cells have been described as being more primitive, proliferative and immunosuppressive than their adult counterparts. Our objective was to examine if this phenomena holds true in dogs. Little information exists regarding canine neonatal MSC characterisation. In this study, we were able to both isolate, phenotype and assess the differentiation and immunomodulatory properties of MSC from canine foetal adnexa allowing us to compare their characteristics to their more well-known bone marrow (BM) cousins. Neonatal tissues, including amnion (AM), placenta (PL), and umbilical cord matrix (UCM) were collected from 6 canine caesarean sections. Primary cells were expanded in vitro for 5 consecutive passages and their proliferation measured. BM-MSC were isolated from 5 control dogs euthanised from other studies and grown in vitro using an identical protocol. All MSC lines were systematically evaluated for their ability to differentiate into 3 mesodermal lineages (adipocyte, osteocyte and chondrocyte) and phenotyped by cytometry and qPCR. In addition, the enzymatic activity of the key immunomodulatory marker indoleamine 2,3-dioxygenase (IDO) was evaluated for each MSC line. MSC displaying a fibroblastic appearance were successfully grown from all neonatal tissues. PL-MSC exhibited significantly higher proliferation rates than AM- and UCM-MSC (p=0.05). Cytometric analysis showed that all MSC express CD90, CD29, and CD44, while no expression of CD45, CD34 and MHC2 was detected. Molecular profiling showed expression of CD105 and CD73 in all MSC. Low levels of SOX2 mRNA was observed in all MSC, while neither NANOG, nor OCT4 were detected. All MSC differentiate into 3 mesodermal

  20. The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells

    PubMed Central

    Suzuki, Daisuke; Sahu, Raju; Leu, N. Adrian; Senoo, Makoto

    2015-01-01

    The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21Waf1/Cip1 expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function. PMID:25503409

  1. Effects of artemether on the proliferation, apoptosis, and differentiation of keratinocytes: potential application for psoriasis treatment

    PubMed Central

    Wu, Jie; Li, Hong; Li, Ming

    2015-01-01

    Artemether exhibits diverse pharmacological effects and has multiple applications. This study aimed to investigate its antiproliferative and apoptogenic effects on HaCaT cells and keratinocyte differentiation-inducing activity in vivo. WST-8 analysis demonstrated that Artemether can inhibit the proliferation of cultured HaCaT cells in a time- and dose-dependent manner. Annexin V/PI dual staining and JC-1 staining further revealed that Artemether can dose-dependently augment HaCaT apoptosis. To investigate the keratinocyte differentiation-inducing activity of Artemether, it was prepared as topical creams at concentrations of 1%, 3%, and 5%. During the 4 weeks of topical treatment, no evidence of irritation was observed in the mouse tail test. Artemether cream dose-dependently increased the degree of orthokeratosis and the relative epidermal thickness of mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present study suggests that Artemether may be a promising antipsoriatic agent worthy of further investigation. PMID:26221244

  2. Potential treatment of inflammatory and proliferative diseases by ultra-low doses of ionizing radiations.

    PubMed

    Sanders, Charles L

    2012-12-01

    Ultra-low doses and dose- rates of ionizing radiation are effective in preventing disease which suggests that they also may be effective in treating disease. Limited experimental and anecdotal evidence indicates that low radiation doses from radon in mines and spas, thorium-bearing monazite sands and enhanced radioactive uranium ore obtained from a natural geological reactor may be useful in treating many inflammatory conditions and proliferative disorders, including cancer. Optimal therapeutic applications were identified via a literature survey as dose-rates ranging from 7 to 11μGy/hr or 28 to 44 times world average background rates. Rocks from an abandoned uranium mine in Utah were considered for therapeutic application and were examined by γ-ray and laser-induced breakdown fluorescence spectroscopy. The rocks showed the presence of transuranics and fission products with a γ-ray energy profile similar to aged spent uranium nuclear fuel (93% dose due to β particles and 7% due to γ rays). Mud packs of pulverized uranium ore rock dust in sealed plastic bags delivering bag surface β,γ dose-rates of 10-450 μGy/h were used with apparent success to treat several inflammatory and proliferative conditions in humans. PMID:23304108

  3. Potential Treatment of Inflammatory and Proliferative Diseases by Ultra-Low Doses of Ionizing Radiations

    PubMed Central

    Sanders, Charles L.

    2012-01-01

    Ultra-low doses and dose- rates of ionizing radiation are effective in preventing disease which suggests that they also may be effective in treating disease. Limited experimental and anecdotal evidence indicates that low radiation doses from radon in mines and spas, thorium-bearing monazite sands and enhanced radioactive uranium ore obtained from a natural geological reactor may be useful in treating many inflammatory conditions and proliferative disorders, including cancer. Optimal therapeutic applications were identified via a literature survey as dose-rates ranging from 7 to 11μGy/hr or 28 to 44 times world average background rates. Rocks from an abandoned uranium mine in Utah were considered for therapeutic application and were examined by γ-ray and laser-induced breakdown fluorescence spectroscopy. The rocks showed the presence of transuranics and fission products with a γ-ray energy profile similar to aged spent uranium nuclear fuel (93% dose due to β particles and 7% due to γ rays). Mud packs of pulverized uranium ore rock dust in sealed plastic bags delivering bag surface β,γ dose-rates of 10–450 μGy/h were used with apparent success to treat several inflammatory and proliferative conditions in humans. PMID:23304108

  4. A Two-Stepped Culture Method for Efficient Production of Trichogenic Keratinocytes.

    PubMed

    Chan, Chih-Chieh; Fan, Sabrina Mai-Yi; Wang, Wei-Hung; Mu, Yi-Fen; Lin, Sung-Jan

    2015-10-01

    Successful hair follicle (HF) neogenesis in adult life depends on the existence of both capable dermal cells and competent epidermal keratinocytes that recapitulate embryonic organogenesis through epithelial-mesenchymal interaction. In tissue engineering, the maintenance of trichogenic potential of adult epidermal cells, while expanding them remains a challenging issue. We found that although HF outer root sheath keratinocytes could be expanded for more than 100 passages as clonogenic cells without losing the proliferative potential with a 3T3J2 fibroblast feeder layer, these keratinocytes were unable to form new HFs when combined with inductive HF dermal papilla (DP) cells. However, when these high-passage keratinocytes were cocultured with HF DP cells for 4 days in vitro, they regained the trichogenic ability to form new HFs after transplantation. We found that the short-term coculture with DP cells enhanced both Wnt/β-catenin signaling, a signaling cascade key to HF development, and upregulated the expression of HF-specific genes, including K6, K16, K17, and K75, in keratinocytes, indicating that these cells were poised toward a HF fate. Hence, efficient production of trichogenic keratinocytes can be obtained by a two-stepped procedure with initial cell expansion with a 3T3J2 fibroblast feeder followed by short-term coculture with DP cells. PMID:25951188

  5. Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines.

    PubMed

    Zanatta, C F; Mitjans, M; Urgatondo, V; Rocha-Filho, P A; Vinardell, M P

    2010-01-01

    Considering the belief that natural lipids are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, we have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect against UVA and UVB irradiation on keratinocytes and fibroblasts. Cell treatment was divided into two steps, prior and after exposition to 30 min of UVA plus UVB radiation or to 60 min of UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing alpha-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the two cell lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection, especially in after sun formulations. PMID:19766688

  6. Curcumin conjugated with PLGA potentiates sustainability, anti-proliferative activity and apoptosis in human colon carcinoma cells.

    PubMed

    Waghela, Bhargav N; Sharma, Anupama; Dhumale, Suhashini; Pandey, Shashibahl M; Pathak, Chandramani

    2015-01-01

    Curcumin, an ingredient of turmeric, exhibits a variety of biological activities such as anti-inflammatory, anti-atherosclerotic, anti-proliferative, anti-oxidant, anti-cancer and anti-metastatic. It is a highly pleiotropic molecule that inhibits cell proliferation and induces apoptosis in cancer cells. Despite its imperative biological activities, chemical instability, photo-instability and poor bioavailability limits its utilization as an effective therapeutic agent. Therefore, enhancing the bioavailability of curcumin may improve its therapeutic index for clinical setting. In the present study, we have conjugated curcumin with a biodegradable polymer Poly (D, L-lactic-co-glycolic acid) and evaluated its apoptotic potential in human colon carcinoma cells (HCT 116). The results show that curcumin-PLGA conjugate efficiently inhibits cell proliferation and cell survival in human colon carcinoma cells as compared to native curcumin. Additionally, curcumin conjugated with PLGA shows improved cellular uptake and exhibits controlled release at physiological pH as compared to native curcumin. The curcumin-PLGA conjugate efficiently activates the cascade of caspases and promotes intrinsic apoptotic signaling. Thus, the results suggest that conjugation potentiates the sustainability, anti-proliferative and apoptotic activity of curcumin. This approach could be a promising strategy to improve the therapeutic index of cancer therapy. PMID:25692854

  7. FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells

    PubMed Central

    Smirnov, Artem; Panatta, Emanuele; Lena, AnnaMaria; Castiglia, Daniele; Di Daniele, Nicola; Melino, Gerry; Candi, Eleonora

    2016-01-01

    Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis. PMID:27385468

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

    PubMed

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

    2016-05-01

    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

  9. 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

  10. Pathogenetic mechanisms of heavy metals effect on proapoptotic and proliferative potential of breast cancer

    PubMed Central

    Romaniuk, Anatolii; Moskalenko, Roman; Kuzenko, Yevhen; Gladchenko, Oksana; Lyndina, Yuliia

    2015-01-01

    Materials and Methods Chemical composition was studied with the help of the scanning electron microscope with energy-dispersion spectrometer. Immunohistochemical reaction showed the p53 and Ki-67 receptors expression. The study of DNA fragmentation was performed in agarose gel. Results There was an interrelation between the accumulations of the trace elements with the degree of cancer malignancy. There were 85% of cases with positive reaction to Ki-67 and 40% cases with positive reaction to p53. We found a moderate correlation between the accumulation of microelements in the breast cancer tissue and the level of proliferative activity. We noted the combination of the increase of DNA fragmentation with the expression of p53 and Ki-67 receptors. Conclusions The trace elements can cause the initiation and the progression of the tumorous growth, which is expressed in the increased proliferation of tumor cells. This leads to the destabilization of the genetic material which can be expressed in the synthesis of mutant p53 protein. Finally, it leads to the block of apoptosis and regulatory effects of cells. This can cause the tumor progression and the destabilization of the genome, which is reflected in the increased DNA fragmentation. PMID:26120478

  11. α6 Integrin and CD44 Enrich for a Primary Keratinocyte Population That Displays Resistance to UV-Induced Apoptosis

    PubMed Central

    Wray, Helen; Mackenzie, Ian C.; Storey, Alan; Navsaria, Harshad

    2012-01-01

    Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrinhigh+/CD44+ sub-population of basal keratinocytes. These α6 integrinhigh+/CD44+ keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrinhigh+/CD44+ cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrinhigh+/CD44+ cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrinhigh+/CD44+ cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents. PMID:23071680

  12. The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling

    PubMed Central

    Engström, Wilhelm; Darbre, Philippa; Eriksson, Staffan; Gulliver, Linda; Hultman, Tove; Karamouzis, Michalis V.; Klaunig, James E.; Mehta, Rekha; Moorwood, Kim; Sanderson, Thomas; Sone, Hideko; Vadgama, Pankaj; Wagemaker, Gerard; Ward, Andrew; Singh, Neetu; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Colacci, Anna Maria; Vaccari, Monica; Mondello, Chiara; Scovassi, A. Ivana; Raju, Jayadev; Hamid, Roslida A.; Memeo, Lorenzo; Forte, Stefano; Roy, Rabindra; Woodrick, Jordan; Salem, Hosni K.; Ryan, Elizabeth; Brown, Dustin G.; Bisson, William H.

    2015-01-01

    The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span. PMID:26106143

  13. Copper-GHK increases integrin expression and p63 positivity by keratinocytes.

    PubMed

    Kang, Youn-A; Choi, Hye-Ryung; Na, Jung-Im; Huh, Chang-Hun; Kim, Min-Ji; Youn, Sang-Woong; Kim, Kyu-Han; Park, Kyoung-Chan

    2009-04-01

    Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin. PMID:19319546

  14. Decorin gene expression and its regulation in human keratinocytes

    SciTech Connect

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico; Kuri-Harcuch, Walid

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  15. Proliferative potential and p53 overexpression in precursor and early stage lesions of bronchioloalveolar lung carcinoma.

    PubMed Central

    Kitamura, H.; Kameda, Y.; Nakamura, N.; Nakatani, Y.; Inayama, Y.; Iida, M.; Noda, K.; Ogawa, N.; Shibagaki, T.; Kanisawa, M.

    1995-01-01

    To elucidate the pathogenesis of bronchioloalveolar lung carcinoma (BAC), we evaluated the lesion size, growth fraction, and p53 overexpression of atypical adenomatous hyperplasia (AAH) and early stage BAC. AAH was classified as showing low grade or high grade atypia. AAH-like carcinoma, presumably very early stage BAC, was distinguished from AAH in that it exhibited remarkable atypia suggestive of malignant potential and from overt BAC in that it lacked unequivocal malignant features, including invasive/destructive growth. The growth fraction was determined immunohistochemically in terms of the Ki-67 labeling index. The overexpression of p53 was evaluated by assessing the nuclear accumulation of immunoreactive p53 protein. Both the lesion size and the growth fraction increased from low grade AAH, to high grade AAH, to AAH-like carcinoma, and to overt adenocarcinoma. The overexpression of p53 in AAH-like carcinoma was similar to that in overt adenocarcinoma and was more frequent than that in AAH. Our findings indicate that AAH, AAH-like carcinoma, and overt BAC represent different categories, although the cellular events occurring in these lesions presumably represent a continuous spectrum of the changes that are reflected in the cytomorphology and lesion size. The findings here suggest that AAH and AAH-like carcinomas constitute a population of heterogeneous lesions representing different steps toward overt BAC. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7717455

  16. Discrimination of the toxic potential of chemically differing topical glucocorticoids using a neutral red release assay with human keratinocytes and fibroblasts.

    PubMed

    Korting, H C; Hülsebus, E; Kerscher, M; Greber, R; Schäfer-Korting, M

    1995-07-01

    In inflammatory skin disease, hydrocortisone and prednisolone double esters are about equipotent to conventional medium potency topical glucocorticoids, such as betamethasone valerate. Local adverse effects, in particular skin atrophy, are a potential problem with topical glucocorticoids. Recently, cell cultures have shown promise as a means of assessing local tolerance. To investigate the toxic potential of hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone aceponate, prednicarbate, triamcinolone acetonide, betamethasone valerate and desoximethasone, human keratinocytes and fibroblasts were exposed to these agents in vitro, using a modified neutral red release assay. In addition, the morphology of these cells was assessed by light microscopy. Although all the topical glucocorticoids tested proved toxic to both cell types, there were major differences between glucocorticoids in their effect on fibroblasts. Hydrocortisone and the non-halogenated double-ester-type glucocorticoids were less toxic than the conventional medium potency topical glucocorticoids tested (betamethasone valerate and desoximethasone). In particular, hydrocortisone aceponate was less toxic than betamethasone valerate (P < or = 0.05). In general, the effect of topical glucocorticoids on the cells, based on neutral red release, was more marked with keratinocytes than with fibroblasts. Although the ranking order with respect to the toxic potential was similar, a clear-cut difference was not observed between non-halogenated double-ester-type glucocorticoids and betamethasone valerate. Morphological changes due to glucocorticoid exposure followed the same pattern with both keratinocytes and fibroblasts. The neutral red release assay is able to discriminate between the cytotoxic effects of chemically differing topical glucocorticoids on human keratinocytes and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7669640

  17. Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats

    PubMed Central

    Pınarlı, Ferda Alpaslan; Ökten, Gülsen; Beden, Ümit; Fışgın, Tunç; Kefeli, Mehmet; Kara, Nurten; Duru, Feride; Tomak, Leman

    2014-01-01

    AIM To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells. METHODS The study included 5 groups each including 10 female “Sprague Dawley” rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction. RESULTS Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea. CONCLUSION We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells. PMID:24790860

  18. Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures.

    PubMed

    Sharma, Ruchi; Barakzai, Safia Z; Taylor, Sarah E; Donadeu, F Xavier

    2016-08-01

    Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23897780

  19. Suppression of Proinflammatory Cytokines in Functionalized Fullerene-Exposed Dermal Keratinocytes

    DOE PAGESBeta

    Gao, Jun; Wang, Hsing-Lin; Iyer, Rashi

    2010-01-01

    Initial experiments using differentially functionalized fullerenes, CD-, hexa-, and tris-, suggested a properties dependent effect on cytotoxic and proliferative responses in human skin keratinocytes. In the present study we investigated the cytokine secretion profile of dermal epithelial cells exposed to functionalized fullerenes. Keratinocyte-derived cytokines affect homing and trafficking of normal and malignant epidermal immune as well as nonimmune cells in vivo. These cytokines are critical for regulating activation, proliferation, and differentiation of epidermal cells. Our results indicate that tris- (size range <100 nm) significantly reduces inflammatory cytokine release in a dose- and time-dependent manner. In contrast CD- demonstrated a relatively pro-inflammatorymore » cytokine response, while hexa- did not significantly perturb cytokine responses. Physical and chemical characterizations of these engineered nanomaterials suggest that the disparate biological responses observed may potentially be a function of the aggregation properties of these fullerenes.« less

  20. Stereotyped distribution of proliferating keratinocytes in disorders affecting the epidermis

    SciTech Connect

    Pierard-Franchimont, C.; Pierard, G.E.

    1989-06-01

    We used the technique of autoradiography after incorporation of tritiated thymidine (/sup 3/H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of /sup 3/H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of /sup 3/H-TdR-labeled keratinocytes in the various layers of the epidermis.

  1. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  2. Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation.

    PubMed

    Amigó, María; Schalkwijk, Joost; Olthuis, Diana; De Rosa, Salvatore; Payá, Miguel; Terencio, María Carmen; Lamme, Evert

    2006-11-17

    Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression. PMID:16973179

  3. Role of PI3K, MAPK/ERK1/2, and p38 in implementation of the proliferative and differentiation potential of erythroid progenitors after blood loss.

    PubMed

    Dygai, A M; Zhdanov, V V; Miroshnichenko, L A; Udut, E V; Zyuz'kov, G N; Simanina, E V; Sherstoboev, E Yu; Chaikovskii, A V; Stavrova, L A; Burmina, Ya V; Khrichkova, T Yu; Reichart, D V; Goldberg, V E

    2015-02-01

    The involvement of PI3K, ERK and p38-dependent signaling system in the regulation of functional activity of erythroid precursors after blood loss (30% of circulating volume) was studied. We demonstrated the important role of PI3K and p38 in the suppression of differentiation of erythroid precursors the contribution of p38 to stimulation of mitotic activity of erythroid CFU, which maintains the growth potential of the precursors at the optimal physiological level. The classical MAPK/ERK-kinase pathway does not determine the proliferative and differentiation status of erythroid CFU. PMID:25711660

  4. 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

  5. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

    PubMed Central

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-01-01

    UVB irradiation induces harmful photochemical reactions, including formation of cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  6. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.

    PubMed

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-12-01

    UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  7. Role of taurine accumulation in keratinocyte hydration.

    PubMed

    Janeke, Guido; Siefken, Wilfried; Carstensen, Stefanie; Springmann, Gunja; Bleck, Oliver; Steinhart, Hans; Höger, Peter; Wittern, Klaus-Peter; Wenck, Horst; Stäb, Franz; Sauermann, Gerhard; Schreiner, Volker; Doering, Thomas

    2003-08-01

    Epidermal keratinocytes are exposed to a low water concentration at the stratum corneum-stratum granulosum interface. When epithelial tissues are osmotically perturbed, cellular protection and cell volume regulation is mediated by accumulation of organic osmolytes such as taurine. Previous studies reported the presence of taurine in the epidermis of several animal species. Therefore, we analyzed human skin for the presence of the taurine transporter (TAUT) and studied the accumulation of taurine as one potential mechanism protecting epidermal keratinocytes from dehydration. According to our results, TAUT is expressed as a 69 kDa protein in human epidermis but not in the dermis. For the epidermis a gradient was evident with maximal levels of TAUT in the outermost granular keratinocyte layer and lower levels in the stratum spinosum. No TAUT was found in the basal layer or in the stratum corneum. Keratinocyte accumulation of taurine was induced by experimental induction of skin dryness via application of silica gel to human skin. Cultured human keratinocytes accumulated taurine in a concentration- and osmolarity-dependent manner. TAUT mRNA levels were increased after exposure of human keratinocytes to hyperosmotic culture medium, indicating osmosensitive TAUT mRNA expression as part of the adaptation of keratinocytes to hyperosmotic stress. Keratinocyte uptake of taurine was inhibited by beta-alanine but not by other osmolytes such as betaine, inositol, or sorbitol. Accumulation of taurine protected cultured human keratinocytes from both osmotically induced and ultraviolet-induced apoptosis. Our data indicate that taurine is an important epidermal osmolyte required to maintain keratinocyte hydration in a dry environment. PMID:12880428

  8. Design, synthesis and QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents.

    PubMed

    Eldehna, Wagdy M; Altoukhy, Ayman; Mahrous, Hoda; Abdel-Aziz, Hatem A

    2015-01-27

    A hybrid pharmacophore approach was adopted to design and synthesize new series of isatin-pyridine hybrids. All the newly prepared hybrids (5a-o, 8 and 11a-d) were in vitro evaluated for their anti-proliferative activity against three human cancer cell lines, namely HepG2 hepatocellular carcinoma, A549 lung cancer and MCF-7 breast cancer. Compound 8 emerged as the most active member against HepG2 cell line (IC50 = 2.5 ± 0.39 μM), with 2.7-fold increased activity than the reference drug, doxorubicin (IC50 = 6.9 ± 2.05 μM). Whilst, compound 11c was found to be the most potent counterpart against A549 and MCF-7 cell lines with IC50 values of 10.8 ± 1.15 and 6.3 ± 0.79, respectively. The weightiness of the utilization of non-cleavable linker, as the chalcone linker, and simplification of the first group, was explored via the SAR study. Furthermore, a QSAR model was built to explore the structural requirements controlling the cytotoxic activities. Notably, the predicted activities by the QSAR model were very close to those experimentally observed, hinting that this model could be safely applied for prediction of more efficacious hits comprising the same skeletal framework. Finally, a theoretical kinetic study was established to predict the ADME of the active hybrids. PMID:25499988

  9. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed Central

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-01

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells. Images PMID:1988936

  10. A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

    SciTech Connect

    Strauss, Bryan E.; Vieira de Carvalho, Anna Carolina; Bajgelman, Marcio C.

    2006-10-06

    We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for this factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.

  11. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

  12. Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

    PubMed Central

    Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Müller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Müller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Günter; Matthäus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai

    2013-01-01

    Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration. PMID:24066165

  13. Acute and chronic wound fluids influence keratinocyte function differently.

    PubMed

    Thamm, Oliver C; Koenen, Paola; Bader, Nicola; Schneider, Alina; Wutzler, Sebastian; Neugebauer, Edmund A M; Spanholtz, Timo A

    2015-04-01

    Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT-PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti-proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP-9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP-9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing. PMID:23517467

  14. Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment.

    PubMed

    Maurelli, Riccardo; Zambruno, Giovanna; Guerra, Liliana; Abbruzzese, Claudia; Dimri, Goberdhan; Gellini, Mara; Bondanza, Sergio; Dellambra, Elena

    2006-07-01

    Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin-dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B-Cell Specific Moloney murine leukemia virus site 1 (Bmi-1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi-1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down-regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining stemness. PMID:16754749

  15. Impaired keratinocyte function on matrix metalloproteinase-1 (MMP-1) damaged collagen

    PubMed Central

    Perone, Patricia; Deming, Monica O’Brien; Warner, Roscoe L.; Aslam, Muhammad N.; Bhagavathula, Narasimharao; Dame, Michael K.; Voorhees, John J.

    2010-01-01

    Healing of superficial skin wounds depends on the proliferation and migration of keratinocytes at the wound margin. When human epidermal keratinocytes were incubated on polymerized type I collagen, they rapidly attached and spread. The cells underwent a proliferative response and, over the subsequent 6-day period, covered the collagen surface with a monolayer of cells. When keratinocytes were plated on collagen that had been fragmented by exposure to matrix metalloproteinase-1 (MMP-1, collagenase-1), the cells attached as readily as to intact collagen but spread more slowly and less completely. Growth was reduced by approximately 50%. Instead of covering the collagen surface, the keratinocytes remained localized to the site of attachment. Keratinocytes on fragmented collagen expressed a more differentiated phenotype as indicated by a higher level of surface E-cadherin. Based on these findings, we suggest that damage to the underlying collagenous matrix may impede efficient keratinocyte function and retard wound closure. PMID:19352688

  16. Proteome profiling of keratinocytes transforming to malignancy.

    PubMed

    Paulitschke, Verena; Gerner, Christopher; Hofstätter, Elisabeth; Mohr, Thomas; Mayer, Rupert Laurenz; Pehamberger, Hubert; Kunstfeld, Rainer

    2015-02-01

    To shed light on the multistep process of squamous cell carcinoma development and the underlying pathologic mechanisms, we performed comparative proteome analysis of keratinocytes, keratinocytes stimulated with Il-1beta, and A431 epidermoid carcinoma cells. Fractionation of the cells into supernatant, nucleus, and cytoplasm was followed by protein separation, proteolytic digest, and nano-LC separation, and fragmentation using an ion trap mass spectrometer. Specific bioinformatics tools were used to generate a list of keratinocyte-specific proteins. Ninety percent of these proteins were found to be upregulated in keratinocytes versus the A431 cells. Classification of the identified proteins by biologic function and gene set enrichment analysis revealed that keratinocytes produced more proteins involved in cell differentiation, cell adhesion, cell junction, calcium ion, calmodulin binding, cytoskeleton organization, and cytokinesis, whereas A431 produced more proteins involved in cell cycle checkpoint, cell cycle process, RNA processing and transport, DNA damage and repair, RNA and DNA binding, and chromatin remodeling. The protein signatures of A431 and normal keratinocytes treated with IL-1beta showed marked similarity, confirming that inflammation is an important step in malignant transformation in nonmelanoma skin cancer. Thus, proteome profiling and bioinformatic processing may support the understanding of the underlying mechanisms, with the potential to facilitate development of early biomarkers and patient-tailored therapy. PMID:25395074

  17. Hyaluronan-phosphatidylethanolamine polymers form pericellular coats on keratinocytes and promote basal keratinocyte proliferation.

    PubMed

    Symonette, Caitlin J; Kaur Mann, Aman; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa(647)-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa(647)-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa(647)-HA-PE penetrated into and was retained within the epidermis than Alexa(647)-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  18. Hyaluronan-Phosphatidylethanolamine Polymers Form Pericellular Coats on Keratinocytes and Promote Basal Keratinocyte Proliferation

    PubMed Central

    Symonette, Caitlin J.; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Turley, Eva A.

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa647-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa647-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa647-HA-PE penetrated into and was retained within the epidermis than Alexa647-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  19. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential

    PubMed Central

    Shia, Wei-Jong; Okumura, Akiko J.; Yan, Ming; Sarkeshik, Ali; Lo, Miao-Chia; Matsuura, Shinobu; Komeno, Yukiko; Zhao, Xinyang; Nimer, Stephen D.; Yates, John R.

    2012-01-01

    Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development. PMID:22498736

  20. Huaier restrains proliferative and invasive potential of human hepatoma SKHEP-1 cells partially through decreased Lamin B1 and elevated NOV.

    PubMed

    Hu, Zhongdong; Yang, Ailin; Su, Guozhu; Zhao, Yunfang; Wang, Ying; Chai, Xingyun; Tu, Pengfei

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cause of malignancy-related mortality worldwide. It is urgently needed to develop potential drugs with good efficacy and low toxicity for HCC treatment. The anti-tumor effect of Traditional Chinese Medicine (TCM) has received increasing attention worldwide. Trametes robiniophila Murr. (Huaier) has been used in TCM for approximately 1,600 years. Clinically, Huaier has satisfactory therapeutic effects in cancer treatment, especially in HCC. However, the mechanisms underlying the anti-cancer effect of Huaier remain ill defined. Herein we have demonstrated that Huaier dramatically inhibited cell proliferation and induced apoptosis in human hepatoma cell line SKHEP-1. Importantly, Huaier restrained the metastatic capability of SKHEP-1 cells. Mechanistically, down-regulation of Lamin B1 and up-regulation of Nephroblastoma overexpressed (NOV) were at least partially responsible for the inhibitory effect of Huaier on the proliferative and invasive capacity of SKHEP-1 cells. Our finding provided new insights into mechanisms of anti-HCC effect of Huaier and suggested a new scientific basis for clinical medication. PMID:27503760

  1. Huaier restrains proliferative and invasive potential of human hepatoma SKHEP-1 cells partially through decreased Lamin B1 and elevated NOV

    PubMed Central

    Hu, Zhongdong; Yang, Ailin; Su, Guozhu; Zhao, Yunfang; Wang, Ying; Chai, Xingyun; Tu, Pengfei

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cause of malignancy-related mortality worldwide. It is urgently needed to develop potential drugs with good efficacy and low toxicity for HCC treatment. The anti-tumor effect of Traditional Chinese Medicine (TCM) has received increasing attention worldwide. Trametes robiniophila Murr. (Huaier) has been used in TCM for approximately 1,600 years. Clinically, Huaier has satisfactory therapeutic effects in cancer treatment, especially in HCC. However, the mechanisms underlying the anti-cancer effect of Huaier remain ill defined. Herein we have demonstrated that Huaier dramatically inhibited cell proliferation and induced apoptosis in human hepatoma cell line SKHEP-1. Importantly, Huaier restrained the metastatic capability of SKHEP-1 cells. Mechanistically, down-regulation of Lamin B1 and up-regulation of Nephroblastoma overexpressed (NOV) were at least partially responsible for the inhibitory effect of Huaier on the proliferative and invasive capacity of SKHEP-1 cells. Our finding provided new insights into mechanisms of anti-HCC effect of Huaier and suggested a new scientific basis for clinical medication. PMID:27503760

  2. Arsenic-exposed Keratinocytes Exhibit Differential microRNAs Expression Profile; Potential Implication of miR-21, miR-200a and miR-141 in Melanoma Pathway

    PubMed Central

    Gonzalez, Horacio; Lema, Carolina; Kirken, Robert A.; Maldonado, Rosa A.; Varela-Ramirez, Armando; Aguilera, Renato J.

    2016-01-01

    Long-term exposure to arsenic has been linked to cancer in different organs and tissues, including skin. Here, non-malignant human keratinocytes (HaCaT) were exposed to arsenic and its effects on microRNAs (miRNAs; miR) expression were analyzed via miRCURY LNA array analyses. A total of 30 miRNAs were found differentially expressed in arsenic-treated cells, as compared to untreated controls. Among the up-regulated miRNAs, miR-21, miR-200a and miR-141, are well known to be involved in carcinogenesis. Additional findings confirmed that those three miRNAs were indeed up-regulated in arsenic-stimulated keratinocytes as demonstrated by quantitative PCR assay. Furthermore, bioinformatics analysis of both potential cancer-related pathways and targeted genes affected by miR-21, miR-200a and/or miR-141 was performed. Results revealed that miR-21, miR-200a and miR-141 are implicated in skin carcinogenesis related with melanoma development. Conclusively, our results indicate that arsenic-treated keratinocytes exhibited alteration in the miRNAs expression profile and that miR-21, miR-200a and miR-141 could be promising early biomarkers of the epithelial phenotype of cancer cells and they could be potential novel targets for melanoma therapeutic interventions. PMID:27054085

  3. Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation

    PubMed Central

    2013-01-01

    Background MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. Results We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. Conclusions Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation

  4. Structure-activity relationship studies of acridones as potential antipsoriatic agents. 2. Synthesis and antiproliferative activity of 10-substituted hydroxy-10H-acridin-9-ones against human keratinocyte growth.

    PubMed

    Putic, Aleksandar; Stecher, Lambert; Prinz, Helge; Müller, Klaus

    2010-11-01

    A series of 10-substituted hydroxy-10H-acridin-9-ones were synthesized and studied as potential antipsoriatic agents. The antiproliferative activity of the novel derivatives, which can be considered as aza-analogues of the antipsoriatic drug anthralin, was determined using the human keratinocyte cell line HaCaT. Structure-activity relationships with respect to the nature of the N-substituent at the acridone scaffold were delineated. Release of lactate dehydrogenase (LDH) was used to exclude non-specific cytotoxic effects. As compared to anthralin, N-substitution of the acridone scaffold in the target compounds provided agents devoid of radical producing properties, which was documented by their ineffectiveness to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. This was in excellent agreement with the data obtained from the LDH assay in which the novel compounds did not induce membrane damage. Benzyl substitution at the 10-position yielded keratinocyte growth inhibitory activity in the low micromolar range. The most potent inhibitor of keratinocyte hyperproliferation was compound 8a having an N-methyl group and a 1,3-dihydroxy arrangement at the acridone scaffold, with an IC(50) value comparable to that of anthralin. PMID:20850910

  5. 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. PMID:27371895

  6. Synthesis and Evaluation of Biological Activity of Antimicrobial – Pro-Proliferative Peptide Conjugates

    PubMed Central

    Langa, Paulina; Trzonkowski, Piotr; Obuchowski, Michał; Lesner, Adam

    2015-01-01

    Skin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations. PMID:26473368

  7. [Proliferative verrucous leukoplakia].

    PubMed

    Lindenmüller, Irène Hitz; Lambrecht, J Thomas

    2006-01-01

    Proliferative verrucous leukoplakia (PVL) is a seldom form of oral leukoplakia (OL) with high transformation tendency. It starts as a bold hyperkeratosis changing into an exophytic verrucous form spreading in the oral cavity. The clinical diagnosis therefore is a retrospective one. PMID:16792055

  8. Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

    PubMed

    Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

    2016-09-01

    The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p < 0.05) on mean absorbance of fibroblasts cultured with 10 and 25 % PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo. PMID:27394438

  9. Epidermal Healing in Burns: Autologous Keratinocyte Transplantation as a Standard Procedure: Update and Perspective

    PubMed Central

    Barrault, Christine; Levard, Guillaume; Morel, Franck; Bernard, François-Xavier; Lecron, Jean-Claude

    2014-01-01

    Background: Treatment of burned patients is a tricky clinical problem not only because of the extent of the physiologic abnormalities but also because of the limited area of normal skin available. Methods: Literature indexed in the National Center (PubMed) has been reviewed using combinations of key words (burns, children, skin graft, tissue engineering, and keratinocyte grafts). Articles investigating the association between burns and graft therapeutic modalities have been considered. Further literature has been obtained by analysis of references listed in reviewed articles. Results: Severe burns are conventionally treated with split-thickness skin autografts. However, there are usually not enough skin donor sites. For years, the question of how covering the wound surface became one of the major challenges in clinical research area and several procedures were proposed. The microskin graft is one of the oldest methods to cover extensive burns. This technique of skin expansion is efficient, but results remain inconsistent. An alternative is to graft cultured human epidermal keratinocytes. However, because of several complications and labor-intensive process of preparing grafts, the initial optimism for cultured epithelial autograft has gradually declined. In an effort to solve these drawbacks, isolated epithelial cells from selecting donor site were introduced in skin transplantation. Conclusions: Cell suspensions transplanted directly to the wound is an attractive process, removing the need for attachment to a membrane before transfer and avoiding one potential source of inefficiency. Choosing an optimal donor site containing cells with high proliferative capacity is essential for graft success in burns. PMID:25426401

  10. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  11. Novel sphingolipid derivatives promote keratinocyte differentiation.

    PubMed

    Paragh, György; Schling, Petra; Ugocsai, Peter; Kel, Alexander E; Liebisch, Gerhard; Heimerl, Susanne; Moehle, Christoph; Schiemann, Yvonne; Wegmann, Michael; Farwick, Mike; Wikonkál, Norbert M; Mandl, József; Langmann, Thomas; Schmitz, Gerd

    2008-12-01

    Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications. PMID:18631249

  12. Beta Adrenergic Receptors in Keratinocytes

    PubMed Central

    Sivamani, Raja K.; Lam, Susanne T.; Isseroff, R. Rivkah

    2007-01-01

    Synopsis Beta2 adrenergic receptors were identified in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo and psoriasis. New studies also indicate that the beta2AR also modulates keratinocyte migration, and thus can function to regulate wound re-epithelialization. This review focuses on the function of these receptors in keratinocytes and their contribution to cutaneous physiology and disease. PMID:17903623

  13. [Proliferative vitreoretinopathy: curative treatment].

    PubMed

    Chiquet, C; Rouberol, F

    2014-10-01

    Proliferative vitreoretinopathy (PVR), which causes contractile fibrocellular membranes that may prevent retinal reattachment, remains one of the most severe complications of rhegmatogenous retinal detachment (RD), with an incidence of 5-11%, and one of the most frequent causes of surgical failure (50-75%). Its severity is due to the complexity of the surgery required to treat patients, and to its uncertain anatomic and functional prognosis. Curative treatment of PVR includes vitrectomy, sometimes associated with phacoemulsification or scleral buckling; systematic peeling of epiretinal membranes, occasionally retinectomy; and systematic retinopexy by endolaser photocoagulation. The current preferred internal tamponade is silicone oil. Silicone oils of various densities are undergoing comparative studies. PMID:24997865

  14. Luteolin-7-glucoside inhibits IL-22/STAT3 pathway, reducing proliferation, acanthosis, and inflammation in keratinocytes and in mouse psoriatic model.

    PubMed

    Palombo, R; Savini, I; Avigliano, L; Madonna, S; Cavani, A; Albanesi, C; Mauriello, A; Melino, G; Terrinoni, A

    2016-01-01

    The epidermis is a dynamic tissue in which keratinocytes proliferate in the basal layer and undergo a tightly controlled differentiation while moving into the suprabasal layers. The balance between keratinocyte proliferation, differentiation, and death is essential, and its perturbation can result in pathological changes. Some common skin diseases, such as psoriasis, are characterized by hyperproliferation accompanied by inflammatory reactions, suggesting that molecules with topical anti-inflammatory and ROS scavenging abilities may be useful for their treatment. Here we investigate the potential of the flavone Luteolin-7-glucoside (LUT-7G) as a treatment for psoriasis. We show that LUT-7G leads to a modification of the cell cycle and the induction of keratinocyte differentiation, with modification of energy, fatty acid, and redox metabolism. LUT-7G treatment also neutralizes the proliferative stimulus induced by the proinflammatory cytokines IL-22 and IL-6 in HEKn. Moreover, in the Imiquimod (IMQ) mouse model of psoriasis, topical administration of LUT-7G leads to a marked reduction of acanthosis and re-expression of epidermal differentiation markers. Dissection of the IL-22 signalling pathway, activated by IMQ treatment, demonstrates that LUT-7G impairs the nuclear translocation of phosphorylated (activated) STAT3, blocking the IL-22 signalling cascade. Thus LUT-7G appears to be a promising compound for the treatment of hyperproliferative and inflammatory skin diseases, such as psoriasis. PMID:27537526

  15. Structure-activity relationship studies of acridones as potential antipsoriatic agents. 1. Synthesis and antiproliferative activity of simple N-unsubstituted 10H-acridin-9-ones against human keratinocyte growth.

    PubMed

    Putic, Aleksandar; Stecher, Lambert; Prinz, Helge; Müller, Klaus

    2010-08-01

    A series of N-unsubstituted hydroxy-10H-acridin-9-ones were synthesized and evaluated for inhibitory action against HaCaT keratinocyte growth, in order to explore their potential as antipsoriatic agents. For structure-activity relationship studies, the number and position of the hydroxyl groups were modified, the oxygen functions substituted or replaced, or additional functional groups were introduced into the acridone scaffold. 1,8-Dihydroxy-10H-acridin-9-one (4), which is an aza-analogue of the antipsoriatic anthralin, was only marginally active. However, 1,3-dihydroxy-substituted 5ee was the most potent acridone within this series and inhibited keratinocyte growth with an IC(50) value comparable to that of anthralin. In contrast to anthralin, nearly all members of the acridone series were devoid of radical generating properties, which were determined by their capability to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. Structures with a phenolic hydroxyl or an aromatic amine arranged ortho or para to the acridone NH group were exceptions. Also in contrast to anthralin, membrane-damaging effects as documented by the release of lactate dehydrogenase into the culture medium were not observed for acridones. PMID:20452101

  16. Microenvironment-induced myofibroblast-like conversion of engrafted keratinocytes.

    PubMed

    Li, MeiRong; Ti, DongDong; Han, WeiDong; Fu, XiaoBing

    2014-02-01

    Myofibroblasts, recognized classically by α-smooth muscle actin (α-SMA) expression, play a key role in the wound-healing process, promoting wound closure and matrix deposition. Although a body of evidence shows that keratinocytes explanted onto a wound bed promote closure of a skin injury, the underlying mechanisms are not well understood. The basal layer of epidermis is rich in undifferentiated keratinocytes (UKs). We showed that UKs injected into granulation tissue could switch into α-SMA positive cells, and accelerate the rate of skin wound healing. In addition, when the epidermis sheets isolated from foreskin cover up the wound bed or are induced in vitro, keratinocytes located at the basal layers or adjacent sites were observed to convert into myofibroblast-like cells. Thus, UKs have a potential for myofibroblastic transition, which provides a novel mechanism by which keratinocyte explants accelerate skin wound healing. PMID:24443179

  17. Identification of a murine high-proliferative-potential colony-forming cell (HPP-CFC) capable of producing a number of megakaryocytes and replating for secondary HPP-CFCs in culture.

    PubMed

    Han, Z C

    1994-04-01

    The high-proliferative-potential colony-forming cell (HPP-CFC) has been shown to be clearly heterogeneous. Hierarchical subpopulations can be identified by analyzing the kinetics of cell regeneration and the specific cellular requirement for cytokines. In this study, a new type of HPP-CFC, termed high-proliferative potential mixed colony-forming unit-megakaryocyte (HPP-mCFU-MK), was detected in murine bone marrow cell cultures. The HPP-mCFU-MK was able to form macroscopic colonies that fit the criteria of the HPP-CFC colony but contained a number of megakaryocytes. Its growth was stimulated by either aplastic anemia serum (AAS) or a combination of three or more recombinant hematopoietic growth factors but was not inhibited by transforming growth factor-beta 1 (TGF-beta 1) and platelet factor 4 (PF4) in vitro. The recloning of the 12-day-old HPP-mCFU-MK colonies picked up from AAS-stimulated primary cultures caused secondary formation of HPP-CFC colonies. These data suggest that HPP-mCFC-MK is a new subset of stem cell characterized by its ability to produce directly a number of megakaryocytes in response to multifactor stimulation, to generate a secondary set of HPP-CFC in replating culture, and to be unaffected by TGF-beta 1 and PF4 treatment. PMID:8145010

  18. Discovery of a novel class anti-proliferative agents and potential inhibitors of EGFR tyrosine kinases based on 4-anilinotetrahydropyrido[4,3-d]pyrimidine scaffold: Design, synthesis and biological evaluations.

    PubMed

    Zhang, Yong; Zhang, Kai; Zhao, Meng; Zhang, Lixia; Qin, Mingze; Guo, Shuchun; Zhao, Yanfang; Gong, Ping

    2015-08-01

    A novel series of 4-arylamino-6/7-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines were designed, synthesized and their biological activities as the potential anti-proliferative agents and EGFR kinase inhibitors were evaluated. Both of N-acrylamide fragment in THPPs and 4-aniline groups with substituents played key roles for their significant anti-proliferative activities against four cancer cell lines (HT29, A549, H460 and H1975). Especially inhibitory activity of Gefitinib-resistant H1975 were showed more favorable, which could be observed from compounds 13b, 13c, 13n, 13o, 13p, 13r, 13s, 13u and 24c obviously. By evaluation of inhibiting EGFR and HER2 kinases, seven compounds (13b, 13g, 13n, 13o, 13p, 13r and 13s) showed stronger EGFR potency with IC50 ⩽ 18 nM, which could also be understood by preliminary docking study of 13b with EGFR kinase. In view of the primary SAR, bisarylaniline derivatives (13o, 13p, 13r and 13s) showed obvious improvements on HER2 inhibition, which indicated their being potential EGFR/HER2 dual kinase inhibitors. PMID:26122771

  19. 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

  20. [Proliferative vitreoretinopathy: prophylactic treatment].

    PubMed

    Chiquet, C; Rouberol, F

    2014-11-01

    Proliferative vitreoretinopathy (PVR) is a complex process. It causes contractile fibrocellular membranes that may prevent retinal reattachment. PVR therefore remains one of the most severe complications of rhegmatogenous retinal detachment (RD), with an incidence of 5-11%, and is among the most frequent causes of surgical failure (50-75%). Its severity derives from the complexity of the surgery required to treat patients and from its uncertain anatomic and functional prognosis. The first step in preventing PVR is to identify patients at risk by means of clinical and/or biological factors such as the characteristics of retinal tears (large size, number) and detachment (preexisting PVR, extent), and the use of cryotherapy. Surgeons must therefore adapt their surgical approach to the risk of PVR. The study of animal models and the natural history of the condition in humans demonstrate the importance of early antiproliferative treatment in the early stage of the disease. Combining 5-fluoro-uracil and heparin in the vitrectomy infusion lowers the rate of postoperative PVR onset in patients with PVR risk factors. The evaluation of new molecules and new dosages will lead to a decisive step in the fight against PVR. PMID:25012973

  1. Primary structure of keratinocyte transglutaminase

    SciTech Connect

    Phillips, M.A.; Stewart, B.E.; Qin, Q.; Rice, R.H. ); Chakravarty, R. ); Floyd, E.E.; Jetten, A.M. )

    1990-12-01

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine {gamma}-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of {approximately}90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unreltaed activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

  2. Ageing and colony-forming efficiency of human hair follicle keratinocytes.

    PubMed

    Lecardonnel, Jennifer; Deshayes, Nathalie; Genty, Gaïanne; Parent, Nathalie; Bernard, Bruno A; Rathman-Josserand, Michelle; Paris, Maryline

    2013-09-01

    The decline of tissue regenerative potential of skin and hair is a hallmark of physiological ageing and may be associated with age-related changes in tissue-specific stem cells and/or their environment. Human hair follicles (hHF) contain keratinocytes having the property of stem cells such as clonogenic potential. Growth capacity of hHF keratinocytes shows that most of the colony-forming cells are classified as holoclones, meroclones or paraclones when analysed in a clonal assay (Cell, Volume 76, page 1063). Despite the well-known impact of ageing on human hair growth, little is known about changes in hHF keratinocyte clonogenic potential with age. This study aimed at assessing the clone-forming efficiency (CFE) of hHF keratinocytes from three age groups of human donors. It demonstrates that ageing affects hHF keratinocyte CFE. PMID:23947676

  3. 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

  4. Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro.

    PubMed

    Firth, James D; Putnins, Edward E

    2004-01-01

    The ability of keratinocyte growth factor 1 to modulate apoptosis in the absence of proliferation was studied in vitro. A HaCaT scrape wound model was developed in which dense monolayers prior to wounding were cultured to quiescence in defined media with hydroxyurea at concentrations that blocked proliferation without loss of cell viability. Scrape wounding was then found to induce apoptosis, originating at the wound edge, but subsequently radiating away over a 24 h period to encompass areas not originally damaged. Keratinocyte growth factor 1 inhibited this radial progression of apoptosis in a concentration-dependent manner up to 20 ng per mL with induced migration present at the wound edge. The extent of this rescue was modulated by the concentration of Ca2+ prior to wounding. In control wound cultures apoptotic bodies were found in cells adjacent to the wound interface but were greatly reduced in keratinocyte-growth-factor-1-treated groups. Keratinocyte growth factor 1 receptor expression was significantly induced within two to three cell widths of the scraped wound edge, at levels far exceeding those found at the leading edge of a nonwounded epithelial sheet. Tumor necrosis factor alpha (1-5 ng per mL) or Escherichia coli lipopolysaccharide (10-50 ng per mL) exacerbated scrape-induced early apoptosis (1-4 h), but was largely ameliorated by coculture with keratinocyte growth factor 1. Keratinocyte growth factor 1 protection was associated with a reduction in both caspase-3 activation and cytokeratin-19 loss. Protected wound edges were also associated with the maintenance of e-cadherin expression and induction of beta1 integrin and actin stress fiber organization. These results suggest that keratinocyte growth factor 1 may play a role in limiting mechanically induced apoptotic processes at the epithelial wound edge in a manner that is distinct from its proliferative function. PMID:14962112

  5. Low-energy helium-neon laser irradiation increases the motility of cultured human keratinocytes

    SciTech Connect

    Haas, A.F.; Isseroff, R.R.; Wheeland, R.G.; Rood, P.A.; Graves, P.J. )

    1990-06-01

    Helium-neon (HeNe) laser irradiation is known to stimulate wound healing. We investigated whether the biostimulatory effects of HeNe irradiation result from enhancement of keratinocyte proliferation or motility. HeNe effects on keratinocyte motility were evaluated by irradiating a wounded culture with 0.8 J/cm2 3 times over a 20-h period. At 20 h post-irradiation, videocinemicroscopy and sequential quantitative measurements of the leading edge were taken over a 6-h period. There was a significant difference in migration of the leading edge in irradiated wounds compared to non-irradiated wounded controls (12.0 microns/h vs 4.0 microns/h, p less than 0.0001). To determine if the increase in migration observed in irradiated cultures resulted from a proliferative effect of HeNe irradiation, subconfluent human keratinocyte cultures were irradiated with single or multiple doses of different fluences of HeNe irradiation (0.4 to 7.2 J/cm2) and evaluated 72 h post-irradiation. Irradiated and non-irradiated keratinocyte cultures grown on a microporous membrane surface were co-cultured with irradiated and non-irradiated fibroblasts to determine if HeNe irradiation induced a paracrine effect on keratinocyte proliferation. No significant increase in keratinocyte proliferation was demonstrated in any of these treatments. The biostimulatory effects of HeNe irradiation may now be extended to include enhancement of keratinocyte motility in vitro; this may contribute to the efficacy of HeNe irradiation in wound healing.

  6. Simarouba amara extract increases human skin keratinocyte differentiation.

    PubMed

    Bonté, F; Barré, P; Pinguet, P; Dusser, I; Dumas, M; Meybeck, A

    1996-08-01

    An aqueous extract of Simarouba amara was studied for its activity on the differentiation of human skin keratinocytes. Submerged and air-exposed treated keratinocyte cultures displayed a more highly differentiated histoarchitecture, with presence of ultrastructural differentiated elements, than untreated controls. Immunohistochemistry of involucrin and activation of transglutaminase activity provided further evidence for the increase in corneocyte envelope formation observed ultrastructurally. Lipid analysis of air-exposed cultures revealed an increase in the cholesterol sulphate, cholesterol and ceramide contents. After 4 weeks of treatment on the hemiface of volunteers, the capacitance and transepidermal water loss evaluation revealed the potential interest of this extract for improvement of skin hydration. Electron microscopic examination of the corneocyte envelope on tape strips confirmed its actions. Taken together these data demonstrated that an aqueous extract of S. amara increases human keratinocyte differentiation. PMID:8844461

  7. 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

  8. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    PubMed

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  9. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation. PMID:25418897

  10. Anti-proliferative and apoptosis-triggering potential of disulfiram and disulfiram-loaded polysorbate 80-stabilized PLGA nanoparticles on hepatocellular carcinoma Hep3B cell line.

    PubMed

    Hoda, Muddasarul; Pajaniradje, Sankar; Shakya, Garima; Mohankumar, Kumaravel; Rajagopalan, Rukkumani

    2016-08-01

    There is an emerging trend to restudy known drugs for their anti-cancer potential. One such anti-alcoholic drug, disulfiram, with significant anti-cancer potential was studied for its efficacy against Hep3B cell lines, an in vitro model of hepatocellular carcinoma. Simultaneously, we intended to study the effect of polysorbate 80-stabilized PLGA nanoparticles and its DSF-loaded counterpart. Cell and nuclear staining, comet assay, flow cytometry and Western blots were performed. Results suggest that cell proliferation was inhibited by DSF and its PLGA nanoparticles through cell cycle arrest, triggering activation of apoptotic pathways that culminates with cell death. DSF loaded nanoparticles when compared with free DSF, showed significantly lesser effect due to its sustained drug-releasing property, while empty nanoparticles showed negligible influence on Hep3B cells. Our results suggest that DSF alone contributes to cell death, while polysorbate 80-stabilized PLGA nanoparticles show sustained drug release patterns that would potentially lower dosage regimens. PMID:27013133

  11. Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro.

    PubMed

    Yu, Hui; Pan, Wei-Kang; Zheng, Bai-Jun; Wang, Huai-Jie; Chen, Xin-Lin; Liu, Yong; Gao, Ya

    2016-05-01

    A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin(+) cells decreased whilst the percentage of GFAP(+) cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. PMID:27068376

  12. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212)

    SciTech Connect

    Tron, V.A.; Coughlin, M.D.; Jang, D.E.; Stanisz, J.; Sauder, D.N. )

    1990-04-01

    Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

  13. UVB-induced Senescence in Human Keratinocytes Requires a Functional Insulin-like Growth Factor-1 Receptor and p53

    PubMed Central

    Lewis, Davina A.; Yi, Qiaofang; Travers, Jeffrey B.

    2008-01-01

    To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence. PMID:18216278

  14. Peripheral Blood Monocytes as Adult Stem Cells: Molecular Characterization and Improvements in Culture Conditions to Enhance Stem Cell Features and Proliferative Potential

    PubMed Central

    Ungefroren, Hendrik; Hyder, Ayman; Schulze, Maren; Fawzy El-Sayed, Karim M.; Grage-Griebenow, Evelin; Nussler, Andreas K.; Fändrich, Fred

    2016-01-01

    Adult stem or programmable cells hold great promise in diseases in which damaged or nonfunctional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells resembling specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion, the monocytes downregulate monocyte/macrophage differentiation markers, being indicative of partial dedifferentiation, and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency and resumption of mitosis. Upregulation of stem cell markers and mitotic activity in the cultures was shown to be controlled by autocrine production/secretion of activin A and transforming growth factor-beta (TGF-β). These reprogrammed monocyte derivatives were termed “programmable cells of monocytic origin” (PCMO). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMO in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies. PMID:26798361

  15. The potential of the essential fatty acid-deficient hairless rat as a psoriasis screening model for topical anti-proliferative drugs.

    PubMed

    Jensen, Monika; Groth, Lotte; Hølmer, Gunhild; Hansen, Harald S; Fullerton, Ann

    2002-01-01

    The objective of this study was to establish essential fatty acid deficiency (EFAD) in hairless rats and investigate the potential of this model as a psoriasis screening model by testing the effects of calcipotriol and dithranol on differentiation and proliferation in the epidermis. Hairless rats were fed with a fat-free diet lacking linoleic acid. The EFAD condition was established within 8 weeks. In order to ensure that this condition had been established, several parameters were measured and observed, i.e. animal weight, water consumption, transepidermal water loss, clinical skin symptoms, histology of the epidermis and fatty acid analysis of serum and skin. Immediately after the EFAD condition had been established, the animals were treated with dithranol ointment or different concentrations of calcipotriol solution. A reduction in epidermal thickness of 15-20% was seen after the treatment with calcipotriol. Dithranol and its coal tar-containing vehicle also showed a reductive effect on epidermal thickness. EFAD hairless rats possess various histological changes resembling psoriasis. These histological changes normalise during treatment with anti-psoriatic drugs as calcipotriol, dithranol and coal tar. The results of the present study indicate that the EFAD rat may be a useful model for studies of anti-psoriatic drugs affecting cell proliferation. PMID:12476014

  16. DNA repair in cultured keratinocytes

    SciTech Connect

    Liu, S.C.; Parsons, S.; Hanawalt, P.C.

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  17. Inhibition of Polyamine Uptake Potentiates the Anti-Proliferative Effect of Polyamine Synthesis Inhibition and Preserves the Contractile Phenotype of Vascular Smooth Muscle Cells.

    PubMed

    Grossi, Mario; Phanstiel, Otto; Rippe, Catarina; Swärd, Karl; Alajbegovic, Azra; Albinsson, Sebastian; Forte, Amalia; Persson, Lo; Hellstrand, Per; Nilsson, Bengt-Olof

    2016-06-01

    Increased vascular smooth muscle cell (VSMC) proliferation is a factor in atherosclerosis and injury-induced arterial (re) stenosis. Inhibition of polyamine synthesis by α-difluoro-methylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, attenuates VSMC proliferation with high sensitivity and specificity. However, cells can escape polyamine synthesis blockade by importing polyamines from the environment. To address this issue, polyamine transport inhibitors (PTIs) have been developed. We investigated the effects of the novel trimer44NMe (PTI-1) alone and in combination with DFMO on VSMC polyamine uptake, proliferation and phenotype regulation. PTI-1 efficiently inhibited polyamine uptake in primary mouse aortic and human coronary VSMCs in the absence as well as in the presence of DFMO. Interestingly, culture with DFMO for 2 days substantially (>95%) reduced putrescine (Put) and spermidine (Spd) contents without any effect on proliferation. Culture with PTI-1 alone had no effect on either polyamine levels or proliferation rate, but the combination of both treatments reduced Put and Spd levels below the detection limit and inhibited proliferation. Treatment with DFMO for a longer time period (4 days) reduced Put and Spd below their detection limits and reduced proliferation, showing that only a small pool of polyamines is needed to sustain VSMC proliferation. Inhibited proliferation by polyamine depletion was associated with maintained expression of contractile smooth marker genes. In cultured intact mouse aorta, PTI-1 potentiated the DFMO-induced inhibition of cell proliferation. The combination of endogenous polyamine synthesis inhibition with uptake blockade is thus a viable approach for targeting unwanted vascular cell proliferation in vivo, including vascular restenosis. PMID:26529275

  18. Lawsonia intracellularis proliferative enteropathy in a foal.

    PubMed

    Feary, D J; Gebhart, C J; Pusterla, N

    2007-03-01

    A weanling foal was diagnosed with proliferative enteropathy caused by Lawsonia intracellularis based on history, clinical findings of depression, anorexia, weight loss, colic, diarrhea, and ventral edema, and a combination of serology and fecal PCR. An epidemiological investigation on the premises revealed that many of the other foals and adult horses were seropositive for L. intracellularis, despite being clinically normal, and identified a dog as a potential carrier and source of infection for the foal. The foal was successfully treated with a combination of azithromycin and rifampin. PMID:17410971

  19. Hair-Growth-Promoting Effect of Conditioned Medium of High Integrin α6 and Low CD 71 (α6bri/CD71dim) Positive Keratinocyte Cells

    PubMed Central

    Won, Chong Hyun; Jeong, Yun-Mi; Kang, Sangjin; Koo, Tae-Sung; Park, So-Hyun; Park, Ki-Young; Sung, Young-Kwan; Sung, Jong-Hyuk

    2015-01-01

    Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs) and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor). A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells. PMID:25706512

  20. Non-Proliferative Diabetic Retinopathy Vision Simulator

    MedlinePlus

    ... Diabetic Retinopathy Vision Simulator Non-Proliferative Diabetic Retinopathy Vision Simulator Mar. 03, 2014 How does non-proliferative diabetic retinopathy affect your vision? Nonproliferative diabetic retinopathy, also known as background retinopathy, ...

  1. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration

    PubMed Central

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J.; O’Neill, Catherine A.

    2015-01-01

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration. PMID:26537246

  2. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    PubMed

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-01-01

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration. PMID:26537246

  3. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    SciTech Connect

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly Melino, G. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Association Cell Death and Differentiation c and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  4. beta-Endorphin enhances lymphocyte proliferative responses.

    PubMed Central

    Gilman, S C; Schwartz, J M; Milner, R J; Bloom, F E; Feldman, J D

    1982-01-01

    The opioid peptides alpha- and beta-endorphin and [D-Ala2, Met5]enkephalin were investigated for their effect on the proliferation of resting and activated rat splenic lymphocytes in vitro. beta-Endorphin enhanced the proliferative response of spleen cells to the T cell mitogens concanavalin A and phytohemagglutinin. The effect of beta-endorphin was dose dependent and occurred at peptide concentrations similar to those found in rat plasma. alpha-Endorphin and [D-Ala2, Met5]enkephalin did not affect the proliferative responses to any mitogen tested. Furthermore, the potentiating effect of beta-endorphin was not reversed by treatment with 10 microM naloxone. None of the peptides had any effect on resting, unstimulated spleen cells or on the response to a mixture of lipopolysaccharide and dextran sulfate, which is specifically mitogenic for B lymphocytes. The pharmacological properties of the beta-endorphin potentiation indicate that the effect may be mediated by a nonopiate but beta-endorphin-specific mechanism. These results suggest a possible role for peripheral beta-endorphin and may provide a link between stress and disease susceptibility. PMID:6287475

  5. Experimental proliferative glomerulonephritis in the cat.

    PubMed

    Bishop, S A; Stokes, C R; Lucke, V M

    1992-01-01

    animal models of IC-mediated proliferative GN, this model has potential for application to the study of human IC disease. PMID:1556257

  6. Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

    PubMed

    Frank, S; Kämpfer, H; Podda, M; Kaufmann, R; Pfeilschifter, J

    2000-03-15

    Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation. PMID:10698699

  7. 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. PMID:23649341

  8. Possible role of epidermal keratinocytes in the construction of acupuncture meridians.

    PubMed

    Denda, Mitsuhiro; Tsutsumi, Moe

    2014-04-01

    Acupuncture meridians consist of a network of acupuncture points on the skin, stimulation of which is well established to have a variety of physiological effects. We have previously demonstrated that epidermal keratinocytes contain multiple sensory systems for temperature, mechanical stimuli, electric potentials and other stimuli. These sensory systems generate changes in the calcium-ion concentration in the epidermis, so epidermal keratinocytes can generate spatially-localized electro-physiological patterns in the skin. We have previously demonstrated signaling between epidermal keratinocytes and peripheral nerve systems. Therefore, stimuli sensed by epidermal keratinocytes might be transferred to the unmyelinated nerve fibers that are known to exist in the epidermis and, thence, to the spinal cord and brain. We propose that epidermal keratinocytes form an information-gathering network in the skin and that this network plays a key role in whole-body homeostasis in response to the changing environment. We also hypothesize that this network corresponds to the acupuncture meridians. As supporting examples, we present some striking calcium propagation patterns observed in cultured human keratinocytes after adenosine-triphosphate (ATP) stimulation. These results support the ideas that keratinocytes can generate spatially-restricted signaling patterns after environmental stimulation and that the cultures might be in-vitro models of meridians as an information-gathering network in skin. PMID:24745868

  9. Dental metal-induced innate reactivity in keratinocytes.

    PubMed

    Rachmawati, Dessy; Buskermolen, Jeroen K; Scheper, Rik J; Gibbs, Susan; von Blomberg, B Mary E; van Hoogstraten, Ingrid M W

    2015-12-25

    Gold, nickel, copper and mercury, i.e. four metals frequently used in dental applications, were explored for their capacity to induce innate immune activation in keratinocytes (KC). Due to their anatomical location the latter epithelial cells are key in primary local irritative responses of skin and mucosa. Fresh foreskin-derived keratinocytes and skin and gingiva KC cell lines were studied for IL-8 release as a most sensitive parameter for NF-kB activation. First, we verified that viral-defense mediating TLR3 is a key innate immune receptor in both skin- and mucosa derived keratinocytes. Second, we found that, in line with our earlier finding that ionized gold can mimic viral dsRNA in triggering TLR3, gold is very effective in KC activation. It would appear that epithelial TLR3 can play a key role in both skin- and mucosa localized irritation reactivities to gold. Subsequently we found that not only gold, but also nickel, copper and mercury salts can activate innate immune reactivity in keratinocytes, although the pathways involved remain unclear. Although current alloys have been optimized for minimal leakage of metal ions, secondary factors such as mechanical friction and acidity may still facilitate such leakage. Subsequently, these metal ions may create local irritation, itching and swelling by triggering innate immune reactions, potentially also facilitating the development of metal specific adaptive immunity. PMID:26456670

  10. Keratinocytes can modulate and directly initiate nociceptive responses

    PubMed Central

    Baumbauer, Kyle M; DeBerry, Jennifer J; Adelman, Peter C; Miller, Richard H; Hachisuka, Junichi; Lee, Kuan Hsien; Ross, Sarah E; Koerber, H Richard; Davis, Brian M; Albers, Kathryn M

    2015-01-01

    How thermal, mechanical and chemical stimuli applied to the skin are transduced into signals transmitted by peripheral neurons to the CNS is an area of intense study. Several studies indicate that transduction mechanisms are intrinsic to cutaneous neurons and that epidermal keratinocytes only modulate this transduction. Using mice expressing channelrhodopsin (ChR2) in keratinocytes we show that blue light activation of the epidermis alone can produce action potentials (APs) in multiple types of cutaneous sensory neurons including SA1, A-HTMR, CM, CH, CMC, CMH and CMHC fiber types. In loss of function studies, yellow light stimulation of keratinocytes that express halorhodopsin reduced AP generation in response to naturalistic stimuli. These findings support the idea that intrinsic sensory transduction mechanisms in epidermal keratinocytes can directly elicit AP firing in nociceptive as well as tactile sensory afferents and suggest a significantly expanded role for the epidermis in sensory processing. DOI: http://dx.doi.org/10.7554/eLife.09674.001 PMID:26329459

  11. High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes

    PubMed Central

    Pan, Fuqiang; Guo, Rui; Cheng, Wenguang; Chai, Linlin; Wang, Wenping; Cao, Chuan; Li, Shirong

    2015-01-01

    Background Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM) on ClC-2 chloride channels and cell migration of keratinocytes. Methods Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies. Results We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM) medium, accompanied by the decline of volume-activated Cl− current (ICl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited. Conclusion Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling. PMID:26355894

  12. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  13. Proliferative verrucous leukoplakia: An update.

    PubMed

    Munde, Anita; Karle, Ravindra

    2016-01-01

    Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia, which was first described in 1985 by Hansen et al. Since then, various published case series have presented PVL as a disease with aggressive biological behavior due to its high probability of recurrence and a high rate of malignant transformation, usually higher than 70%. PVL is a long-term progressive condition, which is observed more frequently in elderly women, over 60 years at the time of diagnosis. The buccal mucosa and tongue are the most frequently involved sites. It develops initially as a white plaque of hyperkeratosis that eventually becomes a multifocal disease with confluent, exophytic and proliferative features with a progressive deterioration of the lesions, making it more and more difficult to control. Tobacco use does not seem to have a significant influence on the appearance or progression of PVL and may occur both in smokers and nonsmokers. Prognosis is poor for this seemingly harmless-appearing white lesion of the oral mucosa. At present, the etiology of PVL remains unclear as well as its management and diagnosis, which is still retrospective, late and poorly defined, lacking consensus criteria. This short review discusses the clinical and histopathological features, diagnosis, traditional treatment and the current management of the disease. PMID:27461595

  14. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  15. Lithium Regulates Keratinocyte Proliferation Via Glycogen Synthase Kinase 3 and NFAT2 (Nuclear Factor of Activated T Cells 2)

    PubMed Central

    Hampton, Philip J; Jans, Ralph; Flockhart, Ross J; Parker, Graeme; Reynolds, Nick J

    2012-01-01

    Certain environmental factors including drugs exacerbate or precipitate psoriasis. Lithium is the commonest cause of drug-induced psoriasis but underlying mechanisms are currently unknown. Lithium inhibits glycogen synthase kinase 3 (GSK-3). As lithium does not exacerbate other T-cell-mediated chronic inflammatory diseases, we investigated whether lithium may be acting directly on epidermal keratinocytes by inhibiting GSK-3. We report that lithium-induced keratinocyte proliferation at therapeutically relevant doses (1–2 mM) and increased the proportion of cells in S phase of the cell cycle. Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation. Nuclear factor of activated T cells (NFAT) is an important substrate for GSK-3 and for cyclosporin, an effective treatment for psoriasis that inhibits NFAT activation in keratinocytes as well as in lymphocytes. Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation. Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model. Taken together, these data identify GSK-3 and NFAT2 as key regulators of keratinocyte proliferation and as potential molecular targets relevant to lithium-provoked psoriasis. J. Cell. Physiol. 227: 1529–1537, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678407

  16. Raman spectroscopic analysis of atypical proliferative lesions of the breast

    NASA Astrophysics Data System (ADS)

    Subramanian, K.; Kendall, C.; Stone, N.; Brown, J. C.; McCarthy, K.; Bristol, J.; Chan, Y. H.

    2006-02-01

    Atypical lesions of the breast have potential to turn malignant. The diagnosis of these lesions has increased considerably with screening mammography. A good understanding of their progression to invasive cancer is yet to be proved. Using Raman spectroscopy to study their chemical finger printing at different stages of proliferation a clear picture of whether a progression exists between lesions could be made. At present there is no clear recognition of the biochemical changes that distinguish between the different proliferative lesions of the breast. Our aim is to understand these changes through Raman mapping studies. Raman spectroscopy is a highly sensitive and specific technique for demonstration of biochemical changes in different atypical proliferative lesions of the breast. The technique could be used to classify the different grades and analyse progression of pathology in the proliferative lesions of the breast. Breast pathologists carefully marked 50 ducts and classified the different pathology on H and E sections from biopsy samples. Raman spectra were measured, using a Renishaw Raman Spectrometer, on a 20-micron thick consecutive frozen section. Principal component analysis was undertaken using Matlab. Pseudocolor maps of the principal components scores have been generated. The peaks of the corresponding loads were identified enabling visualisation of the biochemical changes associated with proliferative lesions. Proliferative lesions of the duct were grouped according to the existing standard pathological classification and formed four major groups-HUT, ADH, DCIS and IDC. Spectra of biochemical constituents were fitted to mean spectra from selected regions, taken from maps of each pathology, to identify the relative concentration of the constituents. The study gave an insight into chemical make up of the ducts in each pathology group and showed similar results to earlier studies in progression but no clear-cut demarcation or continuum of the

  17. Reduced proliferative and differentiative activity of mouse pink-eyed dilution melanoblasts is related to apoptosis.

    PubMed

    Hirobe, Tomohisa; Terunuma, Emi

    2012-11-01

    The mouse pink-eyed dilution (p) locus is known to control the melanin content, melanosome morphology, and tyrosinase activity in melanocytes. However, it is not well known whether the p allele is involved in regulating melanocyte proliferation, differentiation, and death. The aim of this study is to investigate in detail the role of the p allele in melanocyte proliferation, differentiation, and death using a cell culture system. The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mice at the p locus (black, C57BL/10JHir-P/P) and their congenic mutant (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with serum-free melanoblast-proliferation medium (MDMDF) and melanocyte-proliferation medium (MDMD). The proliferation and differentiation of p/p melanoblasts in MDMDF or MDMD were greatly inhibited compared with those of P/P melanoblasts and melanocytes. It is possible that apoptosis is related to the reduced proliferative and differentiative activity of p/p melanoblasts/melanocytes. The addition of apoptosis-inhibitors, such as caspase-9 inhibitor (C9I) and Bax-inhibiting peptide (BIP) into MDMDF or MDMD stimulated the proliferation and differentiation of p/p melanoblasts. In contrast, in P/P melanoblasts and melanocytes, C9I and BIP failed to stimulate their proliferation and differentiation. The number of apoptotic keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. Moreover, expression of C9 and Bax in keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. These results suggest that the increased apoptosis in keratinocytes and melanoblasts/melanocytes is related to the reduced proliferative and differentiative activity of p/p melanoblasts. PMID:23106556

  18. Development of an inducible gene expression system for primary murine keratinocytes

    PubMed Central

    Nagarajan, Priyadharsini

    2008-01-01

    Background The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and / or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. Objective To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. Methods We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. Results Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2α in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet inducibility upon continuous culturing. Conclusion We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies. PMID

  19. High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation.

    PubMed

    Lan, Cheng-Che E; Huang, Shu-Mei; Wu, Ching-Shuang; Wu, Chin-Han; Chen, Gwo-Shing

    2016-03-01

    Diabetes is an important health issue because of its increasing prevalence and association with impaired wound healing. Epidermal keratinocytes with overexpressed antiangiogenic molecule thrombospondin-1 (TSP1) have been shown to impair proper wound healing. This study examined the potential involvement of keratinocyte-derived TSP1 on diabetic wound healing. Cultured human keratinocytes and diabetic rat model were used to evaluate the effect of high-glucose environment on TSP1 expression in epidermal keratinocytes, and the molecular mechanisms involved in the process were also studied. We demonstrated that high-glucose environment increased TSP1 expression in keratinocytes. In addition, increased oxidative stress induced DNA hypomethylation at the TSP1 promoter region in keratinocytes exposed to high-glucose environment. Similar findings were found in our diabetic rat model. Early antioxidant administration normalized TSP1 expression and global DNA methylation status in diabetic rat skin and improved wound healing in vivo. Because oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes as alterations in epigenome may not be reversible by controlling glucose levels during the later stages of disease course. PMID:26678678

  20. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  1. 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

  2. Exosomes released by keratinocytes modulate melanocyte pigmentation.

    PubMed

    Lo Cicero, Alessandra; 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

  3. Increased blood viscosity in diabetic proliferative retinopathy.

    PubMed

    Lowe, G D; Ghafour, I M; Belch, J J; Forbes, C D; Foulds, W S; MacCuish, A C

    1986-02-01

    Blood rheology and haemostasis were assessed in 18 diabetics with proliferative retinopathy and in 18 diabetics without proliferative retinopathy, matched for age, sex, smoking habit and type, duration and treatment of diabetes. Proliferative retinopathy was associated with significantly higher levels of blood viscosity at high and low shear rates, which were related to higher levels of plasma viscosity and fibrinogen. Blood urea, glucose, glycosylated haemoglobin and white cell count were also significantly higher, whereas haematocrit, red cell deformability and several other haematological and biochemical variables did not differ significantly in the 2 groups. In view of these findings, and of our recent demonstration that increased blood viscosity also exists in those patients with retinal vein occlusion who develop a similar proliferative retinopathy, we suggest that hyperviscosity may contribute to retinal ischaemia and hence proliferative retinopathy. PMID:3698481

  4. Epidermal Micrografts Produced via an Automated and Minimally Invasive Tool Form at the Dermal/Epidermal Junction and Contain Proliferative Cells That Secrete Wound Healing Growth Factors

    PubMed Central

    Osborne, Sandra N.; Schmidt, Marisa A.; Derrick, Kathleen; Harper, John R.

    2015-01-01

    ABSTRACT OBJECTIVE: The aim of this scientific study was to assess epidermal micrografts for formation at the dermal-epidermal (DE) junction, cellular outgrowth, and growth factor secretion. Epidermal harvesting is an autologous option that removes only the superficial epidermal layer of the skin, considerably limiting donor site damage and scarring. Use of epidermal grafting in wound healing has been limited because of tedious, time-consuming, and inconsistent methodologies. Recently, a simplified, automated epidermal harvesting tool (CelluTome Epidermal Harvesting System; Kinetic Concepts Inc, San Antonio, Texas) that applies heat and suction concurrently to produce epidermal micrografts has become commercially available. The new technique of epidermal harvesting was shown to create viable micrografts with minimal patient discomfort and no donor-site scarring. DESIGN: This study was a prospective institutional review board–approved healthy human study. SETTING: This study was conducted at the multispecialty research facility, Clinical Trials of Texas, Inc, in San Antonio, Texas. PATIENTS: The participants were 15 healthy human volunteers. RESULTS: Epidermal micrografts formed at the DE junction, and migratory basal layer keratinocytes and melanocytes were proliferative in culture. Basement membrane–specific collagen type IV was also found to be present in the grafts, suggesting that the combination of heat and vacuum might cause partial delamination of the basement membrane. Viable basal cells actively secreted key growth factors important for modulating wound healing responses, including vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, platelet-derived growth factor, and transforming growth factor α. CONCLUSIONS: Harvested epidermal micrografts retained their original keratinocyte structure, which is critical for potential re-epithelialization and repigmentation of a wound environment. PMID:26258460

  5. Increased keratinocyte proliferation initiated through downregulation of desmoplakin by RNA interference

    SciTech Connect

    Wan Hong . E-mail: hong.wan@cancer.org.uk; South, Andrew P.; Hart, Ian R.

    2007-07-01

    The intercellular adhesive junction desmosomes are essential for the maintenance of tissue structure and integrity in skin. Desmoplakin (Dp) is a major obligate plaque protein which plays a fundamental role in anchoring intermediate filaments to desmosomal cadherins. Evidence from hereditary human disease caused by mutations in the gene encoding Dp, e.g. Dp haploinsufficiency, suggests that alterations in Dp expression result not only in the disruption of tissue structure and integrity but also could evoke changes in keratinocyte proliferation. We have used transient RNA interference (RNAi) to downregulate Dp specifically in HaCaT keratinocytes. We showed that this Dp downregulation also caused reduced expression of several other desmosomal proteins. Increased cell proliferation and enhanced G{sub 1}-to-S-phase entry in the cell cycle, as monitored by colonial cellular density and BrdU incorporation, were seen in Dp RNAi-treated cells. These proliferative changes were associated with elevated phospho-ERK1/2 and phospho-Akt levels. Furthermore, this increase in phospho-ERK/1/2 and phospho-Akt levels was sustained in Dp RNAi-treated cells at confluence whereas in control cells there was a significant reduction in phosphorylation of ERK1/2. This study indicates that Dp may participate in the regulation of keratinocyte cell proliferation by, in part at least, regulating cell cycle progression.

  6. Keratinocyte Production of Cathelicidin Provides Direct Activity against Bacterial Skin Pathogens

    PubMed Central

    Braff, Marissa H.; Zaiou, Mohamed; Fierer, Joshua; Nizet, Victor; Gallo, Richard L.

    2005-01-01

    Immune defense at an interface with the external environment reflects the functions of physical and chemical barriers provided by epithelial and immune cells. Resident epithelial cells, such as keratinocytes, produce numerous peptides with direct antimicrobial activity but also provide a physical barrier against invading pathogens and signal the recruitment of circulating immune cells, such as neutrophils. Antimicrobial peptides such as cathelicidin are produced constitutively by neutrophils and are inducible in keratinocytes in response to infection. The multiplicity of antimicrobial peptides and their cellular sources has resulted in an incomplete understanding of the role of cathelicidin production by epithelial cells in cutaneous immune defense. Therefore, this study sought to evaluate keratinocyte antimicrobial activity and the potential contribution of keratinocyte cathelicidin to host protection against two leading human skin pathogens. Wild-type mice and those with a targeted deletion of the cathelicidin gene, Cnlp, were rendered neutropenic prior to cutaneous infection. Interestingly, Cnlp-deficient mice remained more susceptible to group A streptococcus infection than mice with Cnlp intact, suggesting the involvement of epithelial cell-derived cathelicidin in host immune defense. Keratinocytes were then isolated in culture and found to inhibit the growth of Staphylococcus aureus, an effect that was partially dependent on their ability to synthesize and activate cathelicidin. Further, lentivirus-mediated delivery of activated human cathelicidin enhanced keratinocyte antimicrobial activity. Combined, these data illustrate the potential contribution of keratinocyte cathelicidin to the innate immune defense of skin against bacterial pathogens and highlight the need to consider epithelial antimicrobial function in the diagnosis and therapy of skin infection. PMID:16177355

  7. Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

    SciTech Connect

    Kanakura, Y.; Thompson, H.; Nakano, T.; Yamamura, T.; Asai, H.; Kitamura, Y.; Metcalfe, D.D.; Galli, S.J.

    1988-09-01

    Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of (35S) sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate (35S) proteoglycans. When ''MMC-like'' cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1-W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these ''second generation PMC'' formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.

  8. 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

  9. Differentiation of human embryonic stem cells into clinically amenable keratinocytes in an autogenic environment.

    PubMed

    Kidwai, Fahad K; Liu, Hua; Toh, Wei Seong; Fu, Xin; Jokhun, Doorgesh S; Movahednia, Mohammad M; Li, Mingming; Zou, Yu; Squier, Christopher A; Phan, Toan T; Cao, Tong

    2013-03-01

    Human embryonic stem cells (hESCs)-derived keratinocytes hold great clinical and research potential. However, the current techniques are hampered by the use of xenogenic components that limits their clinical application. Here we demonstrated an efficient differentiation of H9 hESCs (H9-hESCs) into keratinocytes (H9-Kert) with the minimum use of animal-derived materials. For differentiation, we established two microenvironment systems originated from H9-hESCs (autogenic microenvironment). These autogenic microenvironment systems consist of an autogenic coculture system (ACC) and an autogenic feeder-free system (AFF). In addition, we showed a stage-specific effect of Activin in promoting keratinocyte differentiation from H9-hESCs while repressing the expression of early neural markers in the ACC system. Furthermore, we also explained the effect of Activin in construction of the AFF system made up of extracellular matrix similar to basement membrane extracted from H9-hESC-derived fibroblasts. H9-Kert differentiated in both systems expressed keratinocyte markers at mRNA and protein levels. H9-Kert were also able to undergo terminal differentiation in high Ca(2+) medium. These findings support the transition toward the establishment of an animal-free microenvironment for successful differentiation of hESCs into keratinocytes for potential clinical application. PMID:23235526

  10. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

    PubMed Central

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  11. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2.

    PubMed

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  12. MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

    PubMed

    Meisgen, Florian; Xu Landén, Ning; Wang, Aoxue; Réthi, Bence; Bouez, Charbel; Zuccolo, Michela; Gueniche, Audrey; Ståhle, Mona; Sonkoly, Enikö; Breton, Lionel; Pivarcsi, Andor

    2014-07-01

    Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation. PMID:24670381

  13. Proliferative retinopathies: animal models and therapeutic opportunities.

    PubMed

    Villacampa, Pilar; Haurigot, Virginia; Bosch, Fatima

    2015-01-01

    Proliferative retinopathies are the leading causes of blindness in Western societies. The development of new, more efficacious treatments that take advantage of recent advances in the fields of gene and cell therapy requires further investigations on the mechanisms underlying disease onset and progression, and adequate animal models that recapitulate the pathogenesis of human proliferative retinopathy and allow evaluation of the long-term therapeutic benefits that these therapies can offer. Unfortunately, most models of retinal neovascularization have short-term evolution and diabetic rodents show a very mild retinal phenotype, limited to non-proliferative changes, and do not develop proliferative retinopathy at all. Transgenic mice overexpressing Insulin-like Growth Factor-I (IGF-I) in the retina (TgIGF-I) constitute the only rodent model currently available that develops most of the retinal alterations observed in diabetic eyes, with a temporal evolution that resembles that of the human disease. TgIGF-I have retinal vascular alterations that progress as animals age from non-proliferative to proliferative disease, making these mice an excellent model of proliferative retinopathy that, due to its slow progression, allows long-term evaluation of novel antiangiogenic therapies. At the molecular level, transgenic retinas recapitulate a variety of changes that are also observed in diabetic retinas, which reinforces the validity of this model. In addition to vascular and glial alterations, Tg-IGF-I mice show progressive neurodegeneration that leads to blindness in old animals. Thus, TgIGF-I are a useful model for testing the long-term efficacy and safety of innovative antiangiogenic, glial-modulating and neuroprotective therapies for the treatment of diabetic retinopathy and other retinal proliferative disorders. PMID:25760215

  14. Characterization of microfluidic human epidermal keratinocyte culture

    PubMed Central

    O’Neill, Adrian T.; Monteiro-Riviere, Nancy A.

    2008-01-01

    Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body’s defensive barrier and are used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0%–99.6% viability at 72 h under medium perfusion ranging from 0.025–0.4 μl min−1. HEK maintained this viability while ∼100% confluent—a level not possible in 96 well plates. Microscale HEK cultures offer the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new discoveries in toxicological screening methods and wound healing techniques. PMID:19002858

  15. Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.

    PubMed

    Boros, Gábor; Karikó, Katalin; Muramatsu, Hiromi; Miko, Edit; Emri, Eszter; Hegedűs, Csaba; Emri, Gabriella; Remenyik, Éva

    2016-01-01

    In vitro-synthesized mRNA containing nucleoside modifications has great therapeutical potential to transiently express proteins with physiological importance. One such protein is photolyase which rapidly removes UV-induced DNA damages, but this enzyme is absent in humans. Here, we apply a novel mRNA-based platform to achieve functional nonhuman photolyase production in cultured human keratinocytes. Transfection of nucleoside-modified mRNA encoding photolyase leads to accelerated repair of DNA photolesions in human keratinocytes. PMID:27236802

  16. Chitin Modulates Innate Immune Responses of Keratinocytes

    PubMed Central

    Koller, Barbara; Müller-Wiefel, Alisa Sophie; Rupec, Rudolph; Korting, Hans Christian; Ruzicka, Thomas

    2011-01-01

    Background Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. Methodology/Principal Findings We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. Conclusions/Significance We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined. PMID:21383982

  17. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin MD.; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  18. On the interaction of alginate-based core-shell nanocarriers with keratinocytes in vitro.

    PubMed

    Nguyen, Hoang Truc Phuong; Allard-Vannier, Emilie; Gaillard, Cédric; Eddaoudi, Imane; Miloudi, Lynda; Soucé, Martin; Chourpa, Igor; Munnier, Emilie

    2016-06-01

    Calcium alginate nanocarriers (CaANCs) were developed as a potential tool for delivery of hydrophobic active molecules such as pharmaceutical and cosmetic active ingredients. In this study, we focused on interactions between CaANCs and keratinocytes in culture and examined toxicity, internalization and drug release. Prior to cellular interactions, cryogenic transmission electron microscopy images showed that CaANCs appear as regular, spherical and dense particles, giving evidence of the surface gelation of CaANCs. Their size, around 200nm, was stable under tested conditions (temperature, culture media, presence of serum and presence of encapsulated dye), and their toxicity on keratinocytes was very low. Flow cytometry assays showed that CaANCs are internalized into keratinocytes by endocytosis with a predominant implication of the caveolae-mediated route. Förster resonance energy transfer (FRET) demonstrated that after a 2h contact, the release of CaANC contents in the cytoplasm of keratinocytes was almost complete. The endocytosis of CaANCs by a lysosome-free pathway, and the rapid release of their contents inside keratinocytes, will allow vectorized molecules to fully exhibit their pharmacological or cosmetic activity. PMID:26962764

  19. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway.

    PubMed

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin Md; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-03-15

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  20. Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes.

    PubMed

    Cavazza, A; Cocchiarella, F; Bartholomae, C; Schmidt, M; Pincelli, C; Larcher, F; Mavilio, F

    2013-09-01

    Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders. PMID:23615186

  1. A Heat-Sensitive TRP Channel Expressed in Keratinocytes

    NASA Astrophysics Data System (ADS)

    Peier, Andrea M.; Reeve, Alison J.; Andersson, David A.; Moqrich, Aziz; Earley, Taryn J.; Hergarden, Anne C.; Story, Gina M.; Colley, Sian; Hogenesch, John B.; McIntyre, Peter; Bevan, Stuart; Patapoutian, Ardem

    2002-06-01

    Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

  2. Skin graft storage and keratinocyte viability.

    PubMed

    Fahmy, F S; Navsaria, H A; Frame, J D; Jones, C R; Leigh, I M

    1993-06-01

    The viability of human split skin grafts stored in four solutions has been assessed by monitoring the percentage of viable keratinocytes in the stored grafts. Skin grafts stored in RM+ (Ready Mix) tissue culture medium remained more viable than those stored in Hartmann's, Marshall's or saline solutions. By day 10 (postoperative), the percentage of viable keratinocytes of those grafts stored in RM+ was around 85%, compared to a value of around 10% for the other media. By day 30, RM+ achieved a value of around 60% keratinocyte viability compared to a value approaching 1% in the other storage media under investigation. RM+ provides mitogens, nutrients, growth factors and physiological pH, all of which are important factors for successful skin graft storage. PMID:8330085

  3. Constitutive phenolics of Harpephyllum caffrum (Anacardiaceae) and their biological effects on human keratinocytes.

    PubMed

    Nawwar, Mahmoud; Hussein, Sahar; Ayoub, Nahla; Hashim, Amani; El-Sharawy, Reham; Lindequist, Urlike; Harms, Manualle; Wende, Kristian

    2011-12-01

    Assessment of the UV protecting potential of an aqueous methanol leaf extract of Harpephyllum caffrum proved that it possesses a distinct radical scavenging effect and inhibits the production of the proinflammatory cytokine IL-6 by human keratinocytes (HaCaT cells) following UV radiation. Phytochemical investigation of this extract led to isolation and structural determination of the hitherto unknown phenolics, kaempferol 3-O-(2″-sulphatogalactopyranoside), its quercetin analogue and 3-methoxyellagic acid 4-O-galactopyranoside in addition to 18 known phenolic compounds. The structures were determined by spectroscopic and conventional methods of analysis. Flavonoid sulphatoglycosides which have been rarely found in nature were major phenolic constituents of this plant, and this is the first report of the isolation of any of them from Anacardiaceae. The extract was found to diminish UV phototoxic reaction of keratinocytes. However, the isolated kaempferol sulphatogalactopyranoside did not interact with UVB triggered IL-6 production of HaCaT keratinocytes. PMID:21907269

  4. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    PubMed

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  5. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    PubMed Central

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  6. Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes

    PubMed Central

    Ventress, Jennifer K.; Partridge, Lynda J.; Read, Robert C.; Cozens, Daniel; MacNeil, Sheila

    2016-01-01

    Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection. PMID:27467693

  7. RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

    SciTech Connect

    Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara

    2010-03-10

    We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

  8. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells.

    PubMed

    Haase, Ingo; Knaup, Renate; Wartenberg, Maria; Sauer, Heinrich; Hescheler, Jürgen; Mahrle, Gustav

    2007-12-01

    Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments. PMID:17716780

  9. A MECHANISM FOR ASYMMETRIC CELL DIVISION RESULTING IN PROLIFERATIVE ASYNCHRONICITY

    PubMed Central

    Dey-Guha, Ipsita; Alves, Cleidson P.; Yeh, Albert C.; Salony; Sole, Xavier; Darp, Revati; Ramaswamy, Sridhar

    2014-01-01

    All cancers contain an admixture of rapidly and slowly proliferating cancer cells. This proliferative heterogeneity complicates the diagnosis and treatment of cancer patients because slow proliferators are hard to eradicate, can be difficult to detect, and may cause disease relapse sometimes years after apparently curative treatment. While clonal selection theory explains the presence and evolution of rapid proliferators within cancer cell populations, the circumstances and molecular details of how slow proliferators are produced is not well understood. Here, a β1-integrin/FAK/mTORC2/AKT1-associated signaling pathway is discovered that can be triggered for rapidly proliferating cancer cells to undergo asymmetric cell division and produce slowly proliferating AKT1low daughter cells. In addition, evidence indicates that the proliferative output of this signaling cascade involves a proteasome-dependent degradation process mediated by the E3 ubiquitin ligase TTC3. These findings reveal that proliferative heterogeneity within cancer cell populations, in part, is produced through a targetable signaling mechanism, with potential implications for understanding cancer progression, dormancy, and therapeutic resistance. PMID:25582703

  10. Combined use of hyperbaric oxygen and sprayed keratinocyte suspension to tackle a difficult wound

    PubMed Central

    Wilks, D; Rawlins, J; Matteucci, PL

    2014-01-01

    This is the first reported case in the literature to combine the use of a well established therapy to achieve wound healing (ie hyperbaric oxygen treatment) and a novel sprayed keratinocyte suspension technique to treat a challenging wound successfully. The merits and potential issues associated with these treatments are outlined and the case is detailed. PMID:25198965

  11. 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...

  12. Loss of proliferative calcium dependence: simple in vitro indicator of tumorigenicity.

    PubMed Central

    Swierenga, S H; Whitfield, J F; Karasaki, S

    1978-01-01

    The proliferative activities of three lines of "normal" epithelioid rat liver cells and six tumorigenic liver cell lines in the presence of a wide range of calcium concentrations were measured by a simple colony forming assay. The proliferative activities of the normal cells and, to a lesser extent, of the cells of a marginally tumorigenic line were directly proportional to the extracellular calcium concentration. The proliferative activities of the cells of the strongly tumorigenic lines, on the other hand, were either uninfluenced by or inversely proportional to the extracellular calcium concentration. Thus, the proliferative response to the extracellular calcium concentration is a sensitive indicator of the carcinogenic potential of liver cells. Images PMID:282624

  13. 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

  14. Sulfation of estradiol in human epidermal keratinocyte.

    PubMed

    Kushida, Akira; Hattori, Kenji; Yamaguchi, Nozomi; Kobayashi, Tetsuyuki; Date, Akira; Tamura, Hiroomi

    2011-01-01

    Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E(2)) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E(2) and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [(3)H] labeled E(2) was converted to E(2) sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E(2) at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E(2) inactivation is primarily mediated by SULT1E1 in keratinocyte and E(2) action is likely suppressed in epidermal differentiation. PMID:21720030

  15. TOXICITY OF AMORPHOUS SILICA NANOPARTICLES IN MOUSE KERATINOCYTES

    SciTech Connect

    Yu, Kyung; Wang, Wei; Gu, Baohua; Hussain, Saber

    2009-01-01

    The present study was designed to examine the uptake, localization and the cytotoxic effects of well-dispersed amorphous silica nanoparticles in mouse keratinocytes (HEL-30). Mouse keratinocytes were exposed for 24h to various concentrations of amorphous silica nanoparticles in homogeneous suspensions of average size distribution (30, 48, 118 and 535 nm SiO2) then assessed for uptake and biochemical changes. Results of transmission electron microscopy revealed all sizes of silica were taken up into the cells and localized into the cytoplasm. The lactate dehydrogenase (LDH) assay shows LDH leakage was dose- and size-dependent with exposure to 30 and 48 nm nanoparticles. However, no LDH leakage was observed for either 118 or 535 nm nanoparticles. The mitochondrial viability assay (MTT) showed significant toxicity for 30 and 48 nm at high concentrations (100 g/mL) compare to the 118 and 535 nm particles. Further studies were carried out to investigate if cellular reduced GSH and mitochondria membrane potential are involved in the mechanism of SiO2 toxicity. The redox potential of cells (GSH) was reduced significantly at concentrations of 50, 100 and 200 g/mL at 30 nm nanoparticle exposures. However, silica nanoparticles larger than 30 nm showed no changes in GSH levels. Reactive oxygen species (ROS) formation did not show any significant change between controls and the exposed cells. In summary, amorphous silica nanoparticles below 100 nm induced cytotoxicity suggest size-of the particles is critical to produce biological effects.

  16. Vitamin D derivatives enhance cytotoxic effects of H2O2 or cisplatin on human keratinocytes.

    PubMed

    Piotrowska, Anna; Wierzbicka, Justyna; Ślebioda, Tomasz; Woźniak, Michał; Tuckey, Robert C; Slominski, Andrzej T; Żmijewski, Michał A

    2016-06-01

    Although the skin production of vitamin D is initiated by ultraviolet radiation type B (UVB), the role vitamin D plays in antioxidative or pro-oxidative responses remains to be elucidated. We have used immortalized human HaCaT keratinocytes as a model of proliferating epidermal cells to test the influence of vitamin D on cellular response to H2O2 or the anti-cancer drug, cisplatin. Incubation of keratinocytes with 1,25(OH)2D3 or its low calcemic analogues, 20(OH)D3, 21(OH)pD or calcipotriol, sensitized cells to ROS resulting in more potent inhibition of keratinocyte proliferation by H2O2 in the presence of vitamin D compounds. These results were supported by cell cycle and apoptosis analyses, and measurement of the mitochondrial transmembrane potentials (MMP), however some unique properties of individual secosteroids were observed. Furthermore, in HaCaT keratinocytes treated with H2O2, 1,25(OH)2D3, 21(OH)pD and calcipotriol stimulated the expression of SOD1 and CAT genes, but not SOD2, indicating a possible role of mitochondria in ROS-modulated cell death. 1,25(OH)2D3 also showed a short-term, protective effect on HaCaT keratinocytes, as exemplified by the inhibition of apoptosis and the maintenance of MMP. However, with prolonged incubation with H2O2 or cisplatin, 1,25(OH)2D3 caused an acceleration in the death of the keratinocytes. Therefore, we propose that lead vitamin D derivatives can protect the epidermis against neoplastic transformation secondary to oxidative or UV-induced stress through activation of vitamin D-signaling. Furthermore, our data suggest that treatment with low calcemic vitamin D analogues or the maintenance of optimal level of vitamin D by proper supplementation, can enhance the anticancer efficacy of cisplatin. PMID:27083311

  17. Effects triggered by platinum nanoparticles on primary keratinocytes

    PubMed Central

    Konieczny, Piotr; Goralczyk, Anna Grazyna; Szmyd, Radoslaw; Skalniak, Lukasz; Koziel, Joanna; Filon, Francesca Larese; Crosera, Matteo; Cierniak, Agnieszka; Zuba-Surma, Ewa K; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Pyza, Elzbieta; Semik, Danuta; Woznicka, Olga; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 μg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents

  18. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    PubMed

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses. PMID:26403416

  19. Health assessment of environmental pollutants; Proliferative and degenerative diseases

    SciTech Connect

    Stuart, B.O. )

    1987-01-01

    The health assessments of environmental air contaminants are at present frequently based upon probability of cancer, if this has been identified as a potential result of prolonged exposure to the particular inhalation hazard. However, for many airborne hazards chronic inhalation exposure may result in morbidity or mortality risks due to chronic degenerative diseases such as emphysema, fibrosis, or chronic obstructive pulmonary disease that may be nearly as great or greater than those of more widely recognized neoplastic or proliferative disease. The relative hazards of environmentally released radioactive and chemical air contaminants, i.e., radon daughters and diesel engine exhaust, are discussed as examples.

  20. Interleukin-22 Promotes Wound Repair in Diabetes by Improving Keratinocyte Pro-Healing Functions.

    PubMed

    Avitabile, Simona; Odorisio, Teresa; Madonna, Stefania; Eyerich, Stefanie; Guerra, Liliana; Eyerich, Kilian; Zambruno, Giovanna; Cavani, Andrea; Cianfarani, Francesca

    2015-11-01

    Impaired re-epithelialization, imbalanced expression of cytokines and growth factors, and vascular disease contribute to healing impairment in diabetes. IL-22, a pro-inflammatory cytokine mediating a cross-talk between immune system and epithelial cells, has been shown to have a role in repair processes. In this study we aimed to investigate IL-22 regenerative potential in the poor healing context of diabetic wounds. By using streptozotocin-induced diabetic mice, we demonstrated that IL-22 wound treatment significantly accelerated the healing process, by promoting re-epithelialization, granulation tissue formation, and vascularization. Improved re-epithelialization was associated with increased keratinocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation. We showed that endogenous IL-22 content was reduced at both mRNA and protein level during the inflammatory phase of diabetic wounds, with fewer IL-22-positive cells infiltrating the granulation tissue. We demonstrated that IL-22 treatment promoted proliferation and injury repair of hyperglycemic keratinocytes and induced activation of STAT3 and extracellular signal-regulated kinase transduction pathways in keratinocytes grown in hyperglycemic condition or isolated from diabetic patients. Finally, we demonstrated that IL-22 treatment was able to inhibit diabetic keratinocyte differentiation while promoting vascular endothelial growth factor release. Our data indicate a pro-healing role of IL-22 in diabetic wounds, suggesting a therapeutic potential for this cytokine in diabetic ulcer management. PMID:26168231

  1. Epithelial expression of keratinocytes growth factor in oral precancer lesions

    PubMed Central

    Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily

    2016-01-01

    Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated. PMID:27274338

  2. Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

    PubMed

    MacNulty, E E; Ryder, N S

    1996-02-01

    Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance. PMID:8605230

  3. The effect of 648 nm diode laser irradiation on second messengers in senescent human keratinocytes

    NASA Astrophysics Data System (ADS)

    Hawkins Evans, D.; Abrahamse, H.

    2009-02-01

    Background/purpose: Stress induced premature senescence (SIPS) is defined as the long-term effect of subcytotoxic stress on proliferative cell types. Cells in SIPS display differences at the level of protein expression which affect energy metabolism, defense systems, redox potential, cell morphology and transduction pathways. This study aimed to determine the effect of laser irradiation on second messengers in senescent cells and to establish if that effect can be directly linked to changes in cellular function such as cell viability or proliferation. Materials and Methods: Human keratinocyte cell cultures were modified to induce premature senescence using repeated sub-lethal stresses of 200 uM H2O2 or 5% OH every day for four days with two days recovery. SIPS was confirmed by senescence-associated β-galactosidase staining. Control conditions included normal, repeated stress of 500 uM H2O2 to induce apoptosis and 200 uM PBN as an anti-oxidant or free radical scavenger. Cells were irradiated with 1.5 J/cm2 on day 1 and 4 using a 648 nm diode laser (3.3 mW/cm2) and cellular responses were measured 1 h post irradiation. The affect on second messengers was assessed by measuring cAMP, cGMP, nitric oxide and intracellular calcium (Ca2+) while functional changes were assessed using cell morphology, ATP cell viability, LDH membrane integrity and WST-1 cell proliferation. Results: Results indicate an increase in NO and a decrease in cGMP and Ca2+ in 200 uM H2O2 irradiated cells while PBN irradiated cells showed a decrease in cAMP and an increase in ATP viability and cell proliferation. Conclusion: Laser irradiation influences cell signaling which ultimately changes the biological function of senescent cells. If laser therapy can stimulate the biological function of senescent cells it may be beneficial to conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing of arteries in patients with advanced heart disease, neurodegenerative disorders and

  4. DIAGNOSTIC CRITERIA FOR PROLIFERATIVE THYROID LESIONS IN BONY FISHES

    EPA Science Inventory

    Thyroid proliferative lesions are rather common in bony fishes but disagreement exists in the fish pathology community concerning diagnostic criteria for hyperplastic versus neoplastic lesions. To simplify the diagnosis of proliferative thyroid lesions and to reduce confusion reg...

  5. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  6. Tualang Honey protects keratinocytes from ultraviolet radiation induced inflammation and DNA damage†

    PubMed Central

    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/cm2) irradiation. We found that treatment of tualang honey inhibited UVB-induced DNA damage, and enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG). Treatment of tualang honey inhibited UVB-induced nuclear translocation of NF-κB, and degradation of IκBα in murine keratinocyte cell line. Treatment of tualang honey also inhibited UVB-induced inflammatory cytokines and inducible nitric oxide synthase protein expression. Furthermore, treatment of tualang honey inhibited UVB-induced COX-2 expression and PGE2 production. Taken together, we provide evidence that 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. PMID:22276569

  7. Histamine induces proliferation in keratinocytes from atopic dermatitis patients

    PubMed Central

    Glatzer, Franziska; Gschwandtner, Maria; Ehling, Sarah; Rossbach, Kristine; Janik, Katrin; Klos, Andreas; Bäumer, Wolfgang; Kietzmann, Manfred; Werfel, Thomas; Gutzmer, Ralf

    2015-01-01

    Background Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in atopic dermatitis and its underlying mechanisms are not completely understood by now. Objective Since elevated levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to H4R activation, on the proliferation of human and murine keratinocytes. Methods The expression of H4R on human and murine keratinocytes was detected by real-time PCR. Keratinocyte proliferation was evaluated by different in vitro cell proliferation assays, scratch assays and measurement of epidermal thickness of murine skin. Results We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis as compared to non-atopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes and H4R transfected HaCaT cells with histamine and H4R agonist resulted in an increase of proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared to control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with lipopolysaccharide and peptidoglycane. Conclusion The H4R is highly expressed on keratinocytes from atopic dermatitis patients and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in atopic dermatitis. PMID:23932072

  8. Human keratinocyte culture from the peritonsillar mucosa.

    PubMed

    Neugebauer, P; Bonnekoh, B; Wevers, A; Michel, O; Mahrle, G; Krieg, T; Stennert, E

    1996-01-01

    Tonsillectomy tissue can be used as a routine source for cultures of oropharyngeal keratinocytes. In so doing, a peritonsillar strip of unaltered mucosa was dissected in the upper submucosa. Subsequent trypsinization yielded 7.0 +/- 3.4 x 10(6) keratinocytes per bilateral tonsillectomy. Keratinocyte attachment and growth in primary culture were promoted by sublethally irradiated 3T3 murine fibroblasts. Three subcultures could be performed without a feeder layer and were characterized by a population doubling time of 4.5 days during log growth phase. Electrophoretic and immunoblot analysis of the third subculture revealed a strong expression of keratin pairs 5/14 and 6/16 as well as keratins 7 and 19, whereas keratins 8/18 were expressed less intensely. The lowest intensity, was found for keratin 13, which is known to be indicative of the differentiated mucosa. The culture technique thus provides an easily available in vitro model for morphological and functional studies on the epithelial compartment of human oropharyngeal mucosa. PMID:8737778

  9. Methicillin-Resistant Staphylococcus aureus Adaptation to Human Keratinocytes

    PubMed Central

    Soong, Grace; Paulino, Franklin; Wachtel, Sarah; Parker, Dane; Wickersham, Matthew; Zhang, Dongni; Brown, Armand; Lauren, Christine; Dowd, Margaret; West, Emily; Horst, Basil; Planet, Paul

    2015-01-01

    ABSTRACT Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. PMID:25900653

  10. The effect of pantothenic acid deficiency on keratinocyte proliferation and the synthesis of keratinocyte growth factor and collagen in fibroblasts.

    PubMed

    Kobayashi, Daisaku; Kusama, Miho; Onda, Masaaki; Nakahata, Norimichi

    2011-01-01

    It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol derivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. PMID:21258175

  11. Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes.

    PubMed Central

    Ollert, M W; Söhnchen, R; Korting, H C; Ollert, U; Bräutigam, S; Bräutigam, W

    1993-01-01

    We established an in vitro adherence model with primarily cultured human keratinocytes as target cells which allows for the investigation of the molecular mechanisms that are responsible for Candida albicans host cell attachment in the initiation of cutaneous candidosis. The extent of C. albicans binding to cultured human keratinocytes was dependent on the yeast inoculum size and the incubation temperature. Heat and paraform-aldehyde treatment of yeasts completely abolished the binding activity of C. albicans. Of the different Candida species tested, C. albicans was by far the most adhesive species. C. albicans adherence was blocked by the acid protease inhibitor pepstatin A and the metabolic inhibitor sodium azide. The latter, however, was much less effective when yeasts were preincubated, suggesting that sodium azide was mainly acting on the keratinocytes. The extracellular matrix protein fibronectin was slightly inhibitory, whereas the fibronectin-derived peptides RGD and RGDS were not able to prevent attachment. PepTite-2000, another RGD-containing synthetic peptide, reduced C. albicans adherence by a margin of 25% (P < 0.005). CDPGYIGSR-NH2, which is a synthetic adhesive peptide derived from the laminin B chain, was much more efficient in its inhibitory activity than the RGD peptides and reduced C. albicans adherence to cultured human keratinocytes up to 76% (P < 0.001). Laminin itself and the synthetic pentapeptide YIGSR were less active. A dose-dependent reduction in adherence was also observed with collagen type III. Additionally, saccharides were tested for their potential to inhibit C. albicans attachment to keratinocytes. The most potent competitive saccharide inhibitors of C. albicans adherence to human keratinocytes were the amino sugars D-(+)-glucosamine and D-(+)-galactosamine with one isolate of C. albicans (4918) and D-(+)-glucosamine and alpha-D-(+)-fucose with another C. albicans isolate (Sp-1). Collectively, our data suggest the existence of

  12. Adipose derived mesenchymal stem cells express keratinocyte lineage markers in a co-culture model.

    PubMed

    Irfan-Maqsood, M; Matin, M M; Heirani-Tabasi, A; Bahrami, M; Naderi-Meshkin, H; Mirahmadi, M; Hassanzadeh, H; Sanjar Moussavi, N; Raza-Shah, H; Raeesolmohaddeseen, M; Bidkhori, H; Bahrami, A R

    2016-01-01

    be concluded that mesenchymal stem cells can regenerate the damaged skin if transplanted to damaged area but for their successful differentiation and enhanced regeneration, they need a population of keratinocytes in situ which need further experiments for validation of co-culture model and its potential for being used in clinics. PMID:27188869

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

    SciTech Connect

    Zhang Kunzhong; Tian Yeping; Yin Liangjie; Zhang Mei; Beck, Lisa A.; Zhang, Bingrong; Okunieff, Paul; Zhang Lurong; Vidyasagar, Sadasivan

    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 by 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 the

  14. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Cook, Anthony L. Snow, Elizabeth T.

    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-mutated (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.

  15. Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

    PubMed Central

    Wiener, Dominique J.; Doherr, Marcus G.; Müller, Eliane J.; Welle, Monika M.

    2016-01-01

    Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients. PMID:26788850

  16. Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog.

    PubMed

    Wiener, Dominique J; Doherr, Marcus G; Müller, Eliane J; Welle, Monika M

    2016-01-01

    Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients. PMID:26788850

  17. Ampelopsis japonica Makino Extract Inhibits the Inflammatory Reaction Induced by Pathogen-Associated Molecular Patterns in Epidermal Keratinocytes

    PubMed Central

    Choi, Mi-Ra; Choi, Dae-Kyoung; Kim, Ki-Duck; Kim, Sue Jeong; Kim, Dong-Il; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok

    2016-01-01

    Background Keratinocytes are the major cells in epidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been known that pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes. Objective The aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes. Methods The effects of AE were determined using poly (I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models. Results In cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated nuclear factor-κB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia. Conclusion These results suggest that AE may be a potential candidate for the treatment of skin inflammation. PMID:27274634

  18. Up-regulation of Interferon-inducible protein 16 contributes to psoriasis by modulating chemokine production in keratinocytes

    PubMed Central

    Cao, Tianyu; Shao, Shuai; Li, Bing; Jin, Liang; Lei, Jie; Qiao, Hongjiang; Wang, Gang

    2016-01-01

    Psoriasis is a common chronic inflammatory skin disease characterized by epidermal hyperplasia and dermal inflammation. Keratinocyte activation is known to play a critical role in psoriasis, but the underlying mechanism remains unclear. Interferon-inducible protein 16 (IFI16), an innate immune system sensor, is reported to affect keratinocyte function. We therefore hypothesized that IFI16 promotes psoriasis by modulating keratinocyte activation. In the present study, we cinfirmed that IFI16 was overexpressed in epidermal keratinocytes of psoriasis patients. In addition, psoriasis-related cytokines, including IFN-γ, TNF-α, IL-17 and IL-22, induced IFI16 up-regulation in keratinocytes via activation of STAT3 signaling. We also observed that IFI16 activated the TBK1-NF-κB signaling, leading to the production of CXCL10 and CCL20. Importantly, knocking down p204, which is reported as the mouse orthologous of human IFI16, inhibited epidermal hyperplasia in mice with imiquimod-induced psoriasiform dermatitis. These findings indicate that IFI16 plays a critical role in the pathogenesis of psoriasis and may be a potential therapeutic target. PMID:27137868

  19. 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. PMID:26191223

  20. The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

    PubMed Central

    Straight, S W; Herman, B; McCance, D J

    1995-01-01

    The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles. PMID:7707548

  1. Oligonucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell type.

    PubMed

    White, P J; Fogarty, R D; McKean, S C; Venables, D J; Werther, G A; Wraight, C J

    1999-05-01

    The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation. PMID:10233759

  2. Lawsonia intracellularis and equine proliferative enteropathy.

    PubMed

    Page, Allen E; Slovis, Nathan M; Horohov, David W

    2014-12-01

    Lawsonia intracellularis is the etiologic agent for equine proliferative enteropathy (EPE), which typically affects weanling and yearling horses. In North America, EPE cases often occur between August and January, although cases outside of this time frame have been reported. Clinical signs of EPE are usually nonspecific and include lethargy, pyrexia, anorexia, peripheral edema, weight loss, colic, and diarrhea. Diagnosis is based on the presence of hypoproteinemia and hypoalbuminemia along with clinical signs and positive commercial serologic and/or molecular testing. Treatment requires the use of antimicrobials with good intracellular penetration and supportive care to prevent or decrease secondary complications. PMID:25300636

  3. Differential effects of detergents on keratinocyte gene expression.

    PubMed

    van Ruissen, F; Le, M; Carroll, J M; van der Valk, P G; Schalkwijk, J

    1998-04-01

    We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on

  4. Confocal imaging of benign and malignant proliferative skin lesions in vivo

    NASA Astrophysics Data System (ADS)

    Gonzalez, Salvador; Rajadhyaksha, Milind M.; Anderson, R. Rox

    1999-06-01

    Near-infrared confocal reflectance microscopy (CM) provides non- invasive real-time images of thin en-face tissue sections with high resolution and contrast. Imaging of cells, nuclei, other organelles, microvessels, and hair follicles has been possible at resolution comparable to standard histology, to a maximum depth of 250-300 μm in human skin in vivo. We have characterized psoriasis as a prototype of benign proliferative skin conditions, and non-pigmented skin malignancies in vivo based on their unstained, native histologic features using CM. Our data shows that reflectance CM may potentially diagnose and morphometrically evaluate proliferative skin lesions in vivo.

  5. Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes.

    PubMed

    Gamady, Anat; Koren, Ruth; Ron, Dina; Liberman, Uri A; Ravid, Amiram

    2003-06-01

    The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury. PMID:12761878

  6. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

    PubMed Central

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-01-01

    ABSTRACT The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  7. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

    PubMed

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-12-15

    The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  8. Survival and proliferative roles of erythropoietin beyond the erythroid lineage

    PubMed Central

    Noguchi, Constance Tom; Wang, Li; Rogers, Heather M.; Teng, Ruifeng; Jia, Yi

    2011-01-01

    Since the isolation and purification of erythropoietin (EPO) in 1977, the essential role of EPO for mature red blood cell production has been well established. The cloning and production of recombinant human EPO led to its widespread use in treating patients with anaemia. However, the biological activity of EPO is not restricted to regulation of erythropoiesis. EPO receptor (EPOR) expression is also found in endothelial, brain, cardiovascular and other tissues, although at levels considerably lower than that of erythroid progenitor cells. This review discusses the survival and proliferative activity of EPO that extends beyond erythroid progenitor cells. Loss of EpoR expression in mouse models provides evidence for the role of endogenous EPO signalling in nonhaematopoietic tissue during development or for tissue maintenance and/or repair. Determining the extent and distribution of receptor expression provides insights into the potential protective activity of erythropoietin in brain, heart and other nonhaematopoietic tissues. PMID:19040789

  9. Analysis and Enhancements of a Prolific Macroscopic Model of Epilepsy

    PubMed Central

    Fietkiewicz, Christopher; Loparo, Kenneth A.

    2016-01-01

    Macroscopic models of epilepsy can deliver surprisingly realistic EEG simulations. In the present study, a prolific series of models is evaluated with regard to theoretical and computational concerns, and enhancements are developed. Specifically, we analyze three aspects of the models: (1) Using dynamical systems analysis, we demonstrate and explain the presence of direct current potentials in the simulated EEG that were previously undocumented. (2) We explain how the system was not ideally formulated for numerical integration of stochastic differential equations. A reformulated system is developed to support proper methodology. (3) We explain an unreported contradiction in the published model specification regarding the use of a mathematical reduction method. We then use the method to reduce the number of equations and further improve the computational efficiency. The intent of our critique is to enhance the evolution of macroscopic modeling of epilepsy and assist others who wish to explore this exciting class of models further. PMID:27144054

  10. Vitrectomy for Proliferative Diabetic Retinopathy Associated with Klinefelter Syndrome

    PubMed Central

    Tajiri, Kensuke; Otsuki, Kohei; Sato, Takaki; Kimura, Daisaku; Kobayashi, Takatoshi; Kida, Teruyo; Sugasawa, Jun; Ikeda, Tsunehiko

    2015-01-01

    Introduction We encountered a patient with Klinefelter syndrome (KS) who experienced poor outcomes after vitrectomy for proliferative diabetic retinopathy (PDR). Case A 44-year-old male with poorly controlled diabetes was diagnosed with KS by chromosome analysis. Ocular findings revealed severe PDR complicated with extensive preretinal hemorrhages and traction retinal detachment in his left eye, and pars plana vitrectomy was subsequently performed for treatment. Results A clotting hemorrhage developed during surgery and proved difficult to control. Due to postoperative bleeding and redetachment, the vitrectomy was repeated. At the second operation, we performed a silicone oil tamponade; however, the retina was redetached under the silicone oil, and the light perception vision ultimately disappeared. Conclusion The patient, despite showing increased blood coagulability due to diabetes, presented severe coagulopathy, likely related to KS. In patients with KS and severe PDR, the potential difficulty of vitrectomy should always be kept in mind. PMID:26955343

  11. Kanglaite attenuates UVB-induced down-regulation of aquaporin-3 in cultured human skin keratinocytes

    PubMed Central

    SHAN, SHI-JUN; XIAO, TING; CHEN, JOHN; GENG, SHI-LING; LI, CHANG-PING; XU, XUEGANG; HONG, YUXIAO; JI, CHAO; GUO, YING; WEI, HUACHEN; LIU, WEI; LI, DAPENG; CHEN, HONG-DUO

    2012-01-01

    Ultraviolet (UV) radiation plays an important role in the pathogenesis of skin photoaging. Depending on the wavelength of UV, the epidermis is affected primarily by UVB. One major characteristic of photoaging is the dehydration of the skin. Membrane-inserted water channels (aquaporins) are involved in this process. In this study we demonstrated that UVB radiation induced aquaporin-3 (AQP3) down-regulation in cultured human skin keratinocytes. Kanglaite is a mixture consisting of extractions of Coix Seed, which is an effective anti-neoplastic agent and can inhibit the activities of protein kinase C and NF-κB. We demonstrated that Kanglaite inhibited UVB-induced AQP3 down-regulation of cultured human skin keratinocytes. Our findings provide a potential new agent for anti-photoaging. The related molecular mechanisms remain to be further elucidated. PMID:22211241

  12. Adolescent Intakes of Vitamin D and Calcium and Incidence of Proliferative Benign Breast Disease

    PubMed Central

    Su, Xuefen; Colditz, Graham A.; Collins, Laura C.; Baer, Heather J.; Sampson, Laura A.; Willett, Walter C.; Berkey, Catherine S.; Schnitt, Stuart J.; Connolly, James L.; Rosner, Bernard A.; Tamimi, Rulla M.

    2013-01-01

    Vitamin D and calcium have been shown to have protective effects against breast cancer development in animal studies. Vitamin D and calcium play important anticarcinogenic roles in animal studies. Exposures between menarche and first birth may be important in breast development and future breast cancer risk. However, the relations between adolescent vitamin D and calcium intake and the risk of proliferative benign breast disease (BBD), a marker of increased breast cancer risk, have not yet been evaluated. We examined these associations in the Nurses’ Health Study II. Among the 29,480 women who completed an adolescent diet questionnaire in 1998, 682 proliferative BBD cases were identified and confirmed by centralized pathology review between 1991 and 2001. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazards regression and adjusted for potential confounders. A suggestive inverse association was observed between adolescent total vitamin D intake and proliferative BBD. Women in the highest quintile of vitamin D intake during adolescence had a 21% lower risk (multivariate HR (95% CI): 0.79 (0.61, 1.01), p-trend = 0.07) of proliferative BBD than women in the lowest quintile. Results were essentially the same when the analysis was restricted to prospective cases (n = 142) diagnosed after return of the adolescent diet questionnaire and independent of adult vitamin D intake. Adolescent total milk intake was positively associated with proliferative BBD (≥3 servings/day vs. <1 serving/day HR (95% CI): 1.41 (0.91, 2.17), p-trend = 0.03), after additional adjustment for total vitamin D. Calcium intake during adolescence was not associated with proliferative BBD (p-trend = 0.91). Vitamin D intake during adolescence may be important in the earlier stage of breast carcinogenesis. These findings, if corroborated, may suggest new pathways and strategies for breast cancer prevention. PMID:22622809

  13. Serum-free primary human fibroblast and keratinocyte coculture.

    PubMed

    Mujaj, Sally; Manton, Kerry; Upton, Zee; Richards, Sean

    2010-04-01

    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins. PMID:19929322

  14. Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

    PubMed Central

    Xie, Ding; Seremwe, Mutsa; Edwards, John G.; Podolsky, Robert; Bollag, Wendy B.

    2014-01-01

    We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation. PMID:25233484

  15. Clues to prolific productivity among prominent scientists.

    PubMed

    Kantha, S S

    1992-10-01

    In a survey based on the biographical sketches, obituary notes and eulogies of notable scientists, eight were identified as belonging to an elite group, having authored more than 1000 research publications, which include books, monographs and patents. They were, in chronological order, Thomas Alva Edison, Paul Karrer, Margaret Mead, Giulio Natta, Hans Selye, Herbert C Brown, Tetsuji Kametani and Carl Djerassi. Among these, Karrer, Natta and Brown were Nobelists in chemistry. Four criteria which can be identified as clues to their prolific productivity are, 1) enthusiasm for compulsive work and eccentric life style, 2) physical and/or environmental handicap, 3) pioneering efforts in a new research field, and 4) selection of research area, predominantly organic chemistry. PMID:1461180

  16. [Proliferative vitreoretinopathy: pathophysiology and clinical diagnosis].

    PubMed

    Rouberol, F; Chiquet, C

    2014-09-01

    Proliferative vitreoretinopathy (PVR) remains one of the most common causes of failed retinal detachment (RD) surgery. Many histological and clinical studies have highlighted the chain of events leading to PVR: cellular migration into the vitreous cavity, cellular differentiation, myofibroblast proliferation and activation, synthesis of extracellular matrix proteins, then contraction of preretinal tissues. The development of PVR can be explained schematically by cellular exposure to growth factors and cytokines (particularly retinal pigment epithelial cells and glial cells), in the context of break-down of the blood-retinal barrier (inflammation, choroidal detachment, iatrogenic effect of cryotherapy and surgery) and of cellular contact with the vitreous. Although the pathophysiology of PVR is now better understood, its severity remains an issue. A systematic search for preoperative PVR risk factors allows the most suitable therapeutic option to be chosen. PMID:24997864

  17. Photoprotective effect of arctiin against ultraviolet B-induced damage in HaCaT keratinocytes is mediated by microRNA expression changes.

    PubMed

    Cha, Hwa Jun; Lee, Ghang Tai; Lee, Kwang Sik; Lee, Kun Kook; Hong, Jin Tae; Lee, Na Kyeong; Kim, Soo-Yeon; Lee, Bo Mi; An, In-Sook; Hahn, Hyung Jin; Ahn, Kyu Joong; Lee, Su-Jae; An, Sungkwan; Bae, Seunghee

    2014-09-01

    Human keratinocytes are located in the outermost skin layer and thus particularly vulnerable to ultraviolet B (UVB) radiation exposure. Previous studies have focused on the cellular and molecular perspectives of UVB-induced keratinocyte damage. In the present study, it was demonstrated that pretreatment with the phytochemical arctiin, one of the lignin compounds, protects human HaCaT keratinocytes from UVB-mediated damage. Biochemical assays revealed that UVB-induced cytotoxicity and cell death were significantly reduced in arctiin-pretreated HaCaT cells. In addition, arctiin promoted the wound healing and DNA repair properties of keratinocytes. The photoprotective effects of arctiin were associated with changes in the expression levels of specific microRNAs (miRNAs) in HaCaT cells. A bioinformatics analysis demonstrated that the miRNAs were functionally involved in cancer, cell cycle, and Wnt and mitogen-activated protein kinase signaling pathways. In the present study, the results from the cellular and molecular assays demonstrated a novel role for arctiin in UVB protection in keratinocytes, which is mediated by miRNA responses and the suppression of UVB-induced cell death. Furthermore, arctiin is implicated as a potential chemopreventive agent through UVB protection of keratinocytes. PMID:24926940

  18. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  19. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.

    PubMed

    Lee, Wonhye; Debasitis, Jason Cushing; Lee, Vivian Kim; Lee, Jong-Hwan; Fischer, Krisztina; Edminster, Karl; Park, Je-Kyun; Yoo, Seung-Schik

    2009-03-01

    We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing. PMID:19108884

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

    SciTech Connect

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    keratinocytes (NHKs). • Chemical allergens stimulate NHKs to produce VEGF. • VEGF production is preceded by IL-8 production in NHKs. • IFNγ, DNCB and formaldehyde increase lymphangiogenic VEGF-C gene transcription. • VEGF production in NHKs may be a biomarker for the prediction of potential contact allergens.

  1. RXRα ablation in epidermal keratinocytes enhances UV radiation induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes

    PubMed Central

    Wang, Zhixing; Coleman, Daniel J.; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2011-01-01

    We show here that keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute ultraviolet radiation (UVR). Keratinocytic RXRα has a protective role on UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress mediated DNA damage and cellular apoptosis. We discovered that keratinocytic RXRα in a cell autonomous manner regulate mitogenic growth responses in skin epidermis via secretion of hbEGF, GMCSF, IL1-α and COX2, and activation of MAPK pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as ET-1, HGF, α–MSH, SCF and FGF2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRαep−/− mice), which in a non-cell autonomous manner modulated melanocyte proliferation and activation after UVR. RXRαep−/− mouse represents a unique animal model where UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, our results suggest that RXR antagonists, together with inhibitors of cell proliferation can be effective to prevent solar UV radiation induced photo-carcinogenesis. PMID:20944655

  2. Homocysteine Serum Levels in Diabetic Patients with Non Proliferative, Proliferative and without Retinopathy

    PubMed Central

    Gagliano, Caterina; Giordano, Maria; Vacante, Marco; Caraci, Filippo; Drago, Filippo; Avitabile, Teresio; Motta, Massimo

    2014-01-01

    Homocysteine has been associated with extracellular matrix changes. The diabetic retinopathy is a neurovascular complication of diabetes mellitus and it is the leading cause of vision loss among working adults worldwide. In this study, we evaluate the role of homocysteine in diabetic retinopathy analyzing the plasma levels of homocysteine in 63 diabetic type 2 patients with nonproliferative retinopathy (NPDR), 62 patients with proliferative diabetic retinopathy (PDR), 50 healthy subjects used as control group, and 75 randomly selected patients. PMID:24877066

  3. Characterization and Functionality of Proliferative Human Sertoli Cells

    PubMed Central

    Chui, Kitty; Trivedi, Alpa; Cheng, C. Yan; Cherbavaz, Diana B.; Dazin, Paul F.; Huynh, Ai Lam Thu; Mitchell, James B.; Rabinovich, Gabriel A.; Noble-Haeusslein, Linda J.; John, Constance M.

    2014-01-01

    It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility

  4. DIAGNOSTIC CRITERIA FOR PROLIFERATIVE THYROID LESIONS IN BONY FISHES II

    EPA Science Inventory

    Thyroid proliferative lesions are rather common in bony fishes but diagnostic terminology and criteria for these lesions are inconsistent in the literature. The diagnosis of proliferative thyroid lesions is especially challenging in fish due to the fact that the thyroid is not a ...

  5. Scanning Ion Conductance Microscopy of Live Keratinocytes

    NASA Astrophysics Data System (ADS)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

  6. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

    SciTech Connect

    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. )

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  7. The impact of UVB exposure and differentiation state of primary keratinocytes on their interaction with quantum dots

    PubMed Central

    Mortensen, Luke J.; Ravichandran, Supriya; DeLouise, Lisa A.

    2013-01-01

    In this study we utilised an in vitro model system to gain insight into the potential cellular interactions that quantum dot (QD) nanoparticles may experience while transiting the viable skin epidermis, and we consider the effects of UVB exposure. UVB skin exposure is known to induce a skin barrier defect that facilitates QD stratum corneum penetration. Primary human keratinocytes were cultured in low and high calcium to induce basal and differentiated phenotypes, respectively. Results suggest that differentiation state plays a role in keratinocyte response to UVB exposure and exposure to negatively charged CdSe/ZnS core/shell QD. QD cell uptake increased with QD dose but association with differentiated cells was significantly lower than the basal keratinocyte phenotype. Differentiated keratinocytes were also less sensitive to the cytotoxic effects of UVB exposure. We did not observe an effect of UVB preexposure on QD cytotoxicity level despite the fact that fluorescent microscopy and flow cytometry data suggest that UVB may slightly increase QD uptake in the basal cell phenotype. The implications of these findings for assessing potential risk of human skin exposure are discussed. PMID:22998293

  8. Fucoxanthin Protects Cultured Human Keratinocytes against Oxidative Stress by Blocking Free Radicals and Inhibiting Apoptosis

    PubMed Central

    Zheng, Jian; Piao, Mei Jing; Keum, Young Sam; Kim, Hye Sun; Hyun, Jin Won

    2013-01-01

    Fucoxanthin is an important carotenoid derived from edible brown seaweeds and is used in indigenous herbal medicines. The aim of the present study was to examine the cytoprotective effects of fucoxanthin against hydrogen peroxide-induced cell damage. Fucoxanthin decreased the level of intracellular reactive oxygen species, as assessed by fluorescence spectrometry performed after staining cultured human HaCaT keratinocytes with 2',7'-dichlorodihydrofl uorescein diacetate. In addition, electron spin resonance spectrometry showed that fucoxanthin scavenged hydroxyl radical generated by the Fenton reaction in a cell-free system. Fucoxanthin also inhibited comet tail formation and phospho-histone H2A.X expression, suggesting that it prevents hydrogen peroxideinduced cellular DNA damage. Furthermore, the compound reduced the number of apoptotic bodies stained with Hoechst 33342, indicating that it protected keratinocytes against hydrogen peroxide-induced apoptotic cell death. Finally, fucoxanthin prevented the loss of mitochondrial membrane potential. These protective actions were accompanied by the down-regulation of apoptosispromoting mediators (i.e., B-cell lymphoma-2-associated x protein, caspase-9, and caspase-3) and the up-regulation of an apoptosis inhibitor (B-cell lymphoma-2). Taken together, the results of this study suggest that fucoxanthin defends keratinocytes against oxidative damage by scavenging ROS and inhibiting apoptosis. PMID:24244811

  9. An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation

    PubMed Central

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  10. An aquaporin 3-notch1 axis in keratinocyte differentiation and inflammation.

    PubMed

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  11. Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts from Diabetic and Nondiabetic Donors.

    PubMed

    Wardecki, Tina; Werner, Philipp; Thomas, Maria; Templin, Markus F; Schmidt, Gudula; Brandner, Johanna M; Merfort, Irmgard

    2016-04-22

    Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, pro-inflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients. PMID:27002382

  12. Asymmetric stem-cell division ensures sustained keratinocyte hyperproliferation in psoriatic skin lesions

    PubMed Central

    JIA, HAI-YAN; SHI, YING; LUO, LONG-FEI; JIANG, GUAN; ZHOU, QIONG; XU, SHI-ZHENG; LEI, TIE-CHI

    2016-01-01

    Excessive expansion of the transit-amplifying (TA) cell compartment is a distinct morphological characteristic of psoriatic epidermal hyperplasia. In order to examine the activation of basal stem cells and how they replenish such an enlarged compartment of TA cells in psoriatic epidermis, we utilized a BrdU labeling method to monitor mitotic stem cells in a mouse model of psoriasiform dermatitis, which was induced by imiquimod. Our results showed that perpendicular and parallel cell division characteristics of dividing stem cells existed in the inflamed epidermis. When we analyzed template-DNA strand segregation in trypsin-dissociated human psoriatic keratinocytes using BrdU pulse-chase labeling, we found that the percentage of asymmetric segregation of BrdU was significantly increased in the cell pairs of psoriatic epidermal cells compared with normal epidermal cells. Furthermore, we also examined the effects of both interleukin (IL)-17A and IL-22 cytokines on the differentiation status of cultured human keratinocytes. The results indicated that both cytokines had synergistic effects on passage-one epidermal cell sheets derived from skin explants and also on cultured keratinocytes, were involved in the maintenance of the undifferentiated stem cell phenotype, and these results suggest an efficient mechanism for preventing the premature loss of basal stem-cell pools in the pro-inflammatory cytokine-enriched milieu of the psoriatic epidermis. Our findings suggest that inhibition of hyperactive stem cells represents a potential therapeutic target to combat recalcitrant epidermal hyperplasia in psoriasis. PMID:26707630

  13. Keratinocyte galvanotaxis in combined DC and AC electric fields supports an electromechanical transduction sensing mechanism.

    PubMed

    Hart, Francis X; Laird, Mhairi; Riding, Aimie; Pullar, Christine E

    2013-02-01

    Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re-epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane-bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage-gated channel model. PMID:22907479

  14. Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes.

    PubMed

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D

    2005-01-01

    Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R. PMID:15654973

  15. Barrier function of human keratinocyte cultures grown at the air-liquid interface.

    PubMed

    Mak, V H; Cumpstone, M B; Kennedy, A H; Harmon, C S; Guy, R H; Potts, R O

    1991-03-01

    Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism. PMID:2002253

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

    PubMed

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

    2015-12-01

    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

  17. The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

    PubMed Central

    Li, Xinping; Liang, Ling; Zhao, Pin; Uchida, Kenzo; Baba, Hisatoshi; Huang, Hong; Bai, Wenfang; Bai, Liming; Zhang, Mingsheng

    2013-01-01

    Epidermal stem cells (ESCs) are characterized as slow-cycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGF-transfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f+/CD71−). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing. PMID:24170090

  18. The Non-Proliferative Nature of Ascidian Folliculogenesis as a Model of Highly Ordered Cellular Topology Distinct from Proliferative Epithelia

    PubMed Central

    Azzag, Karim; Chelin, Yoann; Rousset, François; Le Goff, Emilie; Martinand-Mari, Camille; Martinez, Anne-Marie; Maurin, Bernard; Daujat-Chavanieu, Martine; Godefroy, Nelly; Averseng, Julien; Mangeat, Paul; Baghdiguian, Stephen

    2015-01-01

    Previous studies have addressed why and how mono‐stratified epithelia adopt a polygonal topology. One major additional, and yet unanswered question is how the frequency of different cell shapes is achieved and whether the same distribution applies between non-proliferative and proliferative epithelia. We compared different proliferative and non-proliferative epithelia from a range of organisms as well as Drosophila melanogaster mutants, deficient for apoptosis or hyperproliferative. We show that the distribution of cell shapes in non‐proliferative epithelia (follicular cells of five species of tunicates) is distinctly, and more stringently organized than proliferative ones (cultured epithelial cells and Drosophila melanogaster imaginal discs). The discrepancy is not supported by geometrical constraints (spherical versus flat monolayers), number of cells, or apoptosis events. We have developed a theoretical model of epithelial morphogenesis, based on the physics of divided media, that takes into account biological parameters such as cell‐cell contact adhesions and tensions, cell and tissue growth, and which reproduces the effects of proliferation by increasing the topological heterogeneity observed experimentally. We therefore present a model for the morphogenesis of epithelia where, in a proliferative context, an extended distribution of cell shapes (range of 4 to 10 neighbors per cell) contrasts with the narrower range of 5-7 neighbors per cell that characterizes non proliferative epithelia. PMID:26000769

  19. Cellular and molecular facets of keratinocyte reepithelization during wound healing

    SciTech Connect

    Santoro, Massimo M. . E-mail: msantoro@unipmn.it; Gaudino, Giovanni

    2005-03-10

    Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.

  20. Suprabasin, a novel epidermal differentiation marker and potential cornified envelope precursor.

    PubMed

    Park, Geon Tae; Lim, Susan E; Jang, Shyh-Ing; Morasso, Maria I

    2002-11-22

    The suprabasin gene is a novel gene expressed in mouse and human differentiating keratinocytes. We identified a partial cDNA encoding suprabasin using a suppression subtractive hybridization method between the proliferative basal and differentiating suprabasal populations of the mouse epidermis. A 3' gene-specific probe hybridized to transcripts of 0.7- and 2.2-kb pairs on Northern blots with specific detection in differentiated keratinocytes of stratified epithelia. The mouse gene was mapped to chromosome 7 by fluorescence in situ hybridization. This region is syntenic to human chromosome band 19q13.1, which contained the only region in the data bases with homology to the mouse suprabasin sequence. During embryonic mouse development, suprabasin mRNA was detected at day 15.5, coinciding with epidermal stratification. Suprabasin was detected in the suprabasal layers of the epithelia in the tongue, stomach, and epidermis. Differentiation of cultured primary epidermal keratinocytes with 0.12 mm Ca(2+) or 12-O-tetradecanoylphorbol-13-acetate treatment resulted in the induction of suprabasin. The 2.2-kb cDNA transcript encodes a protein of 72 kDa with a predicted isoelectric point of 6.85. The translated sequence has an amino-terminal domain, a central domain composed of repeats rich in glycine and alanine, and a carboxyl-terminal domain. The alternatively spliced 0.7-kb transcript encodes a smaller protein that shares the NH(2)- and COOH-terminal regions but lacks the repeat domain region. Cross-linking experiments indicate that suprabasin is a substrate for transglutaminase 2 and 3 activity. Altogether, these results indicate that the suprabasin protein potentially plays a role in the process of epidermal differentiation. PMID:12228223

  1. Lithospermum erythrorhizon extract protects keratinocytes and fibroblasts against oxidative stress.

    PubMed

    Yoo, Hee Geun; Lee, Bong Han; Kim, Wooki; Lee, Jong Suk; Kim, Gun Hee; Chun, Ock K; Koo, Sung I; Kim, Dae-Ok

    2014-11-01

    Oxidative stress damages dermal and epidermal cells and degrades extracellular matrix proteins, such as collagen, ultimately leading to skin aging. The present study evaluated the potential protective effect of the aqueous methanolic extract obtained from Lithospermum erythrorhizon (LE) against oxidative stress, induced by H2O2 and ultraviolet (UV) irradiation, on human keratinocyte (HaCaT) and human dermal fibroblast-neonatal (HDF-n) cells. Exposure of cells to H2O2 or UVB irradiation markedly increased oxidative stress and reduced cell viability. However, pretreatment of cells with the LE extract not only increased cell viability (up to 84.5%), but also significantly decreased oxidative stress. Further, the LE extract downregulated the expression of matrix metalloproteinase-1, an endopeptidase that degrades extracellular matrix collagen. In contrast, treatment with the LE extract did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA irradiation. Thirteen phenolic compounds, including derivatives of shikonin and caffeic acid, were identified by ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. These results suggest that LE-derived extracts may protect oxidative-stress-induced skin aging by inhibiting degradation of skin collagen, and that this protection may derive at least in part from the antioxidant phenolics present in these extracts. Further studies are warranted to determine the potential utility of LE-derived extracts in both therapeutic and cosmetic applications. PMID:25136892

  2. Increase developmental plasticity of human keratinocytes with gene suppression.

    PubMed

    Li, Shengwen Calvin; Jin, Yangsun; Loudon, William G; Song, Yahui; Ma, Zhiwei; Weiner, Leslie P; Zhong, Jiang F

    2011-08-01

    Recent evidence indicates that p53 suppression increased the efficiency of induced pluripotent stem cell (iPSC) generation. This occurred even with the enforced expression of as few as two canonical transcription factors, Oct4 and Sox2. In this study, primary human keratinocytes were successfully induced into a stage of plasticity by transient inactivation of p53, without enforced expression of any of the transcription factors previously used in iPSC generation. These cells were later redifferentiated into neural lineages. The gene suppression plastic cells were morphologically indistinguishable from human ES cells. Gene suppression plastic cells were alkaline phosphatase-positive, had normal karyotypes, and expressed p53. Together with the accumulating evidence of similarities and overlapping mechanisms between iPSC generation and cancer formation, this finding sheds light on the emerging picture of p53 sitting at the crossroads between two intricate cellular potentials: stem cell vs. cancer cell generation. This finding further supports the crucial role played by p53 in cellular reprogramming and suggests an alternative method to switch the lineage identity of human cells. This reported method offers the potential for directed lineage switching with the goal of generating autologous cell populations for novel clinical applications for neurodegenerative diseases. PMID:21768375

  3. Functional analysis of ZFP36 proteins in keratinocytes.

    PubMed

    Prenzler, Frauke; Fragasso, Annunziata; Schmitt, Angelika; Munz, Barbara

    2016-08-01

    The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. PMID:27182009

  4. Distinctive molecular responses to ultraviolet radiation between keratinocytes and melanocytes.

    PubMed

    Sun, Xiaoyun; Kim, Arianna; Nakatani, Masashi; Shen, Yao; Liu, Liang

    2016-09-01

    Solar ultraviolet radiation (UVR) is the major risk factor for skin carcinogenesis. To gain new insights into the molecular pathways mediating UVR effects in the skin, we performed comprehensive transcriptomic analyses to identify shared and distinctive molecular responses to UVR between human keratinocytes and melanocytes. Keratinocytes and melanocytes were irradiated with varying doses of UVB (10, 20 and 30 mJ/cm(2) ) then analysed by RNA-Seq at different time points post-UVB radiation (4, 24 and 72 h). Under basal conditions, keratinocytes and melanocytes expressed similar number of genes, although they each expressed a distinctive subset of genes pertaining to their specific cellular identity. Upon UVB radiation, keratinocytes displayed a clear pattern of time- and dose-dependent changes in gene expression that was different from melanocytes. The early UVB-responsive gene set (4 h post-UVR) differed significantly from delayed UVB-responsive gene sets (24 and 72 h). We also identified multiple novel UVB signature genes including PRSS23, SERPINH1, LCE3D and CNFN, which were conserved between melanocyte and keratinocyte lines from different individuals. Taken together, our findings elucidated both common and distinctive molecular features between melanocytes and keratinocytes and uncovered novel UVB signature genes that might be utilized to predict UVB photobiological effects on the skin. PMID:27119462

  5. Polarized Integrin Mediates Human Keratinocyte Adhesion to Basal Lamina

    NASA Astrophysics Data System (ADS)

    de Luca, Michele; Tamura, Richard N.; Kajiji, Shama; Bondanza, Sergio; Rossino, Paola; Cancedda, Ranieri; Carlo Marchisio, Pier; Quaranta, Vito

    1990-09-01

    Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are α_Eβ_4 (also called α_6β_4) and α_2β_1/α_3β_1. The α_Eβ_4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas α_2β_1/ α_3β_1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-β_4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-β_1 antibodies were ineffective. Only anti-β_4 antibodies were able to detach established keratinocyte colonies. These data suggest that α_Eβ_4 mediates keratinocyte adhesion to basal lamina, whereas the β_1 subfamily is involved in cell-cell adhesion of keratinocytes.

  6. Fatal Hemorrhage in Cerebral Proliferative Angiopathy

    PubMed Central

    Maekawa, H.; Tanaka, M.; Hadeishi, H.

    2012-01-01

    Summary Cerebral proliferative angiopathy (CPA) is a rare vascular abnormality with several angiomorphological features that are distinct from brain arteriovenous malformations (AVMs). The natural history of CPAs indicates a lower risk for hemorrhage compared to brain AVMs. A 62-year-old woman presented with gait instability and dysarthria. MRI and angiography revealed a diffuse vascular network involving the tectum and cerebellar vermis with intermingled brain parenchyma. This lesion had no dominant feeder, high-flow arteriovenous shunt, flow-related aneurysm or highly dilated veins on angiogram. These findings were consistent with a diagnosis of CPA. During follow-up, she developed progressive gait instability and eye movement abnormalities, but no remarkable change was detected on the repeated MRI and angiography. Nine years later, she died of mesencephalic hemorrhage originating from the CPA. To the best of our knowledge, this is the first description of a patient with CPA who died as a result of the initial hemorrhage. It is important to recognize that a part of CPAs is aggressive and can be more vulnerable to critical hemorrhage. PMID:22958770

  7. Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes

    SciTech Connect

    Sinitsyna, Nadezda N.; Reznikova, Tatiana V.; Qin Qin; Song, Hyukhwan; Phillips, Marjorie A.; Rice, Robert H.

    2010-03-15

    While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.

  8. Lack of a differential radiation response for proliferative and non-proliferative rat thyroid cells (FRTL-5) in vitro

    SciTech Connect

    Brosing, J.W.; Giese, W.L.; Mulcahy, R.T.

    1989-06-01

    FRTL-5 rat thyroid epithelial cells maintain normal thyroid function and morphology in vitro, exhibit an absolute requirement for thyroid stimulating hormone (TSH) for proliferation and display radiation dose response characteristics indistinguishable from those of rat thyroid epithelial cells in vivo. In TSH-free medium cells remain in a non-proliferative, yet viable, state for prolonged periods of time and respond to TSH re-stimulation by a return to exponential growth. Flow cytometric analysis using two-step acridine orange (AO) staining revealed an accumulation of cells in the G1 phase of the cell cycle accompanied by a pronounced reduction in red fluorescence (indicative of RNA content) in FRTL-5 cells cultured in the absence of TSH. The response of proliferative and non-proliferative FRTL-5 cells to single dose, split dose and fractionated radiation was compared to determine whether proliferative status was an important response determinant. The response of FRTL-5 cells was not influenced by proliferative status at the time of irradiation. Additionally, dose response was not altered by variable (12 hr-8 days) non-proliferative intervals before or after irradiation. As revealed by split dose experiments, the rate and extent of sublethal damage repair was likewise similar for proliferative and non-proliferative cells. Multifraction experiments employing three fractions separated by 6 hr intervals indicate that non-proliferative FRTL-5 cells completely repair sublethal damage between fractions. These results indicate that the radiation response of FRTL-5 cells is not influenced by the proliferative status of the cells prior to or post-irradiation.

  9. Characterization of a Human Skin Equivalent Model to Study the Effects of Ultraviolet B Radiation on Keratinocytes

    PubMed Central

    Van Lonkhuyzen, Derek R.; Dawson, Rebecca A.; Kimlin, Michael G.; Upton, Zee

    2014-01-01

    The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage. PMID:24219750

  10. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

    PubMed

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-01-01

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. PMID:27509895

  11. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration

    PubMed Central

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-01-01

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. PMID:27509895

  12. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

    Saha, Asish K. . E-mail: aksaha@bu.edu; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-10-20

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.

  13. Expression and modulation of IL-1 alpha in murine keratinocytes

    SciTech Connect

    Ansel, J.C.; Luger, T.A.; Lowry, D.; Perry, P.; Roop, D.R.; Mountz, J.D.

    1988-04-01

    Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low (Ca2+) tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high (Ca2+) media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low (Ca2+) conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes.

  14. Multifactorial Analysis of Conditional Reprogramming of Human Keratinocytes

    PubMed Central

    Ligaba, Segni B.; Khurana, Anikita; Graham, Garrett; Krawczyk, Ewa; Jablonski, Sandra; Petricoin, Emanuel F.; Glazer, Robert I.; Upadhyay, Geeta

    2015-01-01

    Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents. PMID:25714835

  15. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  16. Cell wounding activates phospholipase D in primary mouse keratinocytes.

    PubMed

    Arun, Senthil N; Xie, Ding; Howard, Amber C; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L; Bollag, Wendy B

    2013-03-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D₃, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  17. Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

    PubMed Central

    Chavanas, Stéphane; Adoue, Véronique; Méchin, Marie-Claire; Ying, Shibo; Dong, Sijun; Duplan, Hélène; Charveron, Marie; Takahara, Hidenari; Serre, Guy; Simon, Michel

    2008-01-01

    Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease. PMID:18923650

  18. Non-proliferative and Proliferative Lesions of the Cardiovascular System of the Rat and Mouse

    PubMed Central

    Berridge, Brian R.; Mowat, Vasanthi; Nagai, Hirofumi; Nyska, Abraham; Okazaki, Yoshimasa; Clements, Peter J.; Rinke, Matthias; Snyder, Paul W.; Boyle, Michael C.; Wells, Monique Y.

    2016-01-01

    The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Japan (JSTP), Europe (ESTP), Great Britain (BSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The primary purpose of this publication is to provide a standardized nomenclature for characterizing lesions observed in the cardiovascular (CV) system of rats and mice commonly used in drug or chemical safety assessment. The standardized nomenclature presented in this document is also available electronically for society members on the internet (http://goreni.org). Accurate and precise morphologic descriptions of changes in the CV system are important for understanding the mechanisms and pathogenesis of those changes, differentiation of natural and induced injuries and their ultimate functional consequence. Challenges in nomenclature are associated with lesions or pathologic processes that may present as a temporal or pathogenic spectrum or when natural and induced injuries share indistinguishable features. Specific nomenclature recommendations are offered to provide a consistent approach. PMID:27621537

  19. Non-proliferative and Proliferative Lesions of the Cardiovascular System of the Rat and Mouse.

    PubMed

    Berridge, Brian R; Mowat, Vasanthi; Nagai, Hirofumi; Nyska, Abraham; Okazaki, Yoshimasa; Clements, Peter J; Rinke, Matthias; Snyder, Paul W; Boyle, Michael C; Wells, Monique Y

    2016-01-01

    The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Japan (JSTP), Europe (ESTP), Great Britain (BSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The primary purpose of this publication is to provide a standardized nomenclature for characterizing lesions observed in the cardiovascular (CV) system of rats and mice commonly used in drug or chemical safety assessment. The standardized nomenclature presented in this document is also available electronically for society members on the internet (http://goreni.org). Accurate and precise morphologic descriptions of changes in the CV system are important for understanding the mechanisms and pathogenesis of those changes, differentiation of natural and induced injuries and their ultimate functional consequence. Challenges in nomenclature are associated with lesions or pathologic processes that may present as a temporal or pathogenic spectrum or when natural and induced injuries share indistinguishable features. Specific nomenclature recommendations are offered to provide a consistent approach. PMID:27621537

  20. Proliferative and Non-Proliferative Lesions of the Rat and Mouse Integument

    PubMed Central

    Mecklenburg, Lars; Kusewitt, Donna; Kolly, Carine; Treumann, Silke; Adams, E. Terence; Diegel, Kelly; Yamate, Jyoji; Kaufmann, Wolfgang; Müller, Susanne; Danilenko, Dimitry; Bradley, Alys

    2014-01-01

    The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the societies of toxicological pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP). Its aim is to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory rodents. A widely accepted international harmonization of nomenclature in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and will provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. The purpose of this publication is to provide a standardized nomenclature for classifying microscopical lesions observed in the integument of laboratory rats and mice. Example colour images are provided for most lesions. The standardized nomenclature presented in this document and additional colour images are also available electronically at http://www.goreni.org. The nomenclature presented herein is based on histopathology databases from government, academia, and industrial laboratories throughout the world, and covers lesions that develop spontaneously as well as those induced by exposure to various test materials. (DOI: 10.1293/tox.26.27S; J Toxicol Pathol 2013; 26: 27S–57S) PMID:25035577

  1. Impact of blue LED irradiation on proliferation and gene expression of cultured human keratinocytes

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Sticht, Carsten; Dweep, Harsh; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2015-03-01

    Blue light is known for its anti-microbial, anti-proliferative and anti-inflammatory effects. Furthermore, it is already used for the treatment of neonatal jaundice and acne. However, little is known about the exact mechanisms of action on gene expression level. The aim of this study was to assess the impact of blue LED irradiation on the proliferation and gene expression in immortalized human keratinocytes (HaCaT) in vitro. Furthermore its safety was assessed. XTT-tests revealed a decrease in cell proliferation in blue light irradiated cells depending on the duration of light irradiation. Moreover, gene expression analysis demonstrated deregulated genes already 3 hours after blue light irradiation. 24 hours after blue light irradiation the effects seemed to be even more pronounced. The oxidative stress response was significantly increased, pointing to increased ROS production due to blue light, as well as steroid hormone biosynthesis. Downregulated pathways or biological processes were connected to anti-inflammatory response. Interestingly, also the melanoma pathway contained significantly downregulated genes 24 hours after blue light irradiation, which stands in accordance to literature that blue light can also inhibit proliferation in cancer cells. First tests with melanoma cells revealed a decrease in cell proliferation after blue light irradiation. In conclusion, blue light irradiation might open avenues to new therapeutic regimens; at least blue light seems to have no effect that induces cancer growth or formation.

  2. Keratinocyte cytoskeletal roles in cell sheet engineering

    PubMed Central

    2013-01-01

    Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

  3. Human Keratinocytes Radioprotection with Mentha Longifolia

    NASA Astrophysics Data System (ADS)

    Rizzo, Angela Maria; Berselli, P.; Zava, S.; Negroni, M.; Corsetto, P.; Montorfano, G.; Bertolotti, A.; Ranza, E.; Ottolenghi, A.; Berra, B.

    Antioxidants are suggested to act as radioprotectors, and dietary supplements based on antiox-idants have been proposed for astronauts involved in long-term space missions. Plant extracts with antioxidant properties may be used in dietetic supplements for astronauts; in fact recent nutritional guidelines suggest that "fruits and vegetables may become as important on space-going vessels as limes were on the sea-going vessels of old". Mint presents a large variety of biological properties, such as antiallergenic, antibacterial, anti-inflammatory, antitumor, an-tiviral, gastrointestinal protective, hepatoprotective, chemopreventive activities, most of which are attributable to its antioxidant activity. The aim of the present study is to evaluate the antioxidant properties and protective bio-efficacy of a phenol enriched Mentha longifolia ex-tract on gamma rays stressed human keratinocytes (NCTC2544). We assessed first the in vitro antioxidant activity (ABTS and DPPH), and then evaluated different stress markers in order to investigate various oxidative stress targets: cell viability (MTT); retained proliferating ca-pability (CA); DNA damage (histone H2AX) and protein damage (HSP70 induction). Results indicate that this Mint extract has a higher antioxidant activity respect to fresh extracts, that could be responsible of its really interesting radio-protective effects.

  4. Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

    PubMed Central

    Termini, Lara; Boccardo, Enrique; Esteves, Gustavo H; Hirata, Roberto; Martins, Waleska K; Colo, Anna Estela L; Neves, E Jordão; Villa, Luisa Lina; Reis, Luiz FL

    2008-01-01

    Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in depth analysis of the

  5. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

    PubMed

    Di Marco, E; Marchisio, P C; Bondanza, S; Franzi, A T; Cancedda, R; De Luca, M

    1991-11-15

    Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing. PMID:1718982

  6. Induction of differentiation in psoriatic keratinocytes by propylthiouracil and fructose.

    PubMed

    Arul, Santhosh; Dayalan, Haripriya; Jegadeesan, Muhilan; Damodharan, Prabhavathy

    2016-12-01

    Psoriasis is characterized by uncontrolled proliferation and poor differentiation. Sirtuin1 (SIRT1) a class III deacetylase, crucial for differentiation in normal keratinocytes, is reduced in psoriasis. Down regulated SIRT1 levels may contribute to poor differentiation in psoriasis. In addition, the levels of early differentiation factors Keratin1 (K1) and Keratin10 (K10) are depleted in psoriasis. We attempted to study a possible effect of fructose, a SIRT1 upregulator and Propylthiouracil (PTU) to augment differentiation in psoriatic keratinocytes. Keratinocytes were cultured from lesional biopsies obtained from psoriatic patients and control cells were obtained from patients undergoing abdominoplasty. Cells were treated with fructose and PTU individually. K1 and K10 transcript levels were measured to evaluate early differentiation; SIRT1 protein expression was also studied to decipher its role in the mechanism of differentiation. The K1, K10 transcript levels, SIRT1 protein and transcript levels in fructose treated psoriatic keratinocytes were improved. This suggests keratinocyte differentiation was induced by fructose through SIRT1 upregulation. Whereas PTU induced differentiation, as confirmed by improved K1, K10 transcript levels followed a non-SIRT1 mechanism. We conclude that the use of fructose and PTU may be an adjunct to the existing therapies for psoriasis. PMID:27453822

  7. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  8. Transcription Factor MafB Coordinates Epidermal Keratinocyte Differentiation.

    PubMed

    Miyai, Masashi; Hamada, Michito; Moriguchi, Takashi; Hiruma, Junichiro; Kamitani-Kawamoto, Akiyo; Watanabe, Hajime; Hara-Chikuma, Mariko; Takahashi, Kenzo; Takahashi, Satoru; Kataoka, Kohsuke

    2016-09-01

    Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation. PMID:27208706

  9. 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

  10. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    SciTech Connect

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with /sup 14/C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the /sup 14/C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of /sup 14/C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle.

  11. Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes

    PubMed Central

    Wölfle, Ute; Haarhaus, Birgit; Schempp, Christoph M.

    2015-01-01

    Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes. PMID:26600671

  12. Maintenance of donor phenotype after full-thickness skin transplantation from mice with chronic proliferative dermatitis (cpdm/cpdm) to C57BL/Ka and nude mice and vice versa.

    PubMed

    Gijbels, M J; HogenEsch, H; Bruijnzeel, P L; Elliott, G R; Zurcher, C

    1995-12-01

    Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm) and is characterized by epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To elucidate whether these pathologic features are the result of a local (skin) process or a consequence of a systemic disorder, transplantations were performed of full-thickness grafts of affected skin from cpdm/cpdm mice and normal skin from control (C57BL/Ka) mice on the back of cpdm/cpdm, C57BL/Ka and athymic nude mice. After 3 months, the grafts maintained the histologic phenotype of the donor animal. Intercellular adhesion molecule-1 continued to be expressed by basal keratinocytes of the cpdm/cpdm grafts after transplantation. In contrast, the basal keratinocytes of the C57BL/Ka grafts onto cpdm/cpdm mice remained negative for intercellular adhesion molecule-1 3 months after transplantation. An increased number of proliferating keratinocytes was present in the cpdm/cpdm skin-graft transplanted to nudes or to C57BL/Ka mice based on short-term bromodeoxyuridine labeling. The bromodeoxyuridine incorporation in the keratinocytes of the control C57BL/Ka skin grafts transplanted to cpdm/cpdm, nude, or C57BL/Ka mice was the same as in the keratinocytes of normal C57BL/Ka mice. This study demonstrates that the pathologic features found in the cpdm/cpdm mice are the result of a disorder in the epidermis or dermis and not due to a systemic defect. PMID:7490470

  13. Protective properties of ginsenoside Rb3 against UV-B radiation-induced oxidative stress in HaCaT keratinocytes.

    PubMed

    Oh, Sun-Joo; Oh, Yuri; Ryu, In Wang; Kim, Kyunghoon; Lim, Chang-Jin

    2015-01-01

    This work aimed to evaluate the skin anti-photoaging properties of ginsenoside Rb3 (Rb3), one of the main protopanaxdiol-type ginsenosides from ginseng, in HaCaT keratinocytes. The skin anti-photoaging activity was assessed by analyzing the levels of reactive oxygen species (ROS), pro-matrix metalloproteinase-2 (proMMP-2), pro-matrix metalloproteinase-9 (proMMP-9), total glutathione (GSH), and superoxide dismutase (SOD) activity as well as cell viability in HaCaT keratinocytes under UV-B irradiation. When HaCaT keratinocytes were exposed to Rb3 prior to UV-B irradiation, Rb3 exhibited suppressive activities on UV-B-induced ROS, proMMP-2, and proMMP-9 enhancements. On the contrary, Rb3 displayed enhancing activities on UV-B-reduced total GSH and SOD activity levels. Rb3 could not interfere with cell viabilities in UV-B-irradiated HaCaT keratinocytes. Rb3 plays a protective role against UV-B-induced oxidative stress in human HaCaT keratinocytes, proposing its potential skin anti-photoaging properties. PMID:26287932

  14. The role of the skin barrier in modulating the effects of common skin microbial species on the inflammation, differentiation and proliferation status of epidermal keratinocytes

    PubMed Central

    2013-01-01

    Background Skin resident microbial species are often thought of either as pathogenic or commensal. However, little is known about the role of the skin barrier in modulating their potential for causing disease. To investigate this question we measured the effects of three microbial species commonly found on the skin (Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes) on a reconstructed human epidermal model by either applying the bacteria on the model surface (intact barrier) or adding them to the culture medium (simulating barrier breach). Results When added to the medium, all of the tested species induced inflammatory responses and keratinocyte cell death with species-specific potency. P. acnes and S. epidermidis induced specific alterations in the expression of keratinocyte differentiation and proliferation markers, suggesting a barrier reparation response. S. aureus induced complete keratinocyte cell death. On the contrary, topically applied S. epidermidis and P. acnes caused no inflammatory response even when tested at high concentrations, while topical S. aureus induced a weak reaction. None of the tested species were able to alter the expression of keratinocyte differentiation or expression markers, when applied topically. Conclusions We show that the skin barrier prevents the effects of common skin bacteria on epidermal keratinocyte inflammation, differentiation and proliferation and highlight the importance of skin barrier in defending against the pathogenic effects of common skin bacteria. PMID:24245826

  15. Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy.

    PubMed

    Tamiya, Shigeo; Kaplan, Henry J

    2016-01-01

    Proliferative vitreoretinopathy (PVR) is a potentially blinding fibrotic complication. It is caused by the formation and contraction of epiretinal membranes (ERMs) that ultimately lead to retinal folds and traction retinal detachments. While multiple cell types have been identified in ERMs, retinal pigment epithelial (RPE) cells have long been implicated as a key player in the pathophysiology of PVR. Clinical and experimental evidence has shown that RPE cells undergo epithelial-mesenchymal transition (EMT) to adopt a fibroblastic phenotype. Cell-cell adhesions maintained by adherens and tight junctions are important for the maintenance of RPE phenotype, and disruption of these junctional complexes results in EMT via activation of signaling pathways such as β-catenin/Wnt and Hippo signaling, as well as transcription factors involving Zeb1, Snail, and ZONAB. Upon EMT, RPE cells can further differentiate into myofibroblasts in the presence of TGF-β with cytoskeletal tension mediated by RhoGTPase. These fibroblasts and myofibroblasts derived from RPE cells can contribute to ERM formation by cell migration, proliferation and matrix modification, and play a key role in ERM contraction. It is not solely the proliferation of these cells that results in PVR but rather the contraction of these cells in the ERM. PMID:26675400

  16. A review of keratinocyte delivery to the wound bed.

    PubMed

    Chester, D L; Balderson, D S; Papini, R P G

    2004-01-01

    Over the last 20 years, confluent sheets of cultured epithelial autograft have been used for patients with major burns. Problems with the lack of "take" and long-term durability, as well as the time delay to produce such grafts, have led to the development of delivery systems to transfer keratinocytes to the wound bed. This review article describes the problems of using cultured epithelial autograft and the advantages of using preconfluent keratinocytes. Despite the numerous delivery systems that have been reported, most studies are limited to animal wound bed models. There are a few small clinical studies that have demonstrated enhanced healing using mainly subjective methods. There is a need for controlled, randomized clinical trials to prove the efficacy of keratinocyte delivery systems. Proposals for the use of this technology are made. PMID:15273468

  17. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  18. Reconstruction of Rabbit Urethral Epithelium with Skin Keratinocytes

    PubMed Central

    Rogovaya, O. S.; Fayzulin, A. K.; Vasiliev, A. V.; Kononov, A. V.; Terskikh, V. V.

    2015-01-01

    We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3. PMID:25927003

  19. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation.

    PubMed

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-04-12

    Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  20. Reconstruction of rabbit urethral epithelium with skin keratinocytes.

    PubMed

    Rogovaya, O S; Fayzulin, A K; Vasiliev, A V; Kononov, A V; Terskikh, V V

    2015-01-01

    We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3. PMID:25927003

  1. Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies*

    PubMed Central

    Kalantari-Dehaghi, Mina; Chen, Yumay; Deng, Wu; Chernyavsky, Alex; Marchenko, Steve; Wang, Ping H.; Grando, Sergei A.

    2013-01-01

    The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease. PMID:23599429

  2. Optimal Differentiation of In Vitro Keratinocytes Requires Multifactorial External Control

    PubMed Central

    Borowiec, Anne-Sophie; Delcourt, Philippe; Dewailly, Etienne; Bidaux, Gabriel

    2013-01-01

    For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic

  3. Pathogenesis of skin lesions in mice with chronic proliferative dermatitis (cpdm/cpdm).

    PubMed Central

    Gijbels, M. J.; Zurcher, C.; Kraal, G.; Elliott, G. R.; HogenEsch, H.; Schijff, G.; Savelkoul, H. F.; Bruijnzeel, P. L.

    1996-01-01

    Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm), showing alopecia, epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To further elucidate its pathogenesis, organs of 1-, 2-, 3-, 4-, 5-, and 6-week-old cpdm/cpdm mice were examined. At 4 weeks, the epidermal thickness was increased, whereas already at 3 weeks, the bromodeoxyuridine incorporation was increased in the basal keratinocytes. However, already at the age of 1 week, skin, lungs, and lymph nodes were infiltrated by eosinophils although no macroscopic lesions were present. Compared with control animals, 6-week-old cpdm/cpdm mice had decreased serum IgE levels and increased numbers of mast cells. From the age of 1 week these mast cells became increasingly IgE positive. In contrast, the mast cells of the control animals remained IgE negative. Mast cells of control and cpdm/cpdm mice were interleukin-4 and tumor necrosis factor-alpha positive. A likely explanation for the tissue infiltration of eosinophils could be the release of interleukin-4 and tumor necrosis factor-alpha from activated mast cells. Tumor necrosis factor-alpha may lead to the expression of E-selectin on endothelial cells, facilitating interleukin-4-mediated eosinophil transendothelial migration. Although various pathogenetic aspects of the cpdm/cpdm mouse need further elucidation, this model can be a tool to study eosinophil infiltration, leukocyte-endothelial cell interactions, and mast cell proliferation. Furthermore, the cpdm/cpdm mouse can be used to study chronic inflammatory skin disease because of the severe epidermal proliferation. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:8774148

  4. 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.

  5. Lifespan Extension by Preserving Proliferative Homeostasis in Drosophila

    PubMed Central

    Supoyo, Stephen; DeGennaro, Matthew; Lehmann, Ruth; Jasper, Heinrich

    2010-01-01

    Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs) and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK) signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan. PMID:20976250

  6. 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

  7. Oral proliferative verrucous leukoplakia treated with the photodynamic therapy: a case report

    PubMed Central

    Romeo, Umberto; Russo, Nicola; Palaia, Gaspare; Tenore, Gianluca; Del Vecchio, Alessandro

    2014-01-01

    Summary Aims About 60% of the oral cancer arise on a pre-existent potentially malignant disorder of oral mucosa like the oral proliferative verrucous leukoplakia. The treatment with the photodynamic therapy of these lesions represents, in the last years, an innovative, non-invasive and effective therapeutic possibility to achieve the secondary prevention of oral cancer. In the last decade, case reports have described patients with similar treated through a photochemical reaction induced by laser light. The aim of this study is to evaluate the effectiveness of the topical 5-ALA photodynamic therapy in the treatment of a case of Oral proliferative verrucous leukoplakia. Case report A female patient of 80 years old affected by white verrucous plaques on the right buccal mucosa was recruited for our case report. The right side lesion was treated with the photodynamic therapy with topical administered 5-aminolevulinic acid using the 635 nm laser light to activate the photosensitizer. Results The lesion showed complete response after 4 sessions of photodynamic therapy and no recurrence was noticed after 12 months. Conclusions The photodynamic therapy can be considered an effective treatment in the management of oral verrucous proliferative leukoplakia, but more clinical trials, with prolonged follow-up controls, are necessary to evaluate its effectiveness in the mid and long time period. PMID:25002922

  8. 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. PMID:27155970

  9. The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration

    PubMed Central

    Jones, A.M.; Griffiths, J.L.; Sanders, A.J.; Owen, S.; Ruge, F.; Harding, K.G.; Jiang, W.G.

    2016-01-01

    Chronic wounds represent a significant burden to health services and are associated with patient morbidity. Novel methods to diagnose and/or treat problematic wounds are needed. Interleukin (IL)-15 is a cytokine involved in a number of biological processes and disease states such as inflammation, healing and cancer progression. The current study explores the expression profile of IL-15 and IL-15 receptor α (IL-15Rα) in chronic wounds and its impact on keratinocytes. IL-15 and IL-15Rα expression were examined in healing and non-healing chronic wounds using qPCR and immunohistochemical analysis. The impact of recombinant IL-15 (rhIL-15) on human adult low calcium temperature (HaCaT) keratinocyte growth and migratory potential was further examined. IL-15 transcript expression was slightly, though non-significantly elevated in healing chronic wounds compared with non-healing chronic wounds. IL-15 protein staining was minimal in both subtypes of chronic wounds. By contrast, IL-15Rα transcript and protein expression were both observed to be enhanced in non-healing chronic wounds compared with healing chronic wounds. The treatment of HaCaT cells with rhIL-15 generally enhanced cell growth and promoted migration. Analysis with small molecule inhibitors suggested that the pro-migratory effect of rhIL-15 may be associated with ERK, AKT, PLCγ and FAK signalling. IL-15 may promote healing traits in keratinocytes and the differential expression of IL-15Rα is observed in chronic wounds. Together, this may imply a complex role for this interleukin in wound healing. PMID:27460304

  10. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome.

    PubMed

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves; Habauzit, Denis

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells. PMID:27529420

  11. The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration.

    PubMed

    Jones, A M; Griffiths, J L; Sanders, A J; Owen, S; Ruge, F; Harding, K G; Jiang, W G

    2016-09-01

    Chronic wounds represent a significant burden to health services and are associated with patient morbidity. Novel methods to diagnose and/or treat problematic wounds are needed. Interleukin (IL)-15 is a cytokine involved in a number of biological processes and disease states such as inflammation, healing and cancer progression. The current study explores the expression profile of IL-15 and IL-15 receptor α (IL-15Rα) in chronic wounds and its impact on keratinocytes. IL-15 and IL-15Rα expression were examined in healing and non-healing chronic wounds using qPCR and immunohistochemical analysis. The impact of recombinant IL-15 (rhIL-15) on human adult low calcium temperature (HaCaT) keratinocyte growth and migratory potential was further examined. IL-15 transcript expression was slightly, though non-significantly elevated in healing chronic wounds compared with non-healing chronic wounds. IL-15 protein staining was minimal in both subtypes of chronic wounds. By contrast, IL-15Rα transcript and protein expression were both observed to be enhanced in non-healing chronic wounds compared with healing chronic wounds. The treatment of HaCaT cells with rhIL-15 generally enhanced cell growth and promoted migration. Analysis with small molecule inhibitors suggested that the pro-migratory effect of rhIL-15 may be associated with ERK, AKT, PLCγ and FAK signalling. IL-15 may promote healing traits in keratinocytes and the differential expression of IL-15Rα is observed in chronic wounds. Together, this may imply a complex role for this interleukin in wound healing. PMID:27460304

  12. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome

    PubMed Central

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells. PMID:27529420

  13. The Effect of Secretory Factors of Adipose-Derived Stem Cells on Human Keratinocytes

    PubMed Central

    Moon, Kyoung Mi; Park, Ye-Hyoung; Lee, Jae Seol; Chae, Yong-Byung; Kim, Moon-Moo; Kim, Dong-Soo; Kim, Byung-Woo; Nam, Soo-Wan; Lee, Jong-Hwan

    2012-01-01

    The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM) on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC—in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE)—has great merits for skin regeneration. In this study, human primary keratinocytes (HKs), which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 μg/mL) than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05) notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 μg/mL). AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-β3 differing from that shown by 2-D analysis. Conclusion: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration. PMID:22312315

  14. Experimental Modeling of Proliferative Vitreoretinopathy. An Experimental Morphological Study.

    PubMed

    Khoroshilova-Maslova, I P; Leparskaya, N L; Nabieva, M M; Andreeva, L D

    2015-05-01

    A model of proliferative vitreoretinopathy induced by simultaneous intravitreal injection of recombinant IL-1β and platelet concentrate is created and its main morphological manifestations are studied on Chinchilla rabbits. The model reflects pathogenesis of proliferative vitreoretinopathy: epiretinal membrane with the formation of retinal plication, traction detachment of the retina; moderate inflammatory reaction in the uveal tract, in the optic nerve infundibulum, in the vitreous body; intact structural elements of the retina, dissociation of the retinal pigmented epithelium cells with their subsequent migration. The model is adequate to the clinical picture of proliferative vitreoretinopathy in humans, which recommends it for experimental studies of the efficiency of drug therapy and prevention of this disease. PMID:26033599

  15. Evaluation of the anti-proliferative and cytostatic effect of Citrus sinensis (orange) fruit juice

    PubMed Central

    Chinedu, Enegide; Arome, David; Ameh, Solomon F; Ameh, Gift E

    2014-01-01

    Aim: This work has been designed to evaluate the anti-proliferative and cytostatic effects of Citrus sinensis (orange) fruit juice on rapidly proliferating cells. Materials and Methods: The study was carried out on the seeds of Sorghum bicolor for 72 h. The mean radicle length (mm) of the seeds was taken at 48 and 72 h. Result: The result showed that when compared with the control, methotrexate, the standard drug showed a significant (P < 0.001) anti-proliferative effect throughout the experiment. The inhibition of the radicle growth was more after 72 h (87.42%). At a dose of 5% (v/v), the juice showed a slightly significant (P < 0.05) effect affect after 72 h; however, there was no significant effect at 48 h. The juice at doses of 10% and 20% (v/v) showed a highly significant (P < 0.001) anti-proliferative effect throughout the experiment; however, the percentage inhibitions were higher at 72 h. At 72 h, the percentage inhibition for juice at 10% (v/v) was 72.37% and at 20% (v/v) was 91.96%. The concentrations of 40% and 60% (v/v) showed cytostatic effects as no appreciable growth of the radicles of the seeds was observed throughout the experiment. The percentage inhibition for 40% (v/v) was 100% and 99.72% for 48 and 72 h, respectively, while that for the juice concentration of 60% (v/v) was 100% throughout the study. Conclusion: The experiment has shown that C. sinensis fruit juice has a potential for causing both anti-proliferative and cytostatic effects on fast proliferating cells and hence cancerous cells. PMID:25298937

  16. Protective effects of phenolic fraction of blue honeysuckle fruits against UVA-induced damage to human keratinocytes.

    PubMed

    Svobodová, Alena; Rambousková, Jana; Walterová, Daniela; Vostálová, Jitka

    2008-06-01

    In this study, the UVA (photo)protective activity of the phenolic fraction of L. caerulea fruits (PFLC) was assessed in human keratinocytes HaCaT. The keratinocytes were pre- or post-treated with PFLC (1-250 mg/l) and exposed to UVA irradiation (10-30 J/cm(2)). The results showed that both pre- and post-treatment with PFLC significantly suppressed UVA-induced ROS production, which was also revealed as a decrease in intracellular lipid peroxidation and elevation of reduced glutathione. Protection was concentration-dependent with a maximum at 50 mg/l. These results suggest that PFLC attenuates UVA-induced oxidative stress by reduction of ROS generation and ROS-mediated damage. For this reason, PFLC has potentially skin-protective functions against the deleterious effects of sunlight. PMID:18404271

  17. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

    SciTech Connect

    Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

    1989-01-01

    Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

  18. 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

  19. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    SciTech Connect

    Morrison, A.I. ); Keeble, S.; Watt, F.M. )

    1988-08-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of ({sup 14}C)galactose- or ({sup 14}C)mannose- and ({sup 14}C)glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

  20. Absence of premature senescence in Werner's syndrome keratinocytes.

    PubMed

    Ibrahim, Badr; Sheerin, Angela N; Jennert-Burston, Katrin; Bird, Joe L E; Massala, M V; Illsley, Matthew; James, S Elizabeth; Faragher, Richard G A

    2016-10-01

    Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome. PMID:27492502

  1. Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing.

    PubMed

    Rötzer, Vera; Hartlieb, Eva; Winkler, Julia; Walter, Elias; Schlipp, Angela; Sardy, Miklós; Spindler, Volker; Waschke, Jens

    2016-01-01

    The desmosomal transmembrane adhesion molecules desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogen-activated protein kinase (p38MAPK) and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared with control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch-wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration. PMID:26763450

  2. Problems with claims that sanctity leads to 'pro-life' law, and reasons for doubting it to be a convincing 'middle way'.

    PubMed

    Coggon, John

    2008-03-01

    This paper focuses on the idea of pro-life arguments and the sanctity of life doctrine in the context of debates on end-of-life law. Advocates of the sanctity doctrine are often thought of as being pro-life, which has potentially troublesome implications in policy debate. I explore what it means to be pro-life, and consider sanctity's relation to this. I question the coherence and appeal of truly pro-life law, and law that is premised on the sanctity doctrine. The analysis allows me to examine and reject the idea that the sanctity doctrine is reasonably described as a 'middle way' that we should enshrine in policy and practice. PMID:18592892

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

    SciTech Connect

    Lee, San San; Crabb, Simon J.; Janghra, Nari; Carlberg, Carsten; Williams, Ann C.; Cutress, Ramsey I.; Packham, Graham; Hague, Angela

    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 the 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.

  4. Synthesis and Anti-Proliferative Effects of Quercetin Derivatives.

    PubMed

    Al-Jabban, Sami M R; Zhang, Xiaojie; Chen, Guanglin; Mekuria, Ermias Addo; Rakotondraibe, Liva Harinantenaina; Chen, Qiao-Hong

    2015-12-01

    Prostate cancer is the most common diagnosed invasive cancer in American men and is the second leading cause of cancer-related deaths. Although there are several therapies successful in treating early, localized stage prostate cancer, current treatment of advanced metastatic castration-resistant prostate cancer remains ineffective due to inevitable progression of resistance to first-line treatment with docetaxel. The natural product quercetin (3,3',4',5,7-pentahydroxyflavone), a flavonoid compound ubiquitous in dietary plants, possesses evidenced potential in treating advanced metastatic castration-resistant prostate cancer. However, its poor bioavailability and moderate potency hinder its advancement into clinical therapy. In order to engineer quercetin derivatives with improved potency and pharmacokinetic profiles for the treatment of advanced metastatic prostate cancer, we started this study with creating a small library of alkylated derivatives of quercetin for in vitro evaluation. The biological data and chemical reactivity of quercetin and its derivatives reported in literature directed us to design 3,4',7-O-trialkylquercetins as our first batch of targets. Consequently, nine 3,4',7-O-trialkylquercetins, together with four 3,7-O- dialkylquercetins, four 3,3',4',7-tetraalkylquercetins, and one 3,3',4'-O-trialkylquercetin, were prepared by one step O-alkylation of commercially available quercetin mediated by potassium carbonate. Their structures were determined by ID and 2D NMR data, and HRMS. Their anti-proliferative activities towards both androgen-refractory and androgen-sensitive prostate cancer cells were evaluated using WST-1 cell proliferation assay. The acquired structure-activity relationships indicate that 3,7-O-dialkylquercetins rather than 3,4',7-O-trialkylquercetins were much more potent than quercetin towards prostate cancer cells. PMID:26882678

  5. Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway

    SciTech Connect

    Abel, Erika L.; Bubel, Jennifer D.; Simper, Melissa S.; Powell, Leslie; McClellan, S. Alex; Andreeff, Michael; MacLeod, Michael C.; DiGiovanni, John

    2011-09-01

    Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100 years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by {approx} 3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin. - Highlights: > CDDO-Me treatment increased intracellular GSH in human keratinocytes. > CDDO-Me increased cell viability following exposure to the half-mustard, CEES. > The cytoprotective effect of CDDO-Me was likely due to scavenging with endogenous GSH.

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

    NASA Astrophysics Data System (ADS)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory 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 extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts 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 because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  7. Write to the Top! How to Become a Prolific Academic

    ERIC Educational Resources Information Center

    Johnson, W. Brad; Mullen, Carol A.

    2007-01-01

    This concise guide to writing is designed to help any academic become not only productive but truly prolific. It is a pithy, no-nonsense, no-excuses guide to maximizing the quality and quantity of scholarly output. The authors offer an accessible overview of the art of writing efficiently and effectively, provide a one-stop source for the nuts and…

  8. PROPOSED DIAGNOSTIC CRITERIA FOR PROLIFERATIVE THYROID LESIONS IN BONY FISHES

    EPA Science Inventory

    Distinguishing hyperplastic lesions from neoplasia in the thyroid of bony fishes has been debated by scientists for about one hundred years. As early as the first decade of the last century, the histological interpretation of some of the striking proliferative lesions observed in...

  9. Effects of flavonoids on human lymphocyte proliferative responses

    SciTech Connect

    Mookerjee, B.K.; Lee, T.P.; Logue, G.P.; Lippes, H.A.; Middleton, E.

    1986-01-01

    Flavonoids reversibly inhibit lymphocyte proliferative responses to phytomitogens, soluble antigens and phorbol esters by blocking an early event or events that follow stimulation. Quercetin and tangeretin inhibit thymidine transport in stimulated lymphocytes. These flavonoids reversibly inhibit antigen processing by monocytes and inhibit the expression of class II histocompatibility (DR) antigens in PBM cells.

  10. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis.

    PubMed

    Chang, J; Most, D; Bresnick, S; Mehrara, B; Steinbrech, D S; Reinisch, J; Longaker, M T; Turk, A E

    1999-01-01

    Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There

  11. Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

    PubMed

    Zhao, Bin; Liu, Jia-Qi; Zheng, Zhao; Zhang, Jun; Wang, Shu-Yue; Han, Shi-Chao; Zhou, Qin; Guan, Hao; Li, Chao; Su, Lin-Lin; Hu, Da-Hai

    2016-07-01

    Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing. PMID:26888423

  12. Red Light Combined with Blue Light Irradiation Regulates Proliferation and Apoptosis in Skin Keratinocytes in Combination with Low Concentrations of Curcumin.

    PubMed

    Niu, Tianhui; Tian, Yan; Cai, Qing; Ren, Qu; Wei, Lizhao

    2015-01-01

    Curcumin is a widely known natural phytochemical from plant Curcuma longa. In recent years, curcumin has received increasing attention because of its capability to induce apoptosis and inhibit cell proliferation as well as its anti-inflammatory properties in different cancer cells. However, the therapeutic benefits of curcumin are severely hampered due to its particularly low absorption via trans-dermal or oral bioavailability. Phototherapy with visible light is gaining more and more support in dermatological therapy. Red light is part of the visible light spectrum, which is able to deeply penetrate the skin to about 6 mm, and directly affect the fibroblast of the skin dermis. Blue light is UV-free irradiation which is fit for treating chronic inflammation diseases. In this study, we show that curcumin at low concentrations (1.25-3.12 μM) has a strong anti-proliferative effect on TNF-α-induced psoriasis-like inflammation when applied in combination with light-emitting-diode devices. The treatment was especially effective when LED blue light at 405 nm was combined with red light at 630 or 660 nm, which markedly amplified the anti-proliferative and apoptosis-inducing effects of curcumin. The experimental results demonstrated that this treatment reduced the viability of human skin keratinocytes, decreased cell proliferation, induced apoptosis, inhibited NF-κB activity and activated caspase-8 and caspase-9 while preserving the cell membrane integrity. Moreover, the combined treatment also down-regulated the phosphorylation level of Akt and ERK. Taken together, our results indicated that the combination of curcumin with LED blue light united red light irradiation can attain a higher efficiency of regulating proliferation and apoptosis in skin keratinocytes. PMID:26382065

  13. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    SciTech Connect

    Senthilkumar, P.K.; Robertson, L.W.; Ludewig, G.

    2012-02-15

    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 cell 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 and

  14. Effect of Porcine Placenta Extract from Subcritical Water Extraction on Photodamage in Human Keratinocytes

    PubMed Central

    Han, Bok Kyung; Choi, Hyeon-Son; Hong, Yang Hee; Jung, Eun Young

    2015-01-01

    The objective of this study was to evaluated the photoprotective effects of porcine placenta extract (PPE) on ultraviolet B (UVB)-induced oxidative stress in human keratinocytes (HaCaT) to evaluate its functional activities as a skin food ingredient. PPE prepared by subcritical water extraction was termed SPE, and subsequently digested by enzymes to prepare E-SPE. Increased intracellular reactive oxygen species (ROS) levels (192.0%) induced by UVB were decreased by SPE and E-SPE. SPE had more effective ROS scavenging activity than E-SPE treatment. UVB treatment increased expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), and this elevated expression was decreased by E-SPE treatment. High-dose treatment with E-SPE (50 and 100 µg/mL) reduced TIMP-1 expression levels of UVB-C (control) to 33.5 and 34.6%, respectively. In contrast, at low SPE doses (1 and 10 µg/mL), the treatment slightly decreased TIMP-1 expression levels to 73.3% and 71.3% of UVB-C, respectively. In conclusion, the present study demonstrated the protective effect of SPE and E-SPE against UVB damage in keratinocytes via ROS scavenging, down-regulating MMP-2 expression and up-regulating TIMP-1 expression. This highlights the potential for SPE as an ingredient in the preparation of functional food against photoaging. PMID:26761824

  15. 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

    2012-12-01

    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. PMID:23142932

  16. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    PubMed

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases. PMID:26767426

  17. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    SciTech Connect

    Dezitter, Xavier; Hammoudi, Fatma; Belverge, Nicolas; Deloulme, Jean-Christophe; Drobecq, Herve; Masselot, Bernadette; Formstecher, Pierre; Mendy, Denise; Idziorek, Thierry . E-mail: thierry.idziorek@lille.inserm.fr

    2007-08-31

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis.

  18. Analyses of the Secondary Particle Radiation and the DNA Damage it Causes to Human Keratinocytes

    SciTech Connect

    Lebel E. A.; Tafrov S.; Rusek, A.; Sivertz, M. B.; Yip, K.; Thompson, K. H.

    2011-11-01

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  19. Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

    SciTech Connect

    Lebel E.; Rusek A.; Sivertz, M.; Yip, K.; Thompson, K.; Tafrov, S.

    2011-11-22

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  20. Bitter apricot essential oil induces apoptosis of human HaCaT keratinocytes.

    PubMed

    Li, Keyou; Yang, Wenhua; Li, Zhe; Jia, Wangwang; Li, Jiazhou; Zhang, Pengfei; Xiao, Tiancun

    2016-05-01

    Psoriasis is a chronic skin disease that affects approximately 2% of the world's population. Conventional therapeutic approaches are not effective or necessarily safe for treating symptoms due to the serious side effects and resistance to currently prescribed drugs. Traditionally, in oriental medicine, apricot seed (Semen Armeniacae amarum) is used to treat skin diseases. However, the underlying mechanism of action has not been systematically elucidated. In the present study, the anti-proliferative effect of bitter apricot essential oil (BAEO) on cultured HaCaT cells was evaluated and the mechanism of action investigated. BAEO was isolated by hydrodistillation, and gas chromatography-mass spectrometry (GC-MS) analysis identified benzaldehyde (75.35%), benzoic acid (6.21%) and mandelonitrile (5.38%). HaCaT cell growth, measured by sulforhodamine B assay (SRB), was inhibited by BAEO with an IC50 value of 142.45 μg/ml. Apoptosis of HaCaT cells treated with BAEO was detected by cell cycle, flow cytometry, and western blot analyses. These measurements revealed G0/G1 cell cycle arrest, elevated numbers of early and late stage apoptotic cells, and caspases-3/8/9 and PARP activation. Z-VAD-FMK, a broad-spectrum caspase inhibitor, attenuated BAEO-induced apoptosis. Also, increased Bax and decreased Bcl-2 levels suggest that BAEO-induced apoptosis is mediated through both death receptor and mitochondrial pathways. Moreover, reduced Rel/NF-κB levels suggest that BAEO-mediated apoptosis is also associated with inhibition of the NF-κB pathway. These data suggest that BAEO is a naturally occurring material that functions as a potent pro-apoptotic factor for human keratinocytes. Thus, it is a promising candidate to treat psoriasis. PMID:26971222

  1. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    PubMed

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. PMID:27001307

  2. 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. PMID:26550599

  3. Protective Effect of Processed Panax ginseng, Sun Ginseng on UVB-irradiated Human Skin Keratinocyte and Human Dermal Fibroblast

    PubMed Central

    Lee, Hyejin; Lee, Joo Yeop; Song, Kyu Choon; Kim, Jinhee; Park, Jeong Hill; Chun, Kwang-Hoon; Hwang, Gwi Seo

    2012-01-01

    In this study, we investigated the protective effects of processed Panax ginseng, sun ginseng (SG) against the UVB-irradiation on epidermal keratinocytes and dermal fibroblasts. Pretreatment of SG in HaCaT keratinocytes and human dermal fibroblasts reduced UVB-induced cell damage as seen by reduced lactate dehydrogenase release. We also found that SG restored the UVB-induced decrease in anti-apoptotic gene expression (bcl-2 and bcl-xL) in these cells, indicating that SG has an anti-apoptotic effect and thus can protect cells from cell death caused by strong UVB radiation. In addition, SG inhibited the excessive expression of c-jun and c-fos gene by the UVB in HeCaT cells and human dermal fibroblasts. We also demonstrated that SG may exert an anti-inflammatory activity by reducing the nitric oxide production and inducible nitric oxide synthase mRNA synthesis in HaCaT keratinocytes and human dermal fibroblasts. This was further supported by its inhibitory effects on the elevated cyclooxygenase-2 and tumor necrosis factor-α transcription which was induced by UVB-irradiation in HaCaT cells. In addition, SG may have anti-aging property in terms of induction of procollagen gene expression and inhibition of the matrix metalloprotease-1 gene expression caused by UVBexposure. These findings suggest that SG can be a potential agent that may protect against the dermal cell damage caused by UVB. PMID:23717106

  4. High Throughput, High Content Screening for Novel Pigmentation Regulators Using a Keratinocyte/Melanocyte Co-culture System

    PubMed Central

    Lee, Ju Hee; Chen, Hongxiang; Kolev, Vihren; Aull, Katherine H.; Jung, Inhee; Wang, Jun; Miyamoto, Shoko; Hosoi, Junichi; Mandinova, Anna; Fisher, David E.

    2014-01-01

    Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of MITF and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4,000 screened compounds including zoxazolamine, 3-methoxycatechol, and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors, and are worthy of further evaluation for potential translation to clinical use. PMID:24438532

  5. Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner

    PubMed Central

    Bidaux, Gabriel; Borowiec, Anne-sophie; Gordienko, Dmitri; Beck, Benjamin; Shapovalov, George G.; Lemonnier, Loïc; Flourakis, Matthieu; Vandenberghe, Matthieu; Slomianny, Christian; Dewailly, Etienne; Delcourt, Philippe; Desruelles, Emilie; Ritaine, Abigaël; Polakowska, Renata; Lesage, Jean; Chami, Mounia; Skryma, Roman; Prevarskaya, Natalia

    2015-01-01

    Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals’ skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca2+ concentration ([Ca2+]m). In turn, [Ca2+]m modulates ATP and superoxide (O2•−) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2•− levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8’s role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures. PMID:26080404

  6. XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas

    PubMed Central

    Rezvani, Hamid Reza; Kim, Arianna L.; Rossignol, Rodrigue; Ali, Nsrein; Daly, Meaghan; Mahfouf, Walid; Bellance, Nadège; Taïeb, Alain; de Verneuil, Hubert; Mazurier, Frédéric; Bickers, David R.

    2010-01-01

    DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes. PMID:21123941

  7. Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair

    PubMed Central

    Wang, Tao; Long, Shuang; Zhao, Na; Wang, Yu; Sun, Huiqin; Zou, Zhongmin; Wang, Junping; Ran, Xinze; Su, Yongping

    2015-01-01

    Programmed cell death 4 (PDCD4) is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes’ biology and indicate that PDCD4 plays a role in epidermal homeostasis. PMID:26703592

  8. The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: Implications for wound healing

    SciTech Connect

    Geary, Sean M.; Cowin, Allison J.; Copeland, Ben; Baleato, Rosa M.; Miyazaki, Kaoru; Ashman, Leonie K.

    2008-07-01

    Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin {alpha}3{beta}1- and/or {alpha}6{beta}4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-{beta}1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.

  9. Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.

    PubMed

    Bidaux, Gabriel; Borowiec, Anne-sophie; Gordienko, Dmitri; Beck, Benjamin; Shapovalov, George G; Lemonnier, Loïc; Flourakis, Matthieu; Vandenberghe, Matthieu; Slomianny, Christian; Dewailly, Etienne; Delcourt, Philippe; Desruelles, Emilie; Ritaine, Abigaël; Polakowska, Renata; Lesage, Jean; Chami, Mounia; Skryma, Roman; Prevarskaya, Natalia

    2015-06-30

    Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures. PMID:26080404

  10. Decellularized dermis in combination with cultivated keratinocytes in a short- and long-term animal experimental investigation.

    PubMed

    Bannasch, H; Stark, G B; Knam, F; Horch, R E; Föhn, M

    2008-01-01

    Decellularized human dermis as a potentially ideal scaffold for dermal substitution in severe burns was examined in a two-staged animal experiment. In an initial step, an in vitro generated composite graft consisting of human keratinocytes and decellularized dermis (AlloDerm) was transplanted onto nude mice in a short-term trial (n = 20, 14 days). Subsequently, a combined one-step grafting of full thickness wounds with both decellularized dermis (in part preincubated with fibroblasts) and cultivated autologous keratinocytes as a cell suspension in fibrin glue was done in a long-term porcine animal model (n = 10, 6 months). In both series, macroscopic wound healing was evaluated by planimetry. Histological investigations included morphological as well as immunohistochemical parameters. The short-term study showed both successful integration of the composite grafts and reduction of wound contraction compared with the control group (epithelial grafts). The long-term porcine study displayed reduced myofibroblast formation and contraction in the wounds that had been treated with fibroblast-preincubated dermis. After 4 weeks, a decline of the structural integrity of the dermal matrix could be noticed. The utility of decellularized dermis as template for both dermal reconstitution and keratinocyte delivery vehicle was shown. The closure of full thickness wounds by a single-step combination of an autologous keratinocyte fibrin sealant suspension and acellular dermis in a pig animal model could be shown. Incorporation of fibroblasts led to reduced wound contraction but could not prevent the loss of dermal integrity. The engineered 'skin' remained viable and stable over a period of 6 months. PMID:18181972

  11. Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

    PubMed Central

    Birkaya, Barbara; Ortt, Kori; Sinha, Satrajit

    2007-01-01

    Background p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (ΔNp63). Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes. Results To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes. PMID:17521434

  12. Expression profiles of cortisol-inactivating enzyme, 11β-hydroxysteroid dehydrogenase-2, in human epidermal tumors and its role in keratinocyte proliferation.

    PubMed

    Terao, Mika; Itoi, Saori; Murota, Hiroyuki; Katayama, Ichiro

    2013-02-01

    The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzes the interconversion between hormonally active cortisol and inactive cortisone within cells. There are two isozymes: 11β-HSD1 activates cortisol from cortisone and 11β-HSD2 inactivates cortisol to cortisone. 11β-HSD1 was recently discovered in skin, and we subsequently found that the enzyme negatively regulates keratinocyte proliferation. We verified 11β-HSD1 and 11β-HSD2 expression in benign and malignant skin tumors and investigated the role of 11β-HSD in skin tumor pathogenesis. Randomly selected formalin-fixed sections of skin lesions of seborrheic keratosis (SK), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) were stained with 11β-HSD1 and 11β-HSD2 antibodies, and 11β-HSD expression was also evaluated in murine epidermis in which hyperproliferation was induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). We observed that 11β-HSD1 expression was decreased in all SK, SCC, and BCC lesions compared with unaffected skin. Conversely, 11β-HSD2 expression was increased in SK and BCC but not in SCC. Overexpression of 11β-HSD2 in keratinocytes increased cell proliferation. In the murine model, 11β-HSD1 expression was decreased in TPA-treated hyperproliferative skin. Our findings suggest that 11β-HSD1 expression is decreased in keratinocyte proliferative conditions, and 11β-HSD2 expression is increased in basal cell proliferating conditions, such as BCC and SK. Assessing 11β-HSD1 and 11β-HSD2 expression could be a useful tool for diagnosing and characterizing skin tumors. PMID:23362866

  13. Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

    PubMed Central

    Jeon, Young-Joo; Jang, Jeong-Yun; Shim, Jung-Hyun; Myung, Pyung Keun; Chae, Jung-Il

    2015-01-01

    Background: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. Methods: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4′,6-diamidino-2-phenylindole staining and Western blotting. Results: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. Conclusions: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells. PMID:26151043

  14. TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity.

    PubMed

    Galindo-Villegas, Jorge; Montalban-Arques, Ana; Liarte, Sergio; de Oliveira, Sofia; Pardo-Pastor, Carlos; Rubio-Moscardo, Fanny; Meseguer, José; Valverde, Miguel A; Mulero, Victoriano

    2016-01-15

    As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca(2+)/TGF-β-activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response. PMID:26673139

  15. Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation

    SciTech Connect

    Thraves, P.; Salehi, Z.; Dritschilo, A.; Rhim, J.S. )

    1990-02-01

    Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.

  16. Transcriptional Network of p63 in Human Keratinocytes

    PubMed Central

    Pozzi, Silvia; Zambelli, Federico; Merico, Daniele; Pavesi, Giulio; Robert, Amélie; Maltère, Peggy; Gidrol, Xavier; Mantovani, Roberto; Vigano, M. Alessandra

    2009-01-01

    p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin. PMID:19390658

  17. [Cytokinetics and keratins of keratinocytes from skin of the elderly].

    PubMed

    Wevers, A; Schulze, H J; Mahrle, G; Steigleder, G K

    1989-11-15

    Regarding the keratin pattern of non-exposed skin, we found no significant qualitative or quantitative differences between 6 old persons (mean age 85 years) and 4 young adults (mean age 20 years). There was, however, a slight increase of proliferation keratins (K6, K16) in aged skin. In non-exposed skin taken from 6 old (mean age 70 years) and 5 young persons (mean age 37 years), longterm primary submersion cultures of keratinocytes did not show any significant differences as far as the classical parameters of growth behavior were concerned (i.e. plating efficiency, cell count, and labeled thymidine incorporation). In accordance with these findings, daily measurements of the thymidine kinase activity in the supernatants revealed discrete but not significant differences between keratinocytes in aged people and those in young persons. PMID:2481907

  18. 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. PMID:26908057

  19. Autophagy in human keratinocytes: an early step of the differentiation?

    PubMed

    Aymard, Elodie; Barruche, Vincent; Naves, Thomas; Bordes, Sylvie; Closs, Brigitte; Verdier, Mireille; Ratinaud, Marie-Hélène

    2011-03-01

    Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway. PMID:21166723

  20. Epidermal nevi with aberrant epidermal structure in keratinocytes and melanocytes.

    PubMed

    Oiso, Naoki; Sugawara, Koji; Yonamine, Ayano; Tsuruta, Daisuke; Kawada, Akira

    2015-04-01

    Epidermal nevi are congenital cutaneous hamartomas caused by embryonic somatic mutations. Ultrastructural features of adult epidermal nevi have rarely been investigated. Herein, we report a case involving a Japanese adult who had epidermal nevi with right congenital blindness and a right accessory nipple. The histopathologic and ultrastructural studies showed divergent abnormal epidermal structures in both melanocytes and keratinocytes. Our case indicates the need to further investigate histopathologic, ultrastructural, and genetic associations in adult epidermal nevi. PMID:25657059

  1. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  2. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

    PubMed

    Varma, Sandeep R; Sivaprakasam, Thiyagarajan O; Mishra, Abheepsa; Kumar, L M Sharath; Prakash, N S; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  3. Mechanosensory and ATP Release Deficits following Keratin14-Cre-Mediated TRPA1 Deletion Despite Absence of TRPA1 in Murine Keratinocytes

    PubMed Central

    Palygin, Oleg; Weyer, Andy D.; Barabas, Marie E.; Lawlor, Michael W.; Staruschenko, Alexander; Stucky, Cheryl L.

    2016-01-01

    Keratinocytes are the first cells that come into direct contact with external tactile stimuli; however, their role in touch transduction in vivo is not clear. The ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) is essential for some mechanically-gated currents in sensory neurons, amplifies mechanical responses after inflammation, and has been reported to be expressed in human and mouse skin. Other reports have not detected Trpa1 mRNA transcripts in human or mouse epidermis. Therefore, we set out to determine whether selective deletion of Trpa1 from keratinocytes would impact mechanosensation. We generated K14Cre-Trpa1fl/fl mice lacking TRPA1 in K14-expressing cells, including keratinocytes. Surprisingly, Trpa1 transcripts were very poorly detected in epidermis of these mice or in controls, and detection was minimal enough to preclude observation of Trpa1 mRNA knockdown in the K14Cre-Trpa1fl/fl mice. Unexpectedly, these K14Cre-Trpa1fl/fl mice nonetheless exhibited a pronounced deficit in mechanosensitivity at the behavioral and primary afferent levels, and decreased mechanically-evoked ATP release from skin. Overall, while these data suggest that the intended targeted deletion of Trpa1 from keratin 14-expressing cells of the epidermis induces functional deficits in mechanotransduction and ATP release, these deficits are in fact likely due to factors other than reduction of Trpa1 expression in adult mouse keratinocytes because they express very little, if any, Trpa1. PMID:26978657

  4. Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.

    PubMed

    Liu, Yu-Chen; Cai, Zhi-Ming; Zhang, Xue-Jun

    2016-01-01

    The persistence infection of low-risk type (type 6 or type 11) of human papillomavirus (HPV) is the main cause of genital warts. Given the high rate of recurrence after treatment, the use of a new molecular agent is certain to be of value. The aim of this study was to achieve targeted inactivation of viral E 7 gene in keratinocytes using the reprogrammed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system. To accomplish this, a universal CRISPR-Cas9 system for targeting both HPV6/11 E 7 genes was constructed by using a dual guide RNA vector. After transfection of the vector into E 7-transfromed keratinocytes, the expression level of E 7 protein was measured using western-blot analysis and the sequence of the E 7 gene was determined using Sanger sequencing. Cell proliferation was analyzed by CCK-8 assay, and cell apoptosis was evaluated by Hoechst 33258 staining, flow cytometry analysis and ELISA assay. The results indicated that both HPV6/11 E 7 genes can be inactivated by the single CRISPR-Cas9 system. Furthermore, silencing of E 7 led to inhibition of cell proliferation and induction of apoptosis in E 7-transfromed keratinocytes but not in normal keratinocytes. Our data suggested that the reprogrammed CRISPR-Cas9 system has the potential for the development of an adjuvant therapy for genital warts. PMID:26228041

  5. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

    PubMed

    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. PMID:25041929

  6. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    PubMed Central

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  7. Proliferative periostitis of the mandibular ramus and condyle: a case report

    PubMed Central

    Seok, Hyun; Kim, Seong-Gon

    2015-01-01

    Proliferative periostitis is a rare form of osteomyelitis that is characterized by new bone formation with periosteal reaction common causes of proliferative periostitis are dental caries, periodontitis, cysts, and trauma. While proliferative periostitis typically presents as a localized lesion, in this study, we describe an extensive form of proliferative periostitis involving the whole mandibular ramus and condyle. Because the radiographic findings were similar to osteogenic sarcoma, an accurate differential diagnosis was important for proper treatment. PMID:26339579

  8. Proliferative periostitis of the mandibular ramus and condyle: a case report.

    PubMed

    Seok, Hyun; Kim, Seong-Gon; Song, Ji-Young

    2015-08-01

    Proliferative periostitis is a rare form of osteomyelitis that is characterized by new bone formation with periosteal reaction common causes of proliferative periostitis are dental caries, periodontitis, cysts, and trauma. While proliferative periostitis typically presents as a localized lesion, in this study, we describe an extensive form of proliferative periostitis involving the whole mandibular ramus and condyle. Because the radiographic findings were similar to osteogenic sarcoma, an accurate differential diagnosis was important for proper treatment. PMID:26339579

  9. Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

    PubMed Central

    Stoll, Stefan W.; Stuart, Philip E.; Lambert, Sylviane; Gandarillas, Alberto; Rittié, Laure; Johnston, Andrew; Elder, James T.

    2016-01-01

    The EGF receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD, but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies. PMID:26802239

  10. Ultraviolet B suppresses vitamin D receptor gene expression in keratinocytes.

    PubMed

    Courtois, S J; Segaert, S; Degreef, H; Bouillon, R; Garmyn, M

    1998-05-01

    Keratinocytes not only produce vitamin D3 in response to ultraviolet B light (UVB) and convert 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D) but also possess the vitamin D receptor (VDR) and respond to 1,25(OH)2D. We characterized the regulation of the expression of the VDR gene in primary human keratinocytes following UVB irradiation. We report a marked dose-dependent down-regulation of the VDR mRNA and protein within a few hours after irradiation. This occurs independently of de novo protein synthesis and is not due to a change in the half-life of the VDR mRNA. Interestingly, treatment of the cells with sodium salicylate, caffeic acid phenethyl ester and tosylphenylchloromethylketone inhibited this down-regulation. Our results strongly suggest the existence of a feedback mechanism in that UVB initiates vitamin D synthesis in keratinocytes and at the same time limits VDR abundance. They also provide a rational explanation for the reported lack of any additive effect between 1,25(OH)2D and UVB phototherapy in the treatment of psoriasis. PMID:9600069

  11. Posttranslational modifications regulate HIPK2, a driver of proliferative diseases.

    PubMed

    Saul, Vera V; Schmitz, M Lienhard

    2013-09-01

    The serine/threonine kinase homeodomain-interacting protein kinase (HIPK2) is a tumor suppressor and functions as an evolutionary conserved regulator of signaling and gene expression. This kinase regulates a surprisingly vast array of biological processes that range from the DNA damage response and apoptosis to hypoxia signaling and cell proliferation. Recent studies show the tight control of HIPK2 by hierarchically occurring posttranslational modifications such as phosphorylation, small ubiquitin-like modifier modification, acetylation, and ubiquitination. The physiological function of HIPK2 as a regulator of cell proliferation and survival has a downside: proliferative diseases. Dysregulation of HIPK2 can result in increased proliferation of cell populations as it occurs in cancer or fibrosis. We discuss various models that could explain how inappropriate expression, modification, or localization of HIPK2 can be a driver for these proliferative diseases. PMID:23616089

  12. The pathophysiology of proliferative vitreoretinopathy in its management.

    PubMed

    Ryan, S J

    1985-07-15

    Cellular proliferation following retinal reattachment surgery frequently results in contraction and subsequent recurrent detachment of the retina, negating an initial successful reattachment. This process has been called by a variety of names, such as massive vitreous retraction, massive preretinal retraction, and, more recently, proliferative vitreoretinopathy. Although a good start has been made by the Retina Society to classify the various types of proliferative vitreoretinopathy, some modifications in the classification are required. The fundamental problem in the treatment of proliferative vitreoretinopathy is a lack of knowledge regarding the factors that stimulate the proliferation of cells. The vitreoretinal surgeon should recognize in the life cycle of this process that stage which an eye with retinal detachment has reached. If there is no active cellular proliferation, then a scleral buckle will usually suffice. If there is traction from epiretinal membranes which cannot be relieved by a buckle, then vitrectomy and adjunct procedures are necessary. If there is active cellular proliferation and epiretinal membranes, then the arguments related to proper timing of vitrectomy must be considered. In cases where the retinal holes can be identified and closed, scleral buckling may be performed with subsequent delayed vitrectomy. In most cases, in my experience, a combination of revision of the scleral buckle is required at the time of vitrectomy and membrane segmentation for proliferative vitreoretinopathy. Until such time as drugs are available to inhibit cellular proliferation or until our basic understanding of the cell biology of this process allows other means of pharmacologic intervention, mechanical approaches will remain necessary for the treatment of the most advanced cases. PMID:4014372

  13. Magnetic resonance perfusion imaging in proliferative cerebral angiopathy.

    PubMed

    Vargas, María Catalina; Castillo, Mauricio

    2011-01-01

    Cerebral proliferative angiopathy (CPA) is an unusual type of vascular malformation with unique clinical and imaging characteristics that distinguish it from the classic arteriovenous malformations. The features of CPA include absence of dominant arterial feeders or flow-related aneurysms, capillary angioectasia without large draining veins, and presence of intermingled normal brain parenchyma that is hypoperfused. We describe the magnetic resonance imaging findings including perfusion in 3 patients with CPA. PMID:21245687

  14. Proliferative verrucous leukoplakia may initially mimic lichenoid reactions

    PubMed Central

    Lopes, Marcio Ajudarte; Feio, Patricia; Santos-Silva, Alan Roger; Vargas, Pablo Agustin

    2015-01-01

    Proliferative verrucous leukoplakia is an intriguing disease, which occurs particularly in women aged greater than 60 years, is not associated with tobacco and alcohol, and has a high risk of recurrence and malignant transformation. Although it is well known that the typical presentation is characterized by multifocal and verrucous white lesions, there is no description that its initial clinical presentation may simulate a lichenoid reaction. PMID:26488020

  15. Frequent phosphatidylinositol-3-kinase mutations in proliferative breast lesions.

    PubMed

    Ang, Daphne C; Warrick, Andrea L; Shilling, Amy; Beadling, Carol; Corless, Christopher L; Troxell, Megan L

    2014-05-01

    The phosphatidylinositol-3-kinase pathway is one of the most commonly altered molecular pathways in invasive breast carcinoma, with phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) mutations in 25% of invasive carcinomas. Ductal carcinoma in situ (DCIS), benign papillomas, and small numbers of columnar cell lesions harbor an analogous spectrum of PIK3CA and AKT1 mutations, yet there is little data on usual ductal hyperplasia and atypical ductal and lobular neoplasias. We screened 192 formalin-fixed paraffin-embedded breast lesions from 75 patients for point mutations using a multiplexed panel encompassing 643 point mutations across 53 genes, including 58 PIK3CA substitutions. PIK3CA point mutations were identified in 31/62 (50%) proliferative lesions (usual ductal hyperplasia and columnar cell change), 10/14 (71%) atypical hyperplasias (atypical ductal hyperplasia and flat epithelial atypia), 7/16 (44%) lobular neoplasias (atypical lobular hyperplasia and lobular carcinoma in situ), 10/21 (48%) DCIS, and 13/37 (35%) invasive carcinomas. In genotyping multiple lesions of different stage from the same patient/specimen, we found considerable heterogeneity; most notably, in 12 specimens the proliferative lesion was PIK3CA mutant but the concurrent carcinoma was wild type. In 11 additional specimens, proliferative epithelium and cancer contained different point mutations. The frequently discordant genotypes of usual ductal hyperplasia/columnar cell change and concurrent carcinoma support a role for PIK3CA-activating point mutations in breast epithelial proliferation, perhaps more so than transformation. Further, these data suggest that proliferative breast lesions are heterogeneous and may represent non-obligate precursors of invasive carcinoma. PMID:24186142

  16. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

    SciTech Connect

    Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.; Norris, D.A. )

    1990-01-01

    Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs.

  17. Th1 and Th17 Cells Induce Proliferative Glomerulonephritis

    PubMed Central

    Summers, Shaun A.; Steinmetz, Oliver M.; Li, Ming; Kausman, Joshua Y.; Semple, Timothy; Edgtton, Kristy L.; Borza, Dorin-Bogdan; Braley, Hal; Holdsworth, Stephen R.

    2009-01-01

    Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1−/− mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against α3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 ± 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies. PMID:19820122

  18. Th1 and Th17 cells induce proliferative glomerulonephritis.

    PubMed

    Summers, Shaun A; Steinmetz, Oliver M; Li, Ming; Kausman, Joshua Y; Semple, Timothy; Edgtton, Kristy L; Borza, Dorin-Bogdan; Braley, Hal; Holdsworth, Stephen R; Kitching, A Richard

    2009-12-01

    Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies. PMID:19820122

  19. ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

    PubMed Central

    Jardé, Thierry; Kass, Lisa; Staples, Margaret; Lescesen, Helen; Carne, Peter; Oliva, Karen; McMurrick, Paul J.; Abud, Helen E.

    2015-01-01

    Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts. PMID:26367378

  20. ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer.

    PubMed

    Jardé, Thierry; Kass, Lisa; Staples, Margaret; Lescesen, Helen; Carne, Peter; Oliva, Karen; McMurrick, Paul J; Abud, Helen E

    2015-01-01

    Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts. PMID:26367378

  1. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

    PubMed Central

    Sudbeck, B D; Baumann, P; Ryan, G J; Breitkopf, K; Nischt, R; Krieg, T; Mauch, C

    1999-01-01

    Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. PMID:10085241

  2. A Novel Controllable Hydrogen Sulfide-Releasing Molecule Protects Human Skin Keratinocytes Against Methylglyoxal-Induced Injury and Dysfunction

    PubMed Central

    Yang, Chun-tao; Zhao, Yu; Xian, Ming; Li, Jian-hua; Dong, Qi; Bai, Hong-bo; Xu, Ji-de; Zhang, Mei-fen

    2014-01-01

    Background/Aim Delayed wound healing is a common skin complication of diabetes, which is associated with keratinocyte injury and dysfunction. Levels of methylglyoxal (MGO), an α-dicarbonyl compound, are elevated in diabetic skin tissue and plasma, while levels of hydrogen sulfide (H2S), a critical gaseous signaling molecule, are reduced. Interestingly, the gas has shown dermal protection in our previous study. To date, there is no evidence demonstrating whether MGO affects keratinocyte viability and function or H2S donation abolishes these effects and improves MGO-related impairment of wound healing. The current study was conducted to examine the effects of MGO on the injury and function in human skin keratinocytes and then to evaluate the protective action of a novel H2S-releasing molecule. Methods An N-mercapto-based H2S donor (NSHD)-1 was synthesized and its ability to release H2S was observed in cell medium and cells, respectively. HaCaT cells, a cell line of human skin keratinocyte, were exposed to MGO to establish an in vitro diabetic wound healing model. NSHD-1 was added to the cells before MGO exposure and the improvement of cell function was observed in respect of cellular viability, apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and behavioral function. Results Treatment with MGO decreased cell viability, induced cellular apoptosis, increased intracellular reactive oxygen species (ROS) content and depressed MMP in HaCaT cells. The treatment also damaged cell behavioral function, characterized by decreased cellular adhesion and migration. The synthesized H2S-releasing molecule, NSHD-1, was able to increase H2S levels in both cell medium and cells. Importantly, pretreatment with NSHD-1 inhibited MGO-induced decreases in cell viability and MMP, increases in apoptosis and ROS accumulation in HaCaT cells. The pretreatment was also able to improve adhesion and migration function. Conclusion These results demonstrate that the novel

  3. Keratinocyte-Derived Chemokine Induces Prostate Epithelial Hyperplasia and Reactive Stroma in a Novel Transgenic Mouse Model

    PubMed Central

    Schauer, Isaiah G.; Ressler, Steven J.; Rowley, David R.

    2009-01-01

    Background Interleukin-8 (IL-8) is upregulated in fibrotic and malignant diseases and is a key mediator of proliferative responses. Elevated IL-8 was recently correlated with benign prostatic hyperplasia epithelium and a myofibroblast reactive stroma. Thus, we sought to determine whether overexpressed IL-8 and keratinocyte-derived chemokine (KC), the functional murine homolog of IL-8, induce prostate epithelial hyperplasia and a reactive phenotype. Methods Transgenic mice that overexpress KC within prostate epithelia and xenograft models with engineered human cells that overexpress IL-8 were developed. Results Overexpression of KC in transgenic mice produced hyperplastic prostate epithelial acini associated with a periacinar reactive stroma. KC induced an altered epithelial/stroma proliferation index ratio, increased acini diameter, epithelial infolding, and expression of prototypical reactive stroma markers. Overexpression of IL-8 in normal human prostate epithelial xenografts correlated with elevated epithelial proliferation index and altered morphology. Elevated human prostate stromal and epithelial cell proliferation, nodule-like morphology and increased xenograft survival were observed in IL-8-overexpressing orthotopic xenografts. Conclusions Together, these data demonstrate that overexpression of IL-8/KC results in a prostate epithelial hyperplasia with an associated reactive stroma phenotype. The novel transgenic mouse and human xenograft models described here may be useful in dissecting key mechanisms of IL-8 induced prostate hyperplasia and reactive stroma. PMID:19021203

  4. [The effect of low temperatures on the viability of human epidermal keratinocytes found at different stages of differentiation].

    PubMed

    Raĭdan, M; Shubin, N A; Blinova, M I; Prokhorov, G G; Pinaev, G P

    2011-01-01

    The aim of this study was a comparative analysis to the degree of stability of human epidermal cells found at different stages of differentiation to low temperatures. The effect of different subzero temperatures of liquid nitrogen vapor on keratinocytes found both in human skin fragments and as isolated cells extracted from skin fragments has been studied. The degree of stability of epidermal cells low temperatures was evaluated by their ability to form a multilayer stratum in culture; hence this phenomenon explains the survival of a sufficient amount of proliferative cells after exposure to subzero temperatures. Quantitative analysis of the ratio of epidermal stem, transitory and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined using antibodies corresponding to their different stages of differentiation. The results of this research show that the stability of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. Epidermal stem cells and transitory cells are more stable than differentiated cells. PMID:21473115

  5. Evidence of Anti-Proliferative Activities in Blue Mussel (Mytilus edulis) By-Products

    PubMed Central

    Beaulieu, Lucie; Thibodeau, Jacinthe; Bonnet, Claudie; Bryl, Piotr; Carbonneau, Marie-Elise

    2013-01-01

    Shellfish waste components contain significant levels of high quality protein and are therefore a potential source for biofunctional high-value peptides. The feasibility of applying a pilot scale enzymatic hydrolysis process to whole Mytilus edulis and, by fractionation, recover hydrolysates presenting a biological activity of interest, was evaluated. Fractions were tested on four immortalized cancerous cell lines: A549, BT549, HCT15 and PC3. The 50 kDa fraction, enriched in peptides, presented anti-proliferative activity with all cell lines and results suggest a bioactive molecule synergy within the fraction. At a protein concentration of 44 µg/mL, the 50 kDa fraction induced a mortality of 90% for PC3, 89% for A549, 85% for HCT15 and of 81% for BT549 cell lines. At the low protein concentration of only 11 µg/mL the 50 kDa fraction still entails a cell mortality of 76% for A549 and 87% for PC3 cell lines. The 50 kDa fraction contains 56% of proteins, 3% of lipids and 6% of minerals on a dry weight basis and the lowest levels detected of taurine and methionine and highest levels of threonine, proline and glycine amino acids. The enzymatic hydrolysis process suggests that Mytilus edulis by-products should be viewed as high-valued products with strong potential as anti-proliferative agent and promising active ingredients in functional foods. PMID:23535393

  6. Anti-proliferative activity of Fumaria vaillantii extracts on different cancer cell lines

    PubMed Central

    Tabrizi, Fatemeh Haji Abbas; Irian, Saeed; Amanzadeh, Amir; Heidarnejad, Fatemeh; Gudarzi, Hoda; Salimi, Mona

    2016-01-01

    Plant-derived natural products are known to have cancer chemo-preventive and chemo-therapeutic properties. Plant extracts or their active constituents are used as folk medicine in traditional therapies by 80% of the world population. The aim of the present study was to determine the anti-proliferative potential of Fumaria vaillantii extracts on three different cancer cell lines including malignant melanoma SKMEL-3, human breast adenocarcinoma MCF-7 and human myelogenous leukemia K562 as well as human gingival fibroblast (HGF) as normal cell line. Anti-proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and annexin methods. Total phenolics and flavonoids were determined by Folin-Ciocalteu and aluminum chloride methods. Chloroform fraction had the lowest IC50 value at 72 h (0.1 μg/ml) in MCF-7 cells. Flowcytometry and annexin-V analysis indicated that the chloroform fraction induced necrosis in MCF-7 cells. In addition, the colorimetric methods showed that the methanolic fraction possessed the highest amount of total phenolics (33.03 ± 0.75 mg/g of dry powder) and flavonoids (10.5 ± 2.0 mg/g of dry powder). The collective data demonstrated that F. vaillantii chloroform fraction may contain effective compounds with chemo-therapeutic potential act through an apoptotic independent pathway. PMID:27168755

  7. Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

    PubMed

    Blaimauer, Karin; Watzinger, Elisabeth; Erovic, Boban M; Martinek, Helga; Jagersberger, Tamara; Thurnher, Dietmar

    2006-01-01

    Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct. PMID:16804300

  8. Serum amyloid A induces interleukin-1β secretion from keratinocytes via the NACHT, LRR and PYD domains-containing protein 3 inflammasome

    PubMed Central

    Yu, N; Liu, S; Yi, X; Zhang, S; Ding, Y

    2015-01-01

    Interleukin (IL)-1β is now emerging as a critical cytokine in the pathogenesis of T helper type 17 (Th17)-mediated skin diseases, including psoriasis. Psoriatic keratinocytes are a major source of IL-1β; however, the mechanisms triggering IL-1β processing remain unknown. Recently, an acute-phase protein serum amyloid A (SAA) has been identified as a danger signal that triggers inflammasome activation and IL-1β secretion. In this study, we detected increased SAA mRNA and protein expression in psoriatic epidermis. In cultured keratinocytes, SAA up-regulated the expression of pro-IL-1β and secretion of mature IL-1β. On the transcriptional level, blocking Toll-like receptor-2 (TLR-2), TLR-4 or nuclear factor kappa B (NF-κB) attenuated SAA-induced expression of IL-1β mRNA. SAA up-regulated caspase-1 and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) expression in keratinocytes. Inhibiting caspase-1 activity and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome on the post-transcriptional level. The mechanism of SAA-triggered NLRP3 activation and subsequent IL-1β secretion was found to involve the generation of reactive oxygen species. Finally, the expression of SAA by keratinocytes was up-regulated by IL-17A. Taken together, our results indicate that keratinocyte-derived SAA triggers a key inflammatory mediator, IL-1β, via NLRP3 inflammasome activation, providing new potential targets for the treatment of this chronic skin disease. PMID:25231464

  9. Baicalin Scavenged Reactive Oxygen Species and Protected Human Keratinocytes Against UVB-induced Cytotoxicity.

    PubMed

    Chang, Wen-Shin; Lin, En-Yuan; Hsu, Shih-Wei; Hu, Pei-Shin; Chuang, Chin-Liang; Liao, Cheng-Hsi; Fu, Chun-Kai; Su, Chung-Hao; Gong, Chi-Li; Hsiao, Chieh-Lun; Bau, DA-Tian; Tsai, Chia-Wen

    Ultraviolet B (UVB), with a wavelength of 280-320 nm, represents one of the most important environmental factors for skin disorders, including sunburn, hyperpigmentation, solar keratosis, solar elastosis and skin cancer. Therefore, protection against excessive UVA-induced damage is useful for prevention of sunburn and other human diseases. Baicalin, a major component of traditional Chinese medicine Scutellaria baicalensis, has been reported to possess antioxidant and cytostatic capacities. In this study, we examined whether baicalin is also capable of protecting human keratinocytes from UVB irradiation. The results showed that baicalin effectively scavenged reactive oxygen species (ROS) elevated within 4 h after UVB radiation and reversed the UVB-suppressed cell viability and UVB-induced apoptosis after 24 h. Our results demonstrated the utility of baicalin to complement the contributions of traditional Chinese medicine in UVB-induced damage to skin and suggested their potential application as pharmaceutical agents in long-term sun-shining injury prevention. PMID:27566079

  10. Phloxine B phototoxicity: a mechanistic study using HaCaT keratinocytes.

    PubMed

    Inbaraj, Johnson J; Kukielczak, Barbara M; Chignell, Colin F

    2005-01-01

    Phloxine B (PhB) (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein; D&C Red No. 28) is a red dye found in drugs, cosmetics and foods; it is also currently being evaluated as a phototoxin for the potential control of fruit flies. Previous studies have shown that PhB is an efficient photosensitizer of damage to cellular membranes; thus, exposure of the skin to the dye and sunlight or artificial light may result in phototoxicity. Therefore, we have studied the phototoxicity of PhB and its structural analogue 2',7'-dichlorofluorescein (DCF) to HaCaT keratinocytes. Anaerobic visible irradiation (>400 nm) of PhB generated a semiquinone type radical, as detected by direct electron paramagnetic resonance. Aerobic visible irradiation of a reaction mixture containing PhB, the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and nicotinamide adenine dinucleotide (reduced) generated a superoxide dismutase-sensitive DMPO/O(2)(.-) adduct. Irradiation of PhB and DCF in D(2)O generated singlet oxygen with quantum yields of 0.59 and 0.06, respectively. PhB was much more phototoxic than DCF when cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Visible irradiation of HaCaT keratinocytes in the presence of PhB (5 micro M) resulted in a 90% decrease in cell viability. 3beta-Hydroxy-5alpha-cholest-6-ene-5-hydroperoxide, a singlet oxygen photoproduct of cholesterol, was isolated from HaCaT keratinocytes irradiated in the presence of PhB. Furthermore, PhB phototoxicity was inhibited by histidine and cysteine, quenchers of singlet oxygen. PhB (0.5 microM) and light irradiation also resulted in DNA damage, as measured by the Comet assay. The phototoxicity mechanism of PhB most probably initially involves a Type-II reaction with free radicals playing a minor role. However, secondary oxidative species such as radicals generated as a result of lipid peroxidation may serve to further promote oxidative

  11. Keratinocyte Expression of CGRPβ: Implications for Neuropathic and Inflammatory Pain Mechanisms

    PubMed Central

    Hou, Q; Barr, TP; Gee, LE; Vickers, JT; Wymer, JP; Borsani, E; Rodella, LF; Getsios, S; Burdo, TH; Eisenberg, E; Guha, U; Lavker, RM; Kessler, JA; Chittur, SV; Fiorino, DF; Rice, FL; Albrecht, PJ

    2011-01-01

    Calcitonin Gene-Related Peptide (CGRP) is a vasodilatory peptide that has been detected at high levels in the skin, blood, and cerebral spinal fluid under a variety of inflammatory and chronic pain conditions, presumably derived from peptidergic C and Aδ innervation. Herein, CGRP immunolabeling (IL) was detected in epidermal keratinocytes at levels that were especially high and widespread in the skin of humans from locations afflicted with postherpetic neuralgia (PHN) and complex region pain syndrome type 1 (CRPS), of monkeys infected with simian immunodeficiency virus, and of rats subjected to L5/L6 spinal nerve ligation, sciatic nerve chronic constriction, and subcutaneous injection of Complete Freund’s Adjuvant. Increased CGRP-IL was also detected in epidermal keratinocytes of transgenic mice with keratin-14 promoter driven overexpression of noggin, an antagonist to BMP-4 signaling. Transcriptome microarray, qPCR, and Western blot analyses using laser captured mouse epidermis from transgenics, monolayer cultures of human and mouse keratinocytes, and multilayer human keratinocyte organotypic cultures, revealed that keratinocytes express predominantly the beta isoform of CGRP. Cutaneous peptidergic innervation has been shown to express predominantly the alpha isoform of CGRP. Keratinocytes also express the cognate CGRP receptor components, CRLR, RAMP1, and RCP, consistent with known observations that CGRP promotes several functional changes in keratinocytes, including proliferation and cytokine production. Our results indicate that keratinocyte derived CGRPβ may modulate epidermal homeostasis through autocrine/paracrine signaling and may contribute to chronic pain under pathological conditions. PMID:21641113

  12. KERATINOCYTE CELL-MEDIATED MUTAGENESIS ASSAY: CORRELATION WITH IN VIVO TUMOR STUDIES

    EPA Science Inventory

    A murine keratinocyte cell-mediated mutagenesis assay was characterized and examined as an in vitro model system for studying the biotransformation of promutagens/procarcinogens by mouse skin. The assay used living cultured newborn SENCAR keratinocytes for the metabolic activatio...

  13. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocyt...

  14. Efficient keratinocyte differentiation strictly depends on JNK-induced soluble factors in fibroblasts.

    PubMed

    Schumacher, Marion; Schuster, Christian; Rogon, Zbigniew M; Bauer, Tobias; Caushaj, Nevisa; Baars, Sebastian; Szabowski, Sibylle; Bauer, Christine; Schorpp-Kistner, Marina; Hess, Jochen; Holland-Cunz, Stefan; Wagner, Erwin F; Eils, Roland; Angel, Peter; Hartenstein, Bettina

    2014-05-01

    Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors have a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases (JNKs) , JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes, we used a heterologous coculture model combining primary human keratinocytes and murine fibroblasts. Although cocultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors, but is also critically dependent on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the coculture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK has a pivotal role in the paracrine cross talk between dermal fibroblasts and epidermal keratinocytes during wound healing. PMID:24335928

  15. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    PubMed

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing. PMID:25466959

  16. PIKE mediates EGFR proliferative signaling in squamous cell carcinoma cells.

    PubMed

    Xie, Z; Jiang, Y; Liao, E-Y; Chen, Y; Pennypacker, S D; Peng, J; Chang, S M

    2012-12-01

    One of the key drivers for squamous cell carcinoma (SCC) proliferation is activation of the epidermal growth factor receptor (EGFR), a known proto-oncogene. However, the mechanism of EGFR-dependent SCC proliferation remains unclear. Our previous studies indicate that epidermal growth factor (EGF)-induced SCC cell proliferation requires the SH3 domain of phospholipase C-γ1 (PLC-γ1), but not its catalytic activity. The SH3 domain of PLC-γ1 is known to activate the short form of nuclear phosphatidylinositol 3-kinase enhancer (PIKE) that enhances the activity of nuclear class Ia phosphatidylinositol 3-kinase (PI3K) required for proliferation. However, PIKE has been described for more than a decade to be present exclusively in neuronal cells. In the present study, we found that PIKE was highly expressed in malignant human keratinocytes (SCC4 and SCC12B2) but had low expression in normal human keratinocytes. Immunohistochemical analysis showed strong nuclear staining of PIKE in human epidermal and tongue SCC specimens but little staining in the adjacent non-cancerous epithelium. Treatment of SCC4 cells with EGF-induced translocation of PLC-γ1 to the nucleus and binding of PLC-γ1 to the nuclear PIKE. Knockdown of PLC-γ1 or PIKE blocked EGF-induced activation of class Ia PI3K and protein kinase C-ζ and phosphorylation of nucleolin in the nucleus as well as EGF-induced SCC cell proliferation. However, inhibition of the catalytic activity of PLC-γ1 had little effect. These data suggest that PIKE has a critical role in EGF-induced SCC cell proliferation and may function as a proto-oncogene in SCC. PMID:22349826

  17. Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2016-06-01

    Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc. PMID:26917432

  18. Effect of Tamoxifen and Raloxifene on the Proliferative Activity of the Breast Epithelium in Premenopausal Women*

    PubMed Central

    Lucato, Miliana T; Freitas-Junior, Ruffo; Moreira, Marise AR; Bernardes-Junior, Júlio RM; Pinto, Sebastião A; Paulinelli, Regis R; Soares, Leonardo R

    2015-01-01

    OBJECTIVES To compare the effects of tamoxifen and raloxifene on the proliferative activity of normal breast tissue in premenopausal women as measured by Ki-67/MIB-1 expression. STUDY DESIGN A total of 48 women with benign breast nodules and a recommendation for surgical removal of the lesion took part in this study. They were randomized to use tamoxifen or raloxifene for 22 days, after which they were submitted to surgery. During the surgical procedure, a 1-cm fragment of normal breast tissue was removed to study Ki-67 expression. RESULTS The mean percentage ratios between immunolabeled and non-labeled cells were 2.02 ± 1.09 and 3.13 ± 3.23 for the tamoxifen and raloxifene groups, respectively. There was no statistically significant difference between the tamoxifen (n = 16) and raloxifene (n = 14) groups in relation to the immunohistochemical analysis of Ki-67 (P = 0.205). CONCLUSION The results of this study showed no difference between tamoxifen and raloxifene with respect to the potential of these drugs to reduce the proliferative activity of the normal breast epithelium in premenopausal women. PMID:25861235

  19. Anti-proliferative activities of sinigrin on carcinogen-induced hepatotoxicity in rats.

    PubMed

    Jie, Meng; Cheung, Wan Man; Yu, Vivian; Zhou, Yanling; Tong, Pak Ho; Ho, John W S

    2014-01-01

    Liver cancer is one of the leading causes of cancer death worldwide. A very high incidence of new liver cancer cases is diagnosed every year, and metastasis has been found to correlate to poor prognoses in humans. Better treatments for liver cancer are thus clearly needed. Sinigrin is one of the major ingredients present in Brassica nigra, which has been used in combination with other herbs for treatment of various diseases. The anti-proliferative activities of sinigrin were studied in a model of carcinogen-induced hepatotoxicity in rats. Rats were orally administered with sinigrin on a daily basis for three months before sacrifice. Sinigrin was found to significantly inhibit the proliferation of liver tumor cells; the number of surface tumors in the rat liver was dramatically reduced. Sinigrin induced apoptosis of liver cancer cells through up-regulation of p53 and down-regulation of Bcl-2 family members and caspases. Our findings indicated that the liver functions were gradually restored after treatment with sinigrin and that the agent did not cause liver toxicity. Cell cycle analysis indicated that sinigrin caused cell cycle arrest in G0/G1 phase. The results suggest that sinigrin exerts important anti-proliferative activities in carcinogen-induced hepatocarcinogenesis in rats, and highlight the potential of sinigrin as an anti-cancer agent for liver cancer. PMID:25329483

  20. Activated keratinocytes in the epidermis of hypertrophic scars.

    PubMed Central

    Machesney, M.; Tidman, N.; Waseem, A.; Kirby, L.; Leigh, I.

    1998-01-01

    The etiology of hypertrophic scarring, a pathological end point of wound healing, is unknown. The scars most commonly occur when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds. Hypertrophic scars are conventionally described as a dermal pathology in which the epidermis has only a passive role. In this study, the expression of keratin intermediate filament proteins and filaggrin has been investigated in the epidermis of hypertrophic scars and site-matched controls from the same patients. Hypertrophic scar epidermis was found to express the hyperproliferative keratins K6 and K16 in interfollicular epidermis in association with K17 and precocious expression of filaggrin. K16 mRNA was localized by in situ hybridization using a highly specific cRNA probe. In contrast to the immunohistochemical location of K16 protein, the K16 mRNA was found to be expressed in the basal cell layer of normal skin. In hypertrophic scars the mRNA distribution corroborated the abnormal K16 protein distribution. These results suggest the keratinocytes in hypertrophic scar epidermis have entered an alternative differentiation pathway and are expressing an activated phenotype. Activated keratinocytes are a feature of the early stages of wound healing producing growth factors that influence fibroblasts, endothelial cells, and the inflammatory response. We propose that cellular mechanisms in the pathogenesis of hypertrophic scarring are more complex than isolated dermal phenomena. The persistence of activated keratinocytes in hypertrophic scar epidermis implicates abnormal epidermal-mesenchymal interactions. Images Figure 1 Figure 3 PMID:9588880

  1. Lipoxygenation of arachidonic acid by subcellular preparations from murine keratinocytes

    SciTech Connect

    Ziboh, V.A.; Casebolt, T.L.; Marcelo, C.L.; Voorhees, J.J.

    1984-10-01

    In these studies, we examined the possibility that cell-free preparations from murine keratinocytes possess 5-lipoxygenase activity in addition to the well-established cyclooxygenase pathway of arachidonic acid (AA) in these cells. Our data demonstrated that the high-speed (105,000 g) supernatant preparations of the murine keratinocytes metabolized (14C)AA into labeled lipoxygenase products. Portions of these radioactive metabolites cochromatographed and comigrated with 12-HETE (a marker for 12-lipoxygenase pathway) and with authentic LTB4 (a marker for 5-lipoxygenase pathway) on silicic acid column chromatography and by thin-layer chromatography (TLC) in two solvent systems respectively. Identity of the novel 14C which comigrated with LTB4 on both TLC and column chromatography was verified further by cochromatography of the free acid with authentic LTB4 on a reverse phase (RP) and the methyl esters on a straight phase high-pressure liquid chromatography. Incubation of the cell-free preparations with (14C)AA in the presence of ETYA, NDGA (inhibitors of cyclooxygenase and lipoxygenase pathways) as well as with 15-HETE (an inhibitor of lipoxygenase pathway) resulted in decreased formation of (14C) 12-HETE and the (14C)LTB4-like metabolite. On the contrary, incubations of the cell-free extracts with (14C) AA in the presence of indomethacin (a cyclooxygenase inhibitor) resulted in increased biosynthesis of the labeled lipoxygenase metabolites. These data indicate the existence of enzymes in soluble fraction of murine keratinocyte which can catalyze the transformation of (14C) AA into products of both the 12- and 5-lipoxygenase pathways.

  2. 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. PMID:22069492

  3. The p53 status of cultured human premalignant oral keratinocytes.

    PubMed Central

    Burns, J. E.; Clark, L. J.; Yeudall, W. A.; Mitchell, R.; Mackenzie, K.; Chang, S. E.; Parkinson, E. K.

    1994-01-01

    Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples. Images Figure 1 Figure 2 Figure 3 PMID:7917902

  4. Arsenite suppression of BMP signaling in human keratinocytes

    SciTech Connect

    Phillips, Marjorie A.; Qin, Qin; Hu, Qin; Zhao, Bin; Rice, Robert H.

    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, 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 keratinocyte

  5. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

    PubMed

    Rivas, J M; Ullrich, S E

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by

  6. Discerning the differential molecular pathology of proliferative middle ear lesions using Raman spectroscopy

    PubMed Central

    Pandey, Rishikesh; Paidi, Santosh Kumar; Kang, Jeon Woong; Spegazzini, Nicolas; Dasari, Ramachandra Rao; Valdez, Tulio Alberto; Barman, Ishan

    2015-01-01

    Despite its widespread prevalence, middle ear pathology, especially the development of proliferative lesions, remains largely unexplored and poorly understood. Diagnostic evaluation is still predicated upon a high index of clinical suspicion on otoscopic examination of gross morphologic features. We report the first technique that has the potential to non-invasively identify two key lesions, namely cholesteatoma and myringosclerosis, by providing real-time information of differentially expressed molecules. In addition to revealing signatures consistent with the known pathobiology of these lesions, our observations provide the first evidence of the presence of carbonate- and silicate-substitutions in the calcium phosphate plaques found in myringosclerosis. Collectively, these results demonstrate the potential of Raman spectroscopy to not only provide new understanding of the etiology of these conditions by defining objective molecular markers but also aid in margin assessment to improve surgical outcome. PMID:26289566

  7. Arabinogalactan protein from Jatropha curcas L. seeds as TGFβ1-mediated inductor of keratinocyte in vitro differentiation and stimulation of GM-CSF, HGF, KGF and in organotypic skin equivalents.

    PubMed

    Zippel, Janina; Wells, Thomas; Hensel, Andreas

    2010-10-01

    Arabinogalactan protein JC from Jatropha curcas seed endosperm (mean molecular weight 140 kDa) was isolated by cold water extraction and characterized concerning sugar and amino acid composition. At 10 and 100 µg/mL JC stimulated mitochondrial activity (MTT test) of human skin cells (HaCaT keratinocytes, fibroblasts) and the ATP status of primary keratinocytes. JC did not influence the cellular proliferation, while primary keratinocytes were triggered into differentiation status. Investigations on a potential mode of action of JC were performed on complex organotypic skin equivalents. JC induced the production of HGF, KGF and TGFβ, with TGFβ being the main inductor for the differentiation-inducing effect of JC. Also the expression of GM-CSF was stimulated strongly by JC. This in vitro activity profile indicated JC to be a potent inductor of cellular differentiation via stimulation of growth hormones and TGF-β-induced cell signaling. PMID:20385211

  8. 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

  9. Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74.

    PubMed

    Djudjaj, Sonja; Lue, Hongqi; Rong, Song; Papasotiriou, Marios; Klinkhammer, Barbara M; Zok, Stephanie; Klaener, Ole; Braun, Gerald S; Lindenmeyer, Maja T; Cohen, Clemens D; Bucala, Richard; Tittel, Andre P; Kurts, Christian; Moeller, Marcus J; Floege, Juergen; Ostendorf, Tammo; Bernhagen, Jürgen; Boor, Peter

    2016-06-01

    Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN. PMID:26453615

  10. Anti-proliferative Activity of T-bet

    PubMed Central

    Oh, Yeon Ji; Shin, Ji Hyun; Won, Hee Yeon

    2015-01-01

    T-bet is a critical transcription factor that regulates differentiation of Th1 cells from CD4+ precursor cells. Since T-bet directly binds to the promoter of the IFN-γ gene and activates its transcription, T-bet deficiency impairs IFN-γ production in Th1 cells. Interestingly, T-bet-deficient Th cells also display substantially augmented the production of IL-2, a T cell growth factor. Exogenous expression of T-bet in T-bet deficient Th cells rescued the IFN-γ production and suppressed IL-2 expression. IFN-γ and IL-2 reciprocally regulate Th cell proliferation following TCR stimulation. Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions. By using IFN-γ-null mice to eliminate the anti-proliferative effect of IFN-γ, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions. Since the anti-proliferative activity of T-bet may be influenced by IL-2 suppression in Th cells, we examined whether T-bet modulates IL-2-independent cell proliferation in a non-T cell population. We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-γ promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells. Although the molecular mechanisms underlying anti-proliferative activity of T-bet remain to be elucidated, T-bet may directly suppress cell proliferation in an IFN-γ- or an IL-2-independent manner. PMID:26330806

  11. Proliferative Glomerulonephritis with Monoclonal IgG Deposits

    PubMed Central

    Satoskar, Anjali; Markowitz, Glen S.; Valeri, Anthony M.; Appel, Gerald B.; Stokes, Michael B.; Nadasdy, Tibor; D'Agati, Vivette D.

    2009-01-01

    Dysproteinemias that result in monoclonal glomerular deposits of IgG are relatively uncommon. Here, we report the largest series of proliferative glomerulonephritis with monoclonal IgG deposits, a form of renal involvement by monoclonal gammopathy that mimics immune-complex glomerulonephritis. We retrospectively identified 37 patients, most of whom were white (81%), female (62%), or older than 50 yr (65%). At presentation, 49% had nephrotic syndrome, 68% had renal insufficiency, and 77% had hematuria. In 30% of the patients, we identified a monoclonal serum protein with the same heavy- and light-chain isotypes as the glomerular deposits (mostly IgG1 or IgG2), but only one patient had myeloma. Histologic patterns were predominantly membranoproliferative (57%) or endocapillary proliferative (35%) with membranous features. Electron microscopy revealed granular, nonorganized deposits, and immunofluorescence demonstrated glomerular deposits that stained for a single light-chain isotype and a single heavy-chain subtype, most commonly IgG3κ (53%). During an average of 30.3 mo of follow-up for 32 patients with available data, 38% had complete or partial recovery, 38% had persistent renal dysfunction, and 22% progressed to ESRD. Correlates of ESRD on univariate analysis were higher creatinine at biopsy, percentage of glomerulosclerosis, and degree of interstitial fibrosis but not immunomodulatory treatment or presence of a monoclonal spike. On multivariate analysis, higher percentage of glomerulosclerosis was the only independent predictor of ESRD. Only one patient lacking a monoclonal spike at presentation subsequently developed a monoclonal spike and no patient with a monoclonal spike at presentation subsequently developed a hematologic malignancy. We conclude that proliferative glomerulonephritis with monoclonal IgG deposits does not seem to be a precursor of myeloma in the vast majority of patients. PMID:19470674

  12. Proliferative and morphologic changes in rat colon following bypass surgery.

    PubMed Central

    Barkla, D. H.; Tutton, P. J.

    1985-01-01

    In this study the proliferative and morphologic changes that occur in the colon of normal and dimethylhydrazine-treated rats following surgical bypass of the middle third of the colon are reported. Proliferative changes were measured by estimating accumulated mitotic indexes following vinblastine treatment and morphologic changes were observed with the use of light microscopy and scanning electron microscopy. Data were collected on Days 0, 7, 14, 30, and 72 after surgery. The results show that surgical bypass produces contrasting effects in the segments proximal to and distal to the suture line. In the proximal segment there was morphologic evidence of hyperplasia, although proliferative activity was unchanged except for an increase at 7 days in normal rats. In the distal segment there was a long-lived increase in the mitotic index, although morphologic changes were not seen. The results for DMH-treated rats were similar to those in normal rats. Groups of isolated dysplastic epithelial cells were often seen in the submucosa adjacent to sutures up to 72 days after surgery. Increased lymphoid infiltration was seen in segments proximal to but not distal to the suture line. It is hypothesized that the different responses of the proximal and distal segments may be related to the different embryologic origins of those segments. It is also hypothesized that the seeding of the submucosa with epithelial cells during suturing may be a factor in tumor recurrence. Images Figure 19 Figure 20 Figure 21 Figure 22 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 PMID:4014432

  13. Steroid synthesis by primary human keratinocytes; implications for skin disease

    SciTech Connect

    Hannen, Rosalind F.; Michael, Anthony E.; Jaulim, Adil; Bhogal, Ranjit; Burrin, Jacky M.; Philpott, Michael P.

    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 human 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 these data

  14. Surgery for Proliferative Diabetic Retinopathy: New Tips and Tricks

    PubMed Central

    Oellers, Patrick; Mahmoud, Tamer H.

    2016-01-01

    Over the recent years, retina specialists have enjoyed significant improvements in the surgical management of proliferative diabetic retinopathy including improved preoperative planning, vitreoretinal instrumentation and new surgical maneuvers. In this review, we present new tips and tricks such as preoperative pharmacotherapy approaches including pegaptanib injection and biodegradable dexamethasone implantation, bimanual vitrectomy techniques and the concept of mixing small gauges as well as valved cannulas and intraoperative optical coherence tomography. With advanced surgical planning and sophisticated operative maneuvers tailored to the individual patient, excellent outcomes can be achieved even in severe cases of diabetic tractional detachment. PMID:27195092

  15. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

    SciTech Connect

    Tang, X.-H.; Vivero, Marina; Gudas, Lorraine J.

    2008-01-01

    We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 {mu}M, 400 {mu}l for 4 days) by 1.59 {+-} 0.2-fold (p < 0.05). ATRA treatment (10 {mu}M) resulted in a 59.9 {+-} 9.8% increase (p < 0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

  16. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  17. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes.

    PubMed

    Zhang, Leshuai W; Yu, William W; Colvin, Vicki L; Monteiro-Riviere, Nancy A

    2008-04-15

    Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess penetration at 1 microM, 2 microM and 10 microM for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake, cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC) layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD both did not detect Cd nor fluorescence signal in the perfusate at any time point or concentration. In HEK, viability decreased significantly (p<0.05) from 1.25 nM to 10 nM after 24 h and 48 h. There was a significant increase in IL-6 at 1.25 nM to 10 nM, while IL-8 increased from 2.5 nM to 10 nM after 24 h and 48 h. TEM of HEK treated with 10 nM of QD621 at 24 h depicted QD in cytoplasmic vacuoles and at the periphery of the cell membranes. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, yet if the skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated. PMID:18261754

  18. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes

    SciTech Connect

    Zhang, Leshuai W.; Yu, William W.; Colvin, Vicki L.; Monteiro-Riviere, Nancy A.

    2008-04-15

    Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess penetration at 1 {mu}M, 2 {mu}M and 10 {mu}M for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake, cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC) layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD both did not detect Cd nor fluorescence signal in the perfusate at any time point or concentration. In HEK, viability decreased significantly (p < 0.05) from 1.25 nM to 10nM after 24 h and 48 h. There was a significant increase in IL-6 at 1.25 nM to 10 nM, while IL-8 increased from 2.5nM to 10nM after 24 h and 48 h. TEM of HEK treated with 10 nM of QD621 at 24 h depicted QD in cytoplasmic vacuoles and at the periphery of the cell membranes. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, yet if the skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated.

  19. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    PubMed

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage. PMID:21401652

  20. Chemical composition, and anti-proliferative and anti-inflammatory effects of the leaf and wholeplant samples of diploid and tetraploid gynostemma pentaphyllum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaf and whole-plant samples of the diploid and tetraploid Gynostemma pentaphyllum (GP)were investigated and compared for their chemical compositions, and their potential anti-proliferative and anti-inflammatory effects. The highest levels of total flavonoids and phenolics were observed in the diplo...

  1. Sequence stratigraphic control on prolific HC reservoir development, Southwest Iran

    USGS Publications Warehouse

    Lasemi, Y.; Kondroud, K.N.

    2008-01-01

    An important carbonate formation in the Persian Gulf and the onshore oil fields of Southwest Iran is the Lowermost Cretaceous Fahliyan formation. The formation in Darkhowain field consists of unconformity-bounded depositional sequences containing prolific hydrocarbon reservoirs of contrasting origin. Located in the high stand systems tract (HST) of the lower sequence encompassing over 200m of oil column are the most prolific reservoir. Another reservoir is over 80m thick consisting of shallowing-upward cycles that are best developed within the transgressive systems tract of the upper sequence. Vertical facies distribution and their paleobathymetry and geophysical log signatures of the Fahliyan formation in the Darkhowain platform reveal the presence of two unconformity-bounded depositional sequences in Vail et al., Van Wagoner et al., and Sarg. The Fahliyan formation mainly consists of platform carbonates composed of restricted bioclastic lime mudstone to packstone of the platform interior, Lithocodium boundstone or ooid-intraclast-bioclast grainstone of the high energy platform margin and the bioclast packstone to lime mudstone related to the off-platform setting.

  2. CYLD regulates keratinocyte differentiation and skin cancer progression in humans

    PubMed Central

    Alameda, J P; Fernández-Aceñero, M J; Moreno-Maldonado, R; Navarro, M; Quintana, R; Page, A; Ramírez, A; Bravo, A; Casanova, M L

    2011-01-01

    CYLD is a gene mutated in familial cylindromatosis and related diseases, leading to the development of skin appendages tumors. Although the deubiquitinase CYLD is a skin tumor suppressor, its role in skin physiology is unknown. Using skin organotypic cultures as experimental model to mimic human skin, we have found that CYLD acts as a regulator of epidermal differentiation in humans through the JNK signaling pathway. We have determined the requirement of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. We show that CYLD overexpression increases keratinocyte differentiation while CYLD loss of function impairs epidermal differentiation. In addition, we describe the important role of CYLD in the control of human non-melanoma skin cancer progression. Our results show the reversion of the malignancy of human squamous cell carcinomas that express increased levels of CYLD, while its functional inhibition enhances the aggressiveness of these tumors which progress toward spindle cell carcinomas. We have found that the mechanisms through which CYLD regulates skin cancer progression include the control of tumor differentiation, angiogenesis and cell survival. These findings of the role of CYLD in human skin cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies. PMID:21900959

  3. Migration of keratinocytes through tunnels of digested fibrin

    NASA Astrophysics Data System (ADS)

    Ronfard, Vincent; Barrandon, Yann

    2001-04-01

    We report here a hitherto undescribed form of cell migration. When a suspension of human keratinocytes is plated on a fibrin matrix, single cells invade the matrix and progress through it as rounded cells by dissolving the fibrin and thereby creating tunnels. These tunnels are cylindrical or helical, the latter being the result of constant change in the path of cellular advance around the helical axis. Helical tunnel formation is strongly promoted by epidermal growth factor. The rate of migration of the cell through the track of a helical tunnel (up to 2.1 mm per day) is about 7-fold greater than through a cylindrical tunnel. Pericellular fibrinolysis leading to tunnel formation depends on the presence of plasminogen in the medium and its conversion to plasmin by a cellular activator. Formation of tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte; we propose that the tunnel is cylindrical when the site of release of plasmin is located at a fixed point on the cell surface and helical when the site of release precesses.

  4. Selenoproteins are essential for proper keratinocyte function and skin development.

    PubMed

    Sengupta, Aniruddha; Lichti, Ulrike F; Carlson, Bradley A; Ryscavage, Andrew O; Gladyshev, Vadim N; Yuspa, Stuart H; Hatfield, Dolph L

    2010-01-01

    Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development. PMID:20805887

  5. 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. PMID:18939936

  6. Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: bilayered versus single-layered substitute.

    PubMed

    Idrus, Ruszymah Bt Hj; Rameli, Mohd Adha bin P; Low, Kiat Cheong; Law, Jia Xian; Chua, Kien Hui; Latiff, Mazlyzam Bin Abdul; Saim, Aminuddin Bin

    2014-04-01

    Split-skin grafting (SSG) is the gold standard treatment for full-thickness skin defects. For certain patients, however, an extensive skin lesion resulted in inadequacies of the donor site. Tissue engineering offers an alternative approach by using a very small portion of an individual's skin to harvest cells for propagation and biomaterials to support the cells for implantation. The objective of this study was to determine the effectiveness of autologous bilayered tissue-engineered skin (BTES) and single-layer tissue-engineered skin composed of only keratinocytes (SLTES-K) or fibroblasts (SLTES-F) as alternatives for full-thickness wound healing in a sheep model. Full-thickness skin biopsies were harvested from adult sheep. Isolated fibroblasts were cultured using medium Ham's F12: Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, whereas the keratinocytes were cultured using Define Keratinocytes Serum Free Medium. The BTES, SLTES-K, and SLTES-F were constructed using autologous fibrin as a biomaterial. Eight full-thickness wounds were created on the dorsum of the body of the sheep. On 4 wounds, polyvinyl chloride rings were used as chambers to prevent cell migration at the edge. The wounds were observed at days 7, 14, and 21. After 3 weeks of implantation, the sheep were euthanized and the skins were harvested. The excised tissues were fixed in formalin for histological examination via hematoxylin-eosin, Masson trichrome, and elastin van Gieson staining. The results showed that BTES, SLTES-K, and SLTES-F promote wound healing in nonchambered and chambered wounds, and BTES demonstrated the best healing potential. In conclusion, BTES proved to be an effective tissue-engineered construct that can promote the healing of full-thickness skin lesions. With the support of further clinical trials, this procedure could be an alternative to SSG for patients with partial- and full-thickness burns. PMID:24637651

  7. Exogenous stimulation with Eclipta alba promotes hair matrix keratinocyte proliferation and downregulates TGF-β1 expression in nude mice.

    PubMed

    Begum, Shahnaz; Lee, Mi Ra; Gu, Li Juan; Hossain, Jamil; Sung, Chang Keun

    2015-02-01

    Eclipta alba (L.) Hassk (E. alba) is a traditionally acclaimed medicinal herb used for the promotion of hair growth. However, to the best of our knowledge, no report has been issued to date on its effects on genetically distorted hair follicles (HFs). In this study, we aimed to identify an agent (stimuli) that may be beneficial for the restoration of human hair loss and which may be used as an alternative to synthetic drugs. We investigated the effects of petroleum ether extract (PEE) and different solvent fractions of E. alba on HFs of nude mice. Treatment was performed by topical application on the backs of nude mice and the changes in hair growth patterns were evaluated. Histological analysis was carried out to evaluate the HF morphology and the structural differences. Immunohistochemical (IHC) staining was performed to visualize follicular keratinocyte proliferation. The histological assessments revealed that the PEE-treated skin specimens exhibited prominent follicular hypertrophy. Subsequently, IHC staining revealed a significant increase (p<0.001) in the number of follicular keratinocytes in basal epidermal and matrix cells. Our results also demonstrated that PEE significantly (p<0.001) reduced the levels of transforming growth factor-β1 (TGF-β1) expression during early anagen and anagen-catagen transition. Our results suggest that PEE of E. alba acts as an important exogenous mediator that stimulates follicular keratinocyte proliferation and delays terminal differentiation by downregulating TGF-β1 expression. Thus, this study highlights the potential use of PEE of E. alba in the treatment of certain types of alopecia. PMID:25484129

  8. Phytochemical properties and anti-proliferative activity of Olea europaea L. leaf extracts against pancreatic cancer cells.

    PubMed

    Goldsmith, Chloe D; Vuong, Quan V; Sadeqzadeh, Elham; Stathopoulos, Costas E; Roach, Paul D; Scarlett, Christopher J

    2015-01-01

    Olea europaea L. leaves are an agricultural waste product with a high concentration of phenolic compounds; especially oleuropein. Oleuropein has been shown to exhibit anti-proliferative activity against a number of cancer types. However, they have not been tested against pancreatic cancer, the fifth leading cause of cancer related death in Western countries. Therefore, water, 50% ethanol and 50% methanol extracts of Corregiola and Frantoio variety Olea europaea L. leaves were investigated for their total phenolic compounds, total flavonoids and oleuropein content, antioxidant capacity and anti-proliferative activity against MiaPaCa-2 pancreatic cancer cells. The extracts only had slight differences in their phytochemical properties, and at 100 and 200 μg/mL, all decreased the viability of the pancreatic cancer cells relative to controls. At 50 μg/mL, the water extract from the Corregiola leaves exhibited the highest anti-proliferative activity with the effect possibly due to early eluting HPLC peaks. For this reason, olive leaf extracts warrant further investigation into their potential anti-pancreatic cancer benefits. PMID:26193251

  9. Mitogen induced proliferative responses of lymphocytes from spot (Leiostomus xanthurus) exposed to polycyclic aromatic hydrocarbon contaminated environments

    SciTech Connect

    Faisal, M.; Marzouk, M.S.; Smith, C.L.; Huggett, R.J. )

    1991-01-01

    The marine fish spot, Leiostomus xanthurus, was collected from five sites in the lower Chesapeake Bay system representing a gradient of sediment polycyclic aromatic hydrocarbon (PAH) concentrations. The proliferative responses to mitogens by anterior kidney lymphocytes were assessed using (3H)-thymidine uptake by replicating DNA. The data show two different mitogen-dependent lymphocytic responses as the sediment PAH levels increase at the sampling sites; a suppression of the response to the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin, and a sharp augmentation of the response to B cell mitogen, lipopolysaccharide (LPS), as well as to poke weed mitogen and peanut agglutinin. The magnitude of the lymphoproliferative responses correlated strongly with the total sediment PAH concentrations (r2 greater than 0.8). A similar correlation was also observed with 15 selected individual PAH compounds regardless of their molecular weights. By maintaining the fish in clean York River water for up to 24 weeks, it was possible to reverse the augmented proliferative responses to LPS of fish from all sampling sites and to increase the reduced responses to Con A, in fish from three sites, and partially in two sites where sediments were highly contaminated with PAH. These results suggest that the proliferative responses of fish lymphocytes to mitogens may be a potentially sensitive biomarker of exposure to, and effects of xenobiotics.

  10. Protective effects of myricetin against ultraviolet-B-induced damage in human keratinocytes.

    PubMed

    Huang, Jheng-Hua; Huang, Chieh-Chen; Fang, Jia-You; Yang, Cheng; Chan, Chi-Ming; Wu, Nan-Lin; Kang, Shung-Wen; Hung, Chi-Feng

    2010-02-01

    Myricetin is a flavonoid similar to quercetin, which is commonly found in natural foods such as berries, vegetables, teas, wine, and herbs. It is considered to be an antioxidant which is capable of quenching photoaging-causing free radicals within the skin. In this study, we investigated the mechanisms underlying protective effect of myricetin on ultraviolet-B (UVB)-induced damage to keratinocytes. We found that myricetin concentration-dependently attenuated UVB-induced keratinocyte death as determined by a cell viability assay. Pretreatment with myricetin also reduced the UVB-induced malondialdehyde level. Moreover, UVB-induced H(2)O(2) generation in keratinocytes was inhibited by myricetin according to flow cytometry, suggesting that myricetin can act as a free radical scavenger when keratinocytes experience photodamage. Furthermore, UVB-induced activation of c-jun-NH(2) terminal kinase (JNK) in keratinocytes was inhibited by myricetin. UVB-induced pre-G(1) phase arrest leading to apoptotic changes in keratinocytes was blocked by myricetin. Taken together, the protective mechanisms of keratinocyte by myricetin against UVB-induced photodamage occur by the inhibition of UVB-induced intracellular hydrogen peroxide production, lipid peroxidation and JNK activation. Therefore, myricetin is suitable for further development as an anti-aging agent for skin care. PMID:19778600

  11. Galectin-7 regulates keratinocyte proliferation and differentiation through JNK-miR-203-p63 signaling

    PubMed Central

    Chen, Hung-Lin; Chiang, Po-Cheng; Lo, Chia-Hui; Lo, Yuan-Hsin; Hsu, Daniel K.; Chen, Huan-Yuan; Liu, Fu-Tong

    2015-01-01

    Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression up-regulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway. PMID:26763438

  12. Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

    PubMed Central

    ORAZIZADEH, MAHMOUD; HASHEMITABAR, MAHMOUD; BAHRAMZADEH, SOMAYEH; DEHBASHI, FRESHTEH NEJAD; SAREMY, SADEGH

    2015-01-01

    Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2–3 mm pieces and placed in trypsin at 4°C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7–10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2–3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. PMID:26137227

  13. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity. PMID:25690925

  14. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes

    SciTech Connect

    Rivas, J.M.; Ullrich, S.E. )

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, the authors examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure. 44 refs., 3 figs., 2 tabs.

  15. Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes

    PubMed Central

    Copley, Richard; Taylor, Martin S.; Hayden, Patrick J.; Stolper, Gina; Mott, Richard; Hein, Jotun; Moffatt, Miriam F.; Cookson, William O. C. M.

    2009-01-01

    The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease. PMID:19888454

  16. Nitric Oxide Donors Suppress Chemokine Production by Keratinocytes in Vitro and in Vivo

    PubMed Central

    Giustizieri, Maria Laura; Albanesi, Cristina; Scarponi, Claudia; De Pità, Ornella; Girolomoni, Giampiero

    2002-01-01

    Nitric oxide (NO) is involved in the modulation of inflammatory responses. In psoriatic skin, NO is highly produced by epidermal keratinocytes in response to interferon-γ and tumor necrosis factor-α. In this study, we investigated whether the NO donors, S-nitrosoglutathione (GS-NO) and NOR-1, could regulate chemokine production by human keratinocytes activated with interferon-γ and tumor necrosis factor-α. In addition, we studied the effects of the topical application of a GS-NO ointment on chemokine expression in lesional psoriatic skin. NO donors diminished in a dose-dependent manner and at both mRNA and protein levels the IP-10, RANTES, and MCP-1 expression in keratinocytes cultured from healthy patients and psoriatic patients. In contrast, constitutive and induced interleukin-8 production was unchanged. GS-NO-treated psoriatic skin showed reduction of IP-10, RANTES, and MCP-1, but not interleukin-8 expression by keratinocytes. Moreover, the number of CD14+ and CD3+ cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also down-regulated ICAM-1 protein expression without affecting mRNA accumulation in vitro, and suppressed keratinocyte ICAM-1 in vivo. Finally, NO donors inhibited nuclear factor-κB and STAT-1, but not AP-1 activities in transiently transfected keratinocytes. These results define NO donors as negative regulators of chemokine production by keratinocytes. PMID:12368213

  17. Cell-matrix interactions modulate interstitial collagenase expression by human keratinocytes actively involved in wound healing.

    PubMed Central

    Saarialho-Kere, U K; Kovacs, S O; Pentland, A P; Olerud, J E; Welgus, H G; Parks, W C

    1993-01-01

    We reported that interstitial collagenase is produced by keratinocytes at the edge of ulcers in pyogenic granuloma, and in this report, we assessed if production of this metalloproteinase is a common feature of the epidermal response in a variety of wounds. In all samples of chronic ulcers, regardless of etiology, and in incision wounds, collagenase mRNA, localized by in situ hybridization, was prominently expressed by basal keratinocytes bordering the sites of active re-epithelialization indicating that collagenolytic activity is a characteristic response of the epidermis to wounding. No expression of mRNAs for 72- and 92-kD gelatinases or matrilysin was seen in keratinocytes, and no signal for any metalloproteinase was detected in normal epidermis. Immunostaining for type IV collagen showed that collagenase-positive keratinocytes were not in contact with an intact basement membrane and, unlike normal keratinocytes, expressed alpha 5 beta 1 receptors. These observations suggest that cell-matrix interactions influence collagenase expression by epidermal cells. Indeed, as determined by ELISA, primary cultures of human keratinocytes grown on basement membrane proteins (Matrigel; Collaborative Research Inc., Bedford, MA) did not express significant levels of collagenase, whereas cells grown on type I collagen produced markedly increased levels. These results suggest that migrating keratinocytes actively involved in re-epithelialization acquire a collagenolytic phenotype upon contact with the dermal matrix. Images PMID:8254040

  18. Comparative transcriptomic profiling of hydrogen peroxide signaling networks in zebrafish and human keratinocytes: Implications toward conservation, migration and wound healing

    PubMed Central

    Lisse, Thomas S.; King, Benjamin L.; Rieger, Sandra

    2016-01-01

    Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H2O2) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H2O2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H2O2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H2O2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H2O2–dependent pathways, EGF, FOXO1, and IKKα. This study expands on our current understanding of the clinical potential of low-level H2O2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits. PMID:26846883

  19. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 in cultured keratinocytes.

    PubMed

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2012-08-01

    Mutations in the ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mm) or high (1.2 mm) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO-1 did not, however, follow similar expression patterns. Protein levels of occludin, beta-catenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1-deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1-inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  20. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 in cultured keratinocytes

    PubMed Central

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G.; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2013-01-01

    Mutations in the ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mM) or high (1.2 mM) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudin 1 and 4 even in low calcium. ZO-1 did not however follow similar expression patterns. Protein levels of occludin, betacatenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/3 and plakoglobin did not show marked changes in SPCA1 deficient keratinocytes. Indirect immunofluorescence labeling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes, and increased intracellular pools of TJ and desmosomal components in SPCA1 inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  1. Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes*

    PubMed Central

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, KeWei

    2012-01-01

    α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders. PMID:22679014

  2. Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion.

    PubMed

    Prince, Tessa; McBain, Andrew J; O'Neill, Catherine A

    2012-08-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  3. Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion

    PubMed Central

    Prince, Tessa; McBain, Andrew J.

    2012-01-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

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

    PubMed Central

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    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-dependent 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. PMID:24423726

  5. Buformin exhibits anti-proliferative and anti-invasive effects in endometrial cancer cells

    PubMed Central

    Kilgore, Joshua; Jackson, Amanda L; Clark, Leslie H; Guo, Hui; Zhang, Lu; Jones, Hannah M; Gilliam, Timothy P; Gehrig, Paola A; Zhou, Chunxiao; Bae-Jump, Victoria L

    2016-01-01

    Objective: Biguanides are anti-diabetic drugs that are thought to have anti-tumorigenic effects. Most pre-clinical studies have focused on metformin for cancer treatment and prevention; however, buformin may be potentially more potent than metformin. Given this, our goal was to evaluate the effects of buformin on cell growth, adhesion and invasion in endometrial cancer cell lines. Methods: The ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed by MTT assay. Apoptosis and cell cycle analysis was performed by FITC Annexin V assay and propidium iodide staining, respectively. Adhesion was analyzed using the laminin adhesion assay. Invasion was assessed using the transwell invasion assay. The effects of buformin on the AMPK/mTOR pathway were determined by Western immunoblotting. Results: Buformin and metformin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines. IC50s were 1.4-1.6 mM for metformin and 8-150 μM for buformin. Buformin induced cell cycle G1 phase arrest in the ECC-1 cells and G2 phase arrest in the Ishikawa cells. For both ECC-1 and Ishikawa cells, treatment with buformin resulted in induction of apoptosis, reduction in adhesion and invasion, activation of AMPK and inhibition of phosphorylated-S6. Buformin potentiated the anti-proliferative effects of paclitaxel in both cell lines. Conclusion: Buformin has significant anti-proliferative and anti-metastatic effects in endometrial cancer cells through modulation of the AMPK/mTOR pathway. IC50 values were lower for buformin than metformin, suggesting that buformin may be more potent for endometrial cancer treatment and worthy of further investigation. PMID:27398153

  6. Grape Cells (Multinucleated Keratinocytes) in Noninfectious Dermatoses: Case Series and Review of the Literature

    PubMed Central

    Sulit, Daryl J.; Adams, Erin G.; Shvartsman, Katerina R.; Rapini, Ronald P.

    2015-01-01

    Abstract: Multinucleated keratinocytes (also known as multinucleated epidermal giant cells) are a frequently overlooked histological finding in noninfectious inflammatory dermatoses. They are sometimes found in conditions characterized by chronic rubbing and pruritus, such as lichen simplex chronicus or prurigo nodularis, and may be a helpful clue in making the clinical diagnosis. This finding must be differentiated from other conditions characterized by multinucleated keratinocytes on histopathology, specifically herpes simplex, varicella zoster, or measles viral infections. The authors present a case series of 2 patients with unique clinical noninfectious diagnoses but similar histopathologic findings on biopsy. The histopathologic findings on both cases demonstrated multinucleated keratinocytes, which were related to manipulation of the epidermis. PMID:26588345

  7. Maturation of human gingival keratinocytes cultured with fibroblasts from keratinizing and non-keratinizing epithelia.

    PubMed

    Taichman, L; Sciubba, J; Cho, M I

    1982-01-01

    When keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratified squamous epithelium that lacks evidence of orthokeratinization or parakeratinization. We attempted to induce orthokeratinization or parakeratinization in cultured gingival keratinocytes by co-cultivation with fibroblasts from human skin, gingiva and buccal mucosa. Keratinization was defined by the morphological appearance of the cultured cells and by the presence of large molecular weight keratin proteins (63,000 and 67,000 mol. wt). Using these criteria, the chosen fibroblasts failed to induce any alteration in the pattern of keratinization. We conclude that under our present culture conditions, fibroblasts alone cannot induce keratinization in cultured keratinocytes. PMID:6180716

  8. Urethral reconstruction with a 3D porous bacterial cellulose scaffold seeded with lingual keratinocytes in a rabbit model.

    PubMed

    Huang, Jian-Wen; Lv, Xiang-Guo; Li, Zhe; Song, Lu-Jie; Feng, Chao; Xie, Min-Kai; Li, Chao; Li, Hong-Bin; Wang, Ji-Hong; Zhu, Wei-Dong; Chen, Shi-Yan; Wang, Hua-Ping; Xu, Yue-Min

    2015-09-01

    The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N  =  10), 3D porous BC alone (group 2, N  =  10), and 3D porous BC seeded with lingual keratinocytes (group 3, N  =  10) were used to repair rabbit ventral urethral defects (2.0   ×   0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23   ±   1.14 μm and 67.00   ±   6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p  <  0.05). Comparisons of smooth muscle content and endothelia density among the three groups revealed a significant increase at each time point (p  <  0.05). Our results demonstrated that 3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction. PMID:26358641

  9. Bovine mature adipocytes readily return to a proliferative state.

    PubMed

    Wei, S; Duarte, M S; Du, M; Paulino, P V R; Jiang, Z; Albrecht, E; Fernyhough-Culver, M; Zan, L; Hausman, G J; Dodson, M V

    2012-12-01

    The dynamics of human and animal adipogenesis has been defined using several traditional cell systems including stromal vascular cells and adipocyte-related cell lines. But a relatively new cell system using progeny cells stemming from the dedifferentiation of purified cultures of mature adipocytes may be used for studying the development and biology of adipocytes. In this research, we show that isolated (and purified) mature adipocytes derived from Wagyu cattle dedifferentiate into progeny cells, and that these spindle-shaped, proliferative-competent daughter cells possess ability to proliferate. We outline the optimum cell culture system and offer precautionary thoughts for effective mature adipocyte culture. Collectively, this represents a novel cell model which may provide new insights into cell development, physiology and use as a model for animal production/composition, tissue engineering and disease treatment. PMID:22943980

  10. Indirubin and Indirubin Derivatives for Counteracting Proliferative Diseases

    PubMed Central

    Blažević, Tina; Heiss, Elke H.; Atanasov, Atanas G.; Breuss, Johannes M.; Dirsch, Verena M.; Uhrin, Pavel

    2015-01-01

    Indirubin is the active component of Danggui Longhui Wan, a traditional Chinese medicine formulation. The encouraging clinical results from the 1980s obtained in chronic myelocytic leukemia patients treated with indirubin stimulated numerous studies on this compound. These investigations explored the use of indirubin in different types of cancer and reported the synthesis of novel derivatives with improved chemical and pharmacokinetic properties. In this paper, we review the impressive progress that has been made in elucidating the mechanistic understanding of how indirubin and its derivatives affect physiological and pathophysiological processes, mainly by inhibition of cell proliferation and induction of cell death. Furthermore, we survey the therapeutic use of these compounds in combating proliferative diseases such as cancer, restenosis, and psoriasis. PMID:26457112

  11. Proliferative glomerulonephritis with monoclonal immunoglobulin in renal allografts

    PubMed Central

    Al-Rabadi, Laith; Francis, Jean M.; Henderson, Joel; Ghai, Sandeep

    2015-01-01

    Glomerulopathy due to dysproteinemia can have a wide spectrum of pathologic and clinical features based on specific characteristics of the abnormal protein and the response induced within the parenchymal tissue. Monoclonal immunoglobulin G (IgG) deposition can manifest as a different glomerular disease. Proliferative glomerulonephritis (GN) with monoclonal IgG deposits (PGNMID) is a unique entity mimicking immune complex GN that does not conform to any of those subtypes. IgG monoclonal granular deposition in the glomeruli with a pattern similar to immune complex disease suggested by C3 and C1q deposition should prompt consideration of PGNMID. Literature is scarce in terms of recurrence of disease in renal allografts. In this article we present the clinical–pathologic features of three cases of PGNMID in the renal allograft showing the variable course and manifestation of the disease. PMID:26613031

  12. Lawsonia intracellularis infection and proliferative enteropathy in foals.

    PubMed

    Pusterla, Nicola; Gebhart, Connie

    2013-11-29

    Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This organism is unique in that it causes proliferation of infected enterocytes, resulting in thickening of the intestinal epithelium, most often the small intestine. This disease affects mainly weanling foals and causes fever, lethargy, peripheral edema, diarrhea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinemia, thickening of segments of the small intestinal wall observed on abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in feces. The epidemiology and genetic basis for pathogenesis for this disease is beginning to be elucidated. Phenotypic traits, genomic features, and gene expression profiles during L. intracellularis infection in vitro and in vivo are presented. In addition, this article reviews the epidemiology, pathological and clinicopathological findings, diagnosis, and control of EPE. PMID:23871678

  13. Equine proliferative enteropathy--a review of recent developments.

    PubMed

    Pusterla, N; Gebhart, C J

    2013-07-01

    Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This emerging disease affects mainly weanling foals and causes fever, lethargy, peripheral oedema, diarrhoea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinaemia, thickening of segments of the small intestinal wall observed upon abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in faeces. Although the clinical entity, diagnostic approach and treatment of EPE are well established and described, the epidemiology for this disease has remained largely unaddressed. This article focuses on new developments in the field of EPE, including epidemiology, pathophysiology, clinical signs, diagnosis, treatment and prevention. PMID:23662705

  14. Loss of Keratinocytic RXRα Combined with Activated CDK4 or oncogenic NRAS Generates UVB-induced Melanomas via Loss of p53 and PTEN in the Tumor Microenvironment

    PubMed Central

    Coleman, Daniel J.; Chagani, Sharmeen; Hyter, Stephen; Sherman, Anna M.; Löhr, Christiane V.; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K.

    2014-01-01

    Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared to control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared to controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K. PMID:25189354

  15. Tamponade in surgery for retinal detachment associated with proliferative vitreoretinopathy

    PubMed Central

    Schwartz, Stephen G; Flynn, Harry W; Lee, Wen-Hsiang; Wang, Xue

    2014-01-01

    Background Retinal detachment (RD) with proliferative vitreoretinopathy (PVR) often requires surgery to restore normal anatomy and to stabilize or improve vision. PVR usually occurs in association with recurrent RD (that is, after initial retinal re-attachment surgery) but occasionally may be associated with primary RD. Either way, a tamponade agent (gas or silicone oil) is needed during surgery to reduce the rate of postoperative recurrent RD. Objectives The objective of this review was to assess the relative safety and effectiveness of various tamponade agents used with surgery for retinal detachment (RD) complicated by proliferative vitreoretinopathy (PVR). Search methods We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2013, Issue 5), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to June 2013), EMBASE (January 1980 to June 2013), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to June 2013), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 26 June 2013. Selection criteria We included randomized controlled trials (RCTs) of participants undergoing surgery for RD associated with PVR that compared various tamponade agents. Data collection and analysis Two review authors screened the search results independently. We used the standard methodological procedures expected by The Cochrane Collaboration. PMID:24532038

  16. Correlation of proliferative and clonogenic tumor cells in multiple myeloma

    SciTech Connect

    Karp, J.E.; Burke, P.J.; Saylor, P.L.; Humphrey, R.L.

    1984-09-01

    To expand on the findings from previous clinical trials that the growth of residual tumor is increased at a predictable time following initial drug administration, malignant plasma cells from bone marrows of patients with multiple myeloma (MM) were examined for changes in proliferation and clonogenicity induced in vivo by cyclophosphamide and in vitro by drug-induced humoral stimulatory activity. Peak plasma cell (/sup 3/H)thymidine labeling index (LI) occurred predictably following drug and paralleled changes in agar colony formation by marrow cells obtained during therapy. Colony-forming capacity of pretreatment MM marrow populations was enhanced when those cells were cultured with humoral stimulatory activity, similar to the increased colony formation detected in Day 9 postcyclophosphamide marrows at the time of peak plasma cell LI. To further define a relationship between proliferative plasma cells and colony-forming tumor cells, MM marrows were fractionated by sedimentation on an isokinetic gradient. Enrichment of a proliferative tumor cell cohort was achieved, evidenced by (/sup 3/H)thymidine LI. Colony-forming cells were also enriched by isokinetic gradient sedimentation, and agar colony formation by MM marrow cell fractions correlated with the kinetic characteristics of the isolated subpopulations. These studies of whole and fractionated human MM marrow cell populations suggest that the kinetically active cells which are induced to proliferate in vivo and in vitro are closely related to the clonogenic tumor cells which produce colonies in agar and which, like those cells measured by (/sup 3/H)thymidine LI, respond to growth stimulation by drug-induced humoral stimulatory activity.

  17. Human keratinocytes have two interconvertible modes of proliferation.

    PubMed

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-02-01

    Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence. PMID:26641719

  18. Reorganization of the interchromosomal network during keratinocyte differentiation.

    PubMed

    Sehgal, Nitasha; Seifert, Brandon; Ding, Hu; Chen, Zihe; Stojkovic, Branislav; Bhattacharya, Sambit; Xu, Jinhui; Berezney, Ronald

    2016-06-01

    The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression. PMID:26490167

  19. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    PubMed

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  20. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    PubMed Central

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  1. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

    Kim, Reuben H; Kang, Mo K; Kim, Terresa; Yang, Paul; Bae, Susan; Williams, Drake W; Phung, Samantha; Shin, Ki-Hyuk; Hong, Christine; Park, No-Hee

    2015-01-01

    p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16INK4A, induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells. PMID:26138448

  2. Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

    SciTech Connect

    Rosen, C.F.; Gajic, D.; Drucker, D.J. )

    1990-05-01

    UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.

  3. [Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex].

    PubMed

    Kitajima, Y; Jokura, Y; Yaoita, H

    1991-06-01

    The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1720328

  4. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    PubMed

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    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. Immunolocalization 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(+) 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. PMID:27216463

  5. Tumour endothelial marker-8 in wound healing and its impact on the proliferation and migration of keratinocytes

    PubMed Central

    WANG, SHEILA C.; YE, LIN; SANDERS, ANDREW J.; RUGE, FIONA; HARDING, KEITH G.; JIANG, WEN G.

    2016-01-01

    Chronic wound management represents a significant burden on healthcare systems and negatively impacts on the quality of patient life. New strategies to understand and identify wounds that will not heal in a normal manner are required. Tumour endothelial marker-8 (TEM-8) has been implicated in the wound healing and angiogenesis processes. TEM-8 expression was examined at the transcript level in a cohort of acute (n=10) and chronic (n=14) wounds and in normal skin (n=10). Protein analysis of TEM-8 was also undertaken for this cohort using immunohistochemistry (IHC). TEM-8 impact on keratinocyte cell growth and migration was assessed following TEM-8 ribozyme transgene transfection of human HaCaT keratinocytes using cell growth and electric cell-substrate impedance sensing (ECIS)-based assays. Expression of TEM-8 was observed to be increased in acute wounds compared to chronic wounds and normal skin using quantitative polymerase chain reaction transcript analysis and IHC staining of wound tissues. Knockdown of TEM-8 in HaCaT cells, using two independent ribozyme transgenes, resulted in significant decreases in cell growth as well as reductions in the rate of migration assessed using an ECIS-based system. TEM-8 may be differentially expressed between wound types and loss of this molecule impacts HaCaT growth and migration, potentially implicating this molecule as a factor involved in successful progression of wound healing. PMID:26677171

  6. Vascularization of the Dermal Support Enhances Wound Re-Epithelialization by In Situ Delivery of Epidermal Keratinocytes

    PubMed Central

    Lugo, Liana M.; Lei, Pedro

    2011-01-01

    Despite significant advances in management of severe wounds such as burns and chronic ulcers, autologous split-thickness skin grafts are still the gold standard of care. The main problems with this approach include pain and discomfort associated with harvesting autologous tissue, limited availability of donor sites, and the need for multiple surgeries. Although tissue engineering has great potential to provide alternative approaches for tissue regeneration, several problems have hampered progress in translating technological advances to clinical reality. Specifically, engineering of skin substitutes requires long culture times and delayed vascularization after implantation compromises graft survival. To address these issues we developed a novel two-prong strategy for tissue regeneration in vivo: (1) vascularization of acellular dermal scaffolds by infiltration of angiogenic factors; and (2) generation of stratified epidermis by in situ delivery of epidermal keratinocytes onto the prevascularized dermal support. Using athymic mouse as a model system, we found that incorporation of angiogenic factors within acellular human dermis enhanced the density and diameter of infiltrating host blood vessels. Increased vascularization correlated with enhanced proliferation and stratification of the neoepidermis originating from the fibrin-keratinocyte cell suspension. This strategy promoted tissue regeneration in vivo with no need for engineering skin substitutes; therefore, it may be useful for treatment of major wounds when skin donor sites are scarce and rapid wound coverage is required. PMID:20929281

  7. Fibre optic confocal imaging (FOCI) of keratinocytes, blood vessels and nerves in hairless mouse skin in vivo

    PubMed Central

    BUSSAU, L. J.; VO, L. T.; DELANEY, P. M.; PAPWORTH, G. D.; BARKLA, D. H.; KING, R. G.

    1998-01-01

    Fibre optic confocal imaging (FOCI) enabled subsurface fluorescence microscopy of the skin of hairless mice in vivo. Application of acridine orange enabled imaging of the layers of the epidermis. The corneocytes of the stratum corneum, the keratinocytes in the basal layers and redundant hair follicles were visualised at depths greater than 100 μm. Cellular and nuclear membranes of keratinocytes of the skin were visualised by the use of acridine orange and DIOC5(3). Imaging of the skin after injection of FITC-dextran revealed an extensive network of blood vessels with a size range up to 20 μm. Blood cells could be seen moving through dermal vessels and the blood circulation through the dermal vascular bed was video-taped. The fluorescent dye 4-di-2-ASP showed the presence of nerves fibres around the hair follicles and subsurface blood vessels. Comparison was made between images obtained in vivo using FOCI and in vitro scanning electron microscopy and conventional histology. FOCI offers the potential to study dynamic events in vivo, such as blood flow, skin growth, nerve regeneration and many pathological processes, in ways which have not previously been possible. PMID:9643419

  8. Metabolic activation of pyrrolizidine alkaloids leading to phototoxicity and photogenotoxicity in human HaCaT keratinocytes.

    PubMed

    Wang, Chia-Chi; Xia, Qingsu; Li, Meng; Wang, Shuguang; Zhao, Yuewei; Tolleson, William H; Yin, Jun-Jie; Fu, Peter P

    2014-01-01

    Pyrrolizidine alkaloids, produced by a large number of poisonous plants with wide global distribution, are associated with genotoxicity, tumorigenicity, and hepatotoxicity in animals and humans. Mammalian metabolism converts pyrrolizidine alkaloids to reactive pyrrolic metabolites (dehydropyrrolizidine alkaloids) that form covalent protein and DNA adducts. Although a mechanistic understanding is currently unclear, pyrrolizidine alkaloids can cause secondary (hepatogenous) photosensitization and induce skin cancer. In this study, the phototoxicity of monocrotaline, riddelliine, dehydromonocrotaline, dehydroriddelliine, and dehydroretronecine (DHR) in human HaCaT keratinocytes under ultraviolet A (UVA) irradiation was determined. UVA irradiation of HaCaT cells treated with dehydromonocrotaline, dehydroriddelline, and DHR resulted in increased release of lactate dehydrogenase and enhanced photocytotoxicity proportional to the UVA doses. UVA-induced photochemical DNA damage also increased proportionally with dehydromonocrotaline and dehydroriddelline. UVA treatment potentiated the formation of 8-hydroxy-2'-deoxyguanosine DNA adducts induced by dehydromonocrotaline in HaCaT skin keratinocytes. Using electron spin resistance trapping, we found that UVA irradiation of dehydromonocrotaline and dehydroriddelliine generates reactive oxygen species (ROS), including hydroxyl radical, singlet oxygen, and superoxide, and electron transfer reactions, indicating that cytotoxicity and genotoxicity of these compounds could be mediated by ROS. Our results suggest that dehydropyrrolizidine alkaloids formed or delivered to the skin cause pyrrolizidine alkaloid-induced secondary photosensitization and possible skin cancer. PMID:25436474

  9. A model system to analyse the ability of human keratinocytes to form hair follicles.

    PubMed

    Thangapazham, Rajesh L; Klover, Peter; Li, Shaowei; Wang, Ji-An; Sperling, Leonard; Darling, Thomas N

    2014-06-01

    Earlier studies showed that dermal cells lose trichogenic capacity with passage, but studies on the effect of keratinocyte passage on human hair follicle neogenesis and graft quality have been hampered by the lack of a suitable model system. We recently documented human hair follicle neogenesis in grafted dermal-epidermal composites, and in the present study, we determined the effects of keratinocyte passage on hair follicle neogenesis. Dermal equivalents were made with cultured human dermal papilla cells and were overlaid with either primary or passaged human keratinocytes to form dermal-epidermal composites; these were then grafted onto immunodeficient mice. Superior hair follicle neogenesis was observed using early keratinocyte cultures. Characteristics such as formation of hair shafts and sebaceous glands, presence of hair follicles with features of anagen or telogen follicles, and reproducible hair and skin function parameters make this model a tool to study human hair follicle neogenesis and development. PMID:24758480

  10. Abnormal organization of keratin intermediate filaments in cultured keratinocytes of epidermolysis bullosa simplex.

    PubMed

    Kitajima, Y; Inoue, S; Yaoita, H

    1989-01-01

    Distinctive abnormality in the organization of keratin intermediate filaments (KIFs) was found for the first time in cultured epidermal keratinocytes from two patients with hereditary epidermolysis bullosa simplex (EBS), which showed cleavages above the basement membrane zone due to the fragility of basal cells. KIFs in EBS keratinocytes revealed an irregular radial arrangement composed of sparse but thick KIF bundles. Furthermore, these KIF bundles in many cells changed into numerous ball-like keratin aggregates and disappeared beyond these keratin aggregates in the peripheral cytoplasm. Electron microscopy of cultured EBS keratinocytes showed that many ball-like structures consisting of fine filaments or granules or homogeneous substances were scattered in the peripheral regions of the cell attaching to the dish, and intermediate filaments appeared to be emanating from or surrounding the structures. These ball-like keratin aggregates have never been observed in normal human keratinocytes. PMID:2471468

  11. The Use of Cultured Allogenic Keratinocyte Grafting in a Patient with Epidermolysis Bullosa Simplex

    PubMed Central

    Shin, Kee Cheol; Park, Bo Young; Kim, Woo Seob; Bae, Tae Hui

    2011-01-01

    Epidermolysis bullosa (EB) is a rare genetic disease that is known for continuous skin blistering caused by minor trauma. The skin blisters and bullae that develop often cause skin defects. There is no definitive treatment for EB, only symptomatic relief. We report our experience with cultured allogenic keratinocyte grafting in a newborn patient with EB simplex who had unhealed raw surfaces and was not a skin grafting candidate. The skin lesions of the patient were covered with cultured allogenic keratinocyte grafts and re-epithelialized quickly with no scarring. Allogenic keratinocyte grafting reduced pain and produced noticeable improvements in the unhealed wounds. We think that allogenic keratinocyte grafting can play an important role in the management of patients with EB simplex. PMID:22346287

  12. Protective effect of 3,4-dihydroxybenzoic acid isolated from Cladophora wrightiana Harvey against ultraviolet B radiation-induced cell damage in human HaCaT keratinocytes.

    PubMed

    Cha, Ji Won; Piao, Mei Jing; Kim, Ki Cheon; Zheng, Jian; Yao, Cheng Wen; Hyun, Chang Lim; Kang, Hee Kyoung; Yoo, Eun Sook; Koh, Young Sang; Lee, Nam Ho; Ko, Mi Hee; Hyun, Jin Won

    2014-03-01

    The aim of the present study was to elucidate the protective properties of 3,4-dihydroxybenzoic acid (DBA) isolated from Cladophora wrightiana Harvey (a green alga) against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. DBA exhibited scavenging actions against the 1,1-diphenyl-2-picrylhydrazyl radical, the superoxide anion, and the hydroxyl radical. Furthermore, DBA decreased the levels of intracellular reactive oxygen species generated by hydrogen peroxide or UVB treatment of the cells. DBA also decreased the UVB-augmented levels of phospho-histone H2A.X and the extent of comet tail formation, which are both indications of DNA damage. In addition, the compound safeguarded keratinocytes from UVB-induced injury by reversing the production of apoptotic bodies, overturning the disruption of mitochondrial membrane potential, increasing the expression of the anti-apoptotic protein, B-cell lymphoma 2, and decreasing the expression of the pro-apoptotic proteins, Bcl-2-associated X and cleaved caspase-3. Taken together, these results demonstrate that DBA isolated from a green alga protects human keratinocytes against UVB-induced oxidative stress and apoptosis. PMID:24414942

  13. The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

    PubMed

    Rauhala, Leena; Hämäläinen, Lasse; Dunlop, Thomas W; Pehkonen, Petri; Bart, Geneviève; Kokkonen, Maarit; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-12-25

    The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation. PMID:26391144

  14. Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase signaling pathways.

    PubMed

    Fitsialos, Giorgos; Chassot, Anne-Amandine; Turchi, Laurent; Dayem, Manal A; LeBrigand, Kevin; Moreilhon, Chimène; Meneguzzi, Guerrino; Buscà, Roser; Mari, Bernard; Barbry, Pascal; Ponzio, Gilles

    2007-05-18

    Covering denuded dermal surfaces after injury requires migration, proliferation, and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38(MAPK) and phosphatidylinositol 3-kinase (PI3K), demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. p38(MAPK) inhibition only delays "healing," probably in line with the control of genes involved in the propagation of injury-initiated signaling. In contrast, PI3K inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF, and ETS1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38(MAPK) and negative ones triggered by PI3K. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids. PMID:17363378

  15. Pro-Proliferative Function of Mitochondrial Sirtuin Deacetylase SIRT3 in Human Melanoma.

    PubMed

    George, Jasmine; Nihal, Minakshi; Singh, Chandra K; Zhong, Weixiong; Liu, Xiaoqi; Ahmad, Nihal

    2016-04-01

    Melanoma, the most aggressive form of skin cancer, is often fatal if not treated early. Therefore, novel target-based strategies are required to combat this neoplasm. The objective of this study was to determine the role and functional significance of the mitochondrial sirtuin 3 (SIRT3) in melanoma. We found that compared with normal primary and immortalized human melanocytes, SIRT3 is significantly overexpressed in multiple human melanoma cells at mRNA and protein levels. Further, employing human tissue microarray, we found that SIRT3 is significantly upregulated in clinical melanoma tissues, compared with melanocytic nevi tissues. Furthermore, a short hairpin RNA-mediated knockdown of SIRT3 in human melanoma cells resulted in (i) a decrease in cellular proliferation, colony formation, and cellular migration; (ii) induction of senescence as shown by an increase in senescence-associated beta-galactosidase activity and formation of senescence-associated heterochromatin foci as well as an increase in mRNA and protein levels of p16(INK4a) and p21(Waf1); (iii) G1-phase arrest of the cell cycle; and (iv) decreases in mRNA and protein levels of cyclins (D1, E1) and cyclin-dependent kinases (2, 4, and 6). Conversely, forced exogenous overexpression of SIRT3 promoted an increase in proliferative potential of Hs294T melanoma cells and normal immortalized Mel-ST melanocytes. Finally, we found that SIRT3 knockdown significantly inhibited tumorigenesis in a xenograft model in vivo. To our knowledge, this is the first study supporting the pro-proliferative function of SIRT3 in melanoma. PMID:26743598

  16. The anti-proliferative and anti-angiogenic effect of the methanol extract from brittle star

    PubMed Central

    Baharara, Javad; Amini, Elaheh; Mousavi, Marzieh

    2015-01-01

    Background: Anti-angiogenic therapy is a crucial step in cancer treatment. The discovery of new anti-angiogenic compounds from marine organisms has become an attractive concept in anti-cancer therapy. Because little data correlated to the pro- and anti-angiogenic efficacies of Ophiuroidea, which include brittle star, the current study was designed to explore the anti-angiogenic potential of brittle star methanol extract in vitro and in vivo. Methods: The anti-proliferative effect of brittle star extract on A2780cp cells was examined by MTT assays, and transcriptional expression of VEGF and b-FGF was evaluated by RT-PCR. In an in vivo model, 40 fertilized Ross eggs were divided into control and three experimental groups. The experimental groups were incubated with brittle star extract at concentrations of 25, 50 and 100 µg/ml, and photographed by photo-stereomicroscopy. Ultimately, numbers and lengths of vessels were measured by Image J software. Data were analyzed with SPSS software (p<0.05). Results: Results illustrated that the brittle star extract exerted a dose- and time-dependent anti-proliferative effect on A2780cp cancer cells. In addition, VEGF and b-FGF expression decreased with brittle star methanol extract treatment. Macroscopic evaluations revealed significant changes in the second and third experimental group compared to controls (p<0.05). Conclusion: These finding revealed the anti-angiogenic effects of brittle star methanol extract in vitro and in vivo confer novel insight into the application of natural marine products in angiogenesis-related pathologies. PMID:26989740

  17. Pericyte Chemomechanics and the Angiogenic Switch: Insights Into the Pathogenesis of Proliferative Diabetic Retinopathy?

    PubMed Central

    Durham, Jennifer T.; Dulmovits, Brian M.; Cronk, Stephen M.; Sheets, Anthony R.; Herman, Ira M.

    2015-01-01

    Purpose. To establish the regulatory roles that pericytes have in coordinating retinal endothelial cell (EC) growth and angiogenic potential. Methods. Pericytes were derived from donor diabetic (DHuRP) or normal (NHuRP) human retinae, and characterized using vascular markers, coculture, contraction, morphogenesis, and proliferation assays. To investigate capillary “cross-talk,” pericyte-endothelial coculture growth, and connexin-43 (Cx43) expression assays were performed. Paracrine effects were examined via treating EC with pericyte-derived conditioned media (CM) in proliferation, angiogenesis, and angiocrine assays. The effects of sphingosine 1-phosphate (S1P) were assessed using receptor antagonists. Results. The DHuRP exhibit unique proliferative and morphologic properties, reflecting distinctive cytoskeletal and isoactin expression patterns. Unlike NHuRP, DHuRP are unable to sustain EC growth arrest in coculture and display reduced Cx43 expression. Further, CM from DHuRP (DPCM) markedly stimulates EC proliferation and tube formation. Treatment with S1P receptor antagonists mitigates DPCM growth-promotion in EC and S1P-mediated pericyte contraction. Angiocrine assays on normal and diabetic pericyte secretomes reveal factors involved in angiogenic control, inflammation, and metabolism. Conclusions. Effects from the diabetic microenvironment appear sustainable in cell culture: pericytes derived from diabetic donor eyes seemingly possess a “metabolic memory” in vitro, which may be linked to original donor health status. Diabetes- and pericyte-dependent effects on EC growth and angiogenesis may reflect alterations in bioactive lipid, angiocrine, and chemomechanical signaling. Altogether, our results suggest that diabetes alters pericyte contractile phenotype and cytoskeletal signaling, which ultimately may serve as a key, initiating event required for retinal endothelial reproliferation, angiogenic activation, and the pathological neovascularization

  18. Insulin-sensitizing and anti-proliferative effects of Argania spinosa seed extracts.

    PubMed

    Samane, Samira; Noël, Josette; Charrouf, Zoubida; Amarouch, Hamid; Haddad, Pierre Selim

    2006-09-01

    Argania spinosa is an evergreen tree endemic of southwestern Morocco. Many preparations have been used in traditional Moroccan medicine for centuries to treat several illnesses including diabetes. However, scientific evidence supporting these actions is lacking. Therefore, we prepared various extracts of the argan fruit, namely keel, cake and argan oil extracts, which we tested in the HTC hepatoma cell line for their potential to affect cellular insulin responses. Cell viability was measured by Trypan Blue exclusion and the response to insulin evaluated by the activation of the extracellular regulated kinase (ERK1/2), ERK kinase (MEK1/2) and protein kinase B (PKB/Akt) signaling components. None of the extracts demonstrated significant cytotoxic activity. Certain extracts demonstrated a bi-phasic effect on ERK1/2 activation; low doses of the extract slightly increased ERK1/2 activation in response to insulin, whereas higher doses completely abolished the response. In contrast, none of the extracts had any significant effect on MEK whereas only a cake saponin subfraction enhanced insulin-induced PKB/Akt activation. The specific action of argan oil extracts on ERK1/2 activation made us consider an anti-proliferative action. We have thus tested other transformed cell lines (HT-1080 and MSV-MDCK-INV cells) and found similar results. Inhibition of ERK1/2 activation was also associated with decreased DNA synthesis as evidenced by [(3)H]thymidine incorporation experiments. These results suggest that the products of Argania spinosa may provide a new therapeutic avenue against proliferative diseases. PMID:16951716

  19. Insulin-sensitizing and Anti-proliferative Effects of Argania spinosa Seed Extracts

    PubMed Central

    Samane, Samira; Noël, Josette; Charrouf, Zoubida; Amarouch, Hamid; Haddad, Pierre Selim

    2006-01-01

    Argania spinosa is an evergreen tree endemic of southwestern Morocco. Many preparations have been used in traditional Moroccan medicine for centuries to treat several illnesses including diabetes. However, scientific evidence supporting these actions is lacking. Therefore, we prepared various extracts of the argan fruit, namely keel, cake and argan oil extracts, which we tested in the HTC hepatoma cell line for their potential to affect cellular insulin responses. Cell viability was measured by Trypan Blue exclusion and the response to insulin evaluated by the activation of the extracellular regulated kinase (ERK1/2), ERK kinase (MEK1/2) and protein kinase B (PKB/Akt) signaling components. None of the extracts demonstrated significant cytotoxic activity. Certain extracts demonstrated a bi-phasic effect on ERK1/2 activation; low doses of the extract slightly increased ERK1/2 activation in response to insulin, whereas higher doses completely abolished the response. In contrast, none of the extracts had any significant effect on MEK whereas only a cake saponin subfraction enhanced insulin-induced PKB/Akt activation. The specific action of argan oil extracts on ERK1/2 activation made us consider an anti-proliferative action. We have thus tested other transformed cell lines (HT-1080 and MSV-MDCK-INV cells) and found similar results. Inhibition of ERK1/2 activation was also associated with decreased DNA synthesis as evidenced by [3H]thymidine incorporation experiments. These results suggest that the products of Argania spinosa may provide a new therapeutic avenue against proliferative diseases. PMID:16951716

  20. Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis.

    PubMed

    Urgard, Egon; Lorents, Annely; Klaas, Mariliis; Padari, Kärt; Viil, Janeli; Runnel, Toomas; Langel, Kent; Kingo, Külli; Tkaczyk, Eric; Langel, Ülo; Maimets, Toivo; Jaks, Viljar; Pooga, Margus; Rebane, Ana

    2016-08-10

    The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases. PMID:27269729