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Sample records for alters keratinocytes expression

  1. Altered expression of keratinocyte growth factor and its receptor in psoriasis.

    PubMed

    Finch, P W; Murphy, F; Cardinale, I; Krueger, J G

    1997-12-01

    One of the biological characteristics of psoriasis is excessive flaking of the skin. This is directly related to the marked hyperplasia of epidermal keratinocytes and to incomplete epidermal differentiation. Keratinocyte growth factor (KGF), a potent mitogen for human keratinocytes, is expressed by stromal cells. Alterations in the KGF signaling pathway might account for the epidermal hyperplasia associated with psoriasis. To test this hypothesis, we investigated the expression of KGF and its receptor (KGFR) in psoriasis tissue. KGF and KGFR mRNA levels were found to be frequently elevated in psoriatic skin specimens as compared with normal skin. Increased KGF transcript expression was localized to the dermal layer of the involved skin specimen using in situ hybridization. In contrast, KGFR transcript and protein expression was localized to the basal layer of keratinocytes in normal skin and to the basal and suprabasal layers of the psoriatic epidermis, coincident with the expanded proliferative keratinocyte pool. To identify molecules that might regulate KGFR expression we investigated the effects of various pharmacological agents and cytokines on KGFR synthesis by keratinocytes. Phorbol ester, interleukin-6, interferon-gamma, and ultraviolet B (UVB) treatment all led to substantial down-regulation of KGFR expression. The down-regulation of KGFR synthesis by UVB suggests a possible mechanism for the antiproliferative action of this agent in the treatment of psoriasis. Taken together, these results suggest that increased KGFR-mediated signaling in keratinocytes in the lesional epidermis might account in part for the epidermal hyperplasia in psoriasis.

  2. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed Central

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-01-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease. Images PMID:1534817

  3. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-06-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

  4. Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

    PubMed

    Diaz-Guerra, M; Haddow, S; Bauluz, C; Jorcano, J L; Cano, A; Balmain, A; Quintanilla, M

    1992-02-01

    Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.

  5. Wound healing in the alpha2beta1 integrin-deficient mouse: altered keratinocyte biology and dysregulated matrix metalloproteinase expression.

    PubMed

    Grenache, David G; Zhang, Zhonghua; Wells, Laura E; Santoro, Samuel A; Davidson, Jeffrey M; Zutter, Mary M

    2007-02-01

    The alpha2beta1 integrin, a collagen/laminin receptor, is expressed at high level in the basal cell layer of the epidermis. To define the role of the alpha2beta1 integrin in wound healing, wound repair was extensively evaluated in wild-type and alpha2-null mice in vivo. In addition, the impact of alpha2beta1 integrin-deficiency on the function of primary murine keratinocytes in vitro was analyzed. Our in vivo findings demonstrate that genetic deletion of the alpha2beta1 integrin does not significantly alter the rate of re-epithelialization, collagen deposition, or tensile strength during wound closure in mice. In marked contrast to the observed similarities in wound healing, deletion of the alpha2beta1 integrin resulted in a dramatic increase in neoangiogenesis in the wound microenvironment. In contrast to in vivo studies, primary keratinocytes from alpha2-null mice adhered poorly and displayed impaired migration on type I collagen in vitro. We demonstrate that alpha2beta1 integrin-ligation negatively regulates expression of genes including matrix metalloproteinases both in vivo and in vitro. Furthermore, the changes in gene expression could potentially account for relatively normal wound healing in the alpha2-deficient mouse and our recent observation that suggests an antiangiogenic role for the alpha2beta1 integrin in vivo.

  6. Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

    PubMed Central

    Utheim, Tor Paaske; Islam, Rakibul; Fostad, Ida G.; Eidet, Jon R.; Sehic, Amer; Olstad, Ole K.; Dartt, Darlene A.; Messelt, Edward B.; Griffith, May; Pasovic, Lara

    2016-01-01

    Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell

  7. Bathing in carbon dioxide-enriched water alters protein expression in keratinocytes of skin tissue in rats

    NASA Astrophysics Data System (ADS)

    Kälsch, Julia; Pott, Leona L.; Takeda, Atsushi; Kumamoto, Hideo; Möllmann, Dorothe; Canbay, Ali; Sitek, Barbara; Baba, Hideo A.

    2016-10-01

    Beneficial effects of balneotherapy using naturally occurring carbonated water (CO2 enriched) have been known since the Middle Ages. Although this therapy is clinically applied for peripheral artery disease and skin disorder, the underlying mechanisms are not fully elucidated. Under controlled conditions, rats were bathed in either CO2-enriched water (CO2 content 1200 mg/L) or tap water, both at 37 °C, for 10 min daily over 4 weeks. Proliferation activity was assessed by Ki67 immunohistochemistry of the epidermis of the abdomen. The capillary density was assessed by immunodetection of isolectin-positive cells. Using cryo-fixed abdominal skin epidermis, follicle cells and stroma tissue containing capillaries were separately isolated by means of laser microdissection and subjected to proteomic analysis using label-free technique. Differentially expressed proteins were validated by immunohistochemistry. Proliferation activity of keratinocytes was not significantly different in the epidermis after bathing in CO2-enriched water, and also, capillary density did not change. Proteomic analysis revealed up to 36 significantly regulated proteins in the analyzed tissue. Based on the best expression profiles, ten proteins were selected for immunohistochemical validation. Only one protein, far upstream element binding protein 2 (FUBP2), was similarly downregulated in the epidermis after bathing in CO2-enriched water with both techniques. Low FUBP2 expression was associated with low c-Myc immune-expression in keratinocytes. Long-term bathing in CO2-enriched water showed a cellular protein response of epithelial cells in the epidermis which was detectable by two different methods. However, differences in proliferation activity or capillary density were not detected in the normal skin.

  8. 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.

  9. Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

    PubMed

    Ivanova, Petya; Atanasova, Ganka; Poumay, Yves; Mitev, Vanyo

    2008-03-01

    Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

  10. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  11. Melanocytes expressing MC1R polymorphisms associated with red hair color have altered MSH-ligand activated pigmentary responses in coculture with keratinocytes.

    PubMed

    Roberts, Donald W; Newton, Richard A; Leonard, J Helen; Sturm, Richard A

    2008-05-01

    The occurrence of red hair and pale skin in individuals, which is associated with UV-radiation sensitivity and increased skin cancer risk, is mainly due to polymorphisms in the melanocortin-1 receptor (MC1R) expressed in melanocytes. We have established a serum free human melanocyte-keratinocyte coculture system to study the behavior and functional abilities of melanocytes expressing MC1R red hair color (RHC) variants in order to identify differences from their wild type (WT) counterparts. This model revealed the importance of elevated calcium levels in promoting strong melanocyte interaction with the surrounding keratinocytes and resulted in a dendritic melanocyte morphology similar to that in skin. However, the dendricity response following agonist activation of the MC1R receptor by NDP-MSH peptide, was markedly enhanced in WT melanocytes in comparison to RHC strains. Analysis of mRNA expression and protein levels of the major pigmentation markers following NDP-MSH treatment distinguished the enzyme dopachrome tautomerase as preferentially upregulated in cocultures of WT strains, with negligible or a much reduced response in melanocytes with RHC variant alleles. These results highlight the use of the coculture system in determining fundamental differences in the physiology of melanocytes expressing RHC MC1R receptors and those of WT genotype, which are likely to contribute to the increased skin cancer risk for individuals that carry these variants.

  12. 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.

  13. Matrix metalloproteinase-19 expression in keratinocytes is repressed by transcription factors Tst-1 and Skn-1a: implications for keratinocyte differentiation.

    PubMed

    Beck, Inken M; Müller, Matthias; Mentlein, Rolf; Sadowski, Thorsten; Mueller, Markus S; Paus, Ralf; Sedlacek, Radislav

    2007-05-01

    Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.

  14. Erk5 Controls Slug Expression and Keratinocyte Activation during Wound Healing

    PubMed Central

    Arnoux, Valerie; Nassour, Mayssaa; L'Helgoualc'h, Annie; Hipskind, Robert A.

    2008-01-01

    Reepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell–cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF. PMID:18716062

  15. Keratinocyte growth factor improves alterations of lung permeability and bronchial epithelium in allergic rats.

    PubMed

    Tillie-Leblond, I; Gosset, P; Le Berre, R; Janin, A; Prangère, T; Tonnel, A B; Guery, B P H

    2007-07-01

    Chronic allergic asthma is associated with marked inflammatory reaction, microvascular leakage and epithelium injury. As previously shown in a rat model of chronic asthma, these alterations increase lung permeability and distal airway fluid clearance. Keratinocyte growth factor (KGF) has been shown to induce epithelial cell proliferation and to protect from acute lung injuries. Therefore, the current authors evaluated the potential role of KGF treatment on lung permeability and airway inflammation in rats with chronic asthma. KGF (1 mg x kg(-1)) was administered intravenously before the last ovalbumin (OVA) challenge in sensitised rats. Permeability was assessed by the leak of radiolabelled albumin from the alveolar and systemic compartments. Histopathological analysis was also performed. Treatment with KGF decreased the leak of both markers and decreased the level of extravascular lung water in sensitised rats challenged with OVA. KGF treatment also reduced the inflammatory cell number in bronchoalveolar lavage fluid but not in bronchial mucosa. KGF markedly limited the allergen-induced alterations in epithelium integrity and the expression of the intercellular junction proteins beta-catenin and zonula occludens protein-1. In conclusion, keratinocyte growth factor administration markedly limits lung permeability and airway inflammation, an effect associated with a decrease in epithelium alterations during chronic allergic asthma. These data open new prospects in the therapeutic strategy of asthma.

  16. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

    PubMed

    Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav

    2003-12-01

    Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

  17. UVB Radiation-Induced β-catenin Signaling is Enhanced by COX-2 Expression in Keratinocytes

    PubMed Central

    Smith, Kimberly A.; Tong, Xin; Abu-Yousif, Adnan O.; Mikulec, Carol C.; Gottardi, Cara J.; Fischer, Susan M.; Pelling, Jill C.

    2014-01-01

    UVB radiation is the major carcinogen responsible for skin carcinogenesis, thus elucidation of the molecular pathways altered in skin in response to UVB would reveal novel targets for therapeutic intervention. It is well established that UVB leads to upregulation of cyclooxygenase 2 (COX-2) in the skin which contributes to skin carcinogenesis. Overexpression of COX-2 has been shown to promote colon cancer cell growth through β-catenin signaling, however, little is known about the connection between UVB, COX-2 and β-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces β-catenin signaling in keratinocytes, which is modulated by COX-2 expression. Exposure of the mouse 308 keratinocyte cell line (308 cells) and primary normal human epidermal keratinocytes (NHEKs) to UVB resulted in increased protein levels of both N-terminally unphosphorylated and total β-catenin. In addition, we found that UVB enhanced β-catenin-dependent TOPflash reporter activity and expression of a downstream β-catenin target gene. We demonstrated that UVB-induced β-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of β-catenin. Additionally, β-catenin target gene expression was reduced in UVB-treated COX-2 knockout (KO) MEFs compared to wild-type (WT) MEFs. Furthermore, epidermis from UVB-exposed SKH-1 mice exhibited increased N-terminally unphosphorylated and total β-catenin protein levels and increased staining for total β-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces β-catenin signaling in keratinocytes which is enhanced by COX-2 expression. PMID:21853475

  18. Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

    PubMed

    Lanzafame, Manuela; Botta, Elena; Teson, Massimo; Fortugno, Paola; Zambruno, Giovanna; Stefanini, Miria; Orioli, Donata

    2015-04-01

    Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

  19. A heat-sensitive TRP channel expressed in keratinocytes.

    PubMed

    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

    2002-06-14

    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.

  20. 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

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

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Laskin, Debra L. Heck, Diane E.; Laskin, Jeffrey D.

    2008-09-15

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

  2. Low dose chronic treatment of human keratinocytes with inorganic arsenic causes hyperproliferation and altered protein phosphorylation

    SciTech Connect

    Steinberg, M.L.; Su, L.; Snow, E.T. |

    1997-10-01

    Chronic exposure to arsenate [As(V)] or arsenite [As(III)] causes hyperproliferation of normal and SV40-transformed human epidermal keratinocytes. Line 327 SV40-infected human keratinocytes were grown in the presence of either As(III) or As(V) (0.01 to 10 {mu}M) in complete medium for seven days prior to harvesting and counting. Both As(III) and As(V) were cytotoxic at micromolar concentrations, however submicromolar arsenic caused a significant increase in cell growth. Cell numbers in cultures exposed to As(V) were increased more than 186% relative to controls, and an even larger stimulation in cell growth was observed after treatment with 50 nM As(III). Normal non-SV40 T-antigen. Preliminary cell cycle analysis using unselected, log-phase cultures of arsenic-treated keratinocytes shows an increased proportion of cells in S- and G2/M-phase. Isoelectric focusing of phosphotyrosine-containing proteins from cells labeled with {sup 32}P-inorganic phosphate showed that the hyperproliferation of keratinocytes grown in low concentrations of arsenic is accompanied by altered tyrosine-specific protein phosphorylation. A number of phosphorylated proteins were observed in As-treated cells that were not observed in the controls; and minor bands at IEPs of 3.0, 4.2, 7.2, 7.5 and 8.2. These results, together with the lack of direct enzyme inhibition by arsenic shown by Su et al., this volume, suggest that arsenic-induced skin lesions and carcinogenesis may be the result of altered cell cycle control rather than DNA damage or reduced DNA repair.

  3. Clinicopathological Features and Immunohistochemical Alterations of Keratinocyte Proliferation, Melanocyte Density, Smooth Muscle Hyperplasia and Nerve Fiber Distribution in Becker's Nevus

    PubMed Central

    Sheng, Ping; Cheng, Yun-Long; Cai, Chuan-Chuan; Guo, Wei-Jin; Zhou, Ying; Shi, Ge

    2016-01-01

    Background Although Becker's nevus (BN) is a relatively common disease, the systematic studies of clinicopathological and immunohistochemical results are poorly reported. Objective To investigate the clinicopathological features and immunohistochemical alterations of keratinocyte proliferation, melanocyte density, smooth muscle hyperplasia and nerve fiber distribution in BN. Methods Clinical and pathological data were collected in 60 newly-diagnosed BN cases. Immunohistochemical stain of Ki-67, Melan-A, keratin 15, smooth muscle actin and protein gene product 9.5 was performed in 21 cases. Results The median diagnostic and onset age was 17 and 12 years, respectively. Skin lesions usually appeared on the upper trunk and upper limbs. The pathological features included the rete ridge elongation and fusion and basal hyperpigmentation. Epidermal Ki-67, Melan-A and keratin 15 expression and dermal nerve fiber length were significantly higher in lesional and perilesional skin than in normal skin (p<0.05~0.01), while smooth muscle actin expression was upregulated only in skin lesion (p<0.05). Conclusion Although the clinical diagnosis of BN is often straightforward, histopathology is helpful to differentiate from other pigmentary disorders. The hyperproliferation of keratinocytes, melanocytes, arrector pili muscle and dermal nerve fibers could be involved in the pathogenesis of BN. PMID:27904268

  4. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

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

    SciTech Connect

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-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. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  6. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract

    PubMed Central

    Chittur, Sridar; Parr, Brian; Marcovici, Geno

    2011-01-01

    Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities. PMID:19692448

  7. Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract.

    PubMed

    Chittur, Sridar; Parr, Brian; Marcovici, Geno

    2011-01-01

    Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

  8. Expression and functional role of Sox9 in human epidermal keratinocytes.

    PubMed

    Shi, Ge; Sohn, Kyung-Cheol; Li, Zhengjun; Choi, Dae-Kyoung; Park, Young Min; Kim, Jin-Hwa; Fan, Yi-Ming; Nam, Yong Hee; Kim, Sooyeon; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok; Lee, Jeung-Hoon

    2013-01-01

    In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

  9. FOXO1 expression in keratinocytes promotes connective tissue healing

    PubMed Central

    Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

    2017-01-01

    Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

  10. Regulation of Srpr Expression by miR-330-5p Controls Proliferation of Mouse Epidermal Keratinocyte

    PubMed Central

    Kim, Bong-Kyu; Yoo, Hye-In; Choi, Keonwoo; Lee, Ah-Reum; Yoon, Sungjoo Kim

    2016-01-01

    Srpr is a gene encoding α subunit of the signal recognition particle receptor which is involved in the targeting and translocation of nascent secretory and membrane proteins to the endoplasmic reticulum. Previous studies showed aberrant expression of Srpr in several cell types with abnormal growth rate. Although Srpr is expressed in various tissues including skin, the role of Srpr in keratinocytes and regulation of its expression by miRNAs have not been studied. In this study, we investigated the role of SRPR and regulation of its expression by miRNA in skin keratinocytes. We found that SRPR was highly expressed in epidermal keratinocytes and regulated keratinocyte proliferation by affecting cell cycle progression. We also demonstrated that miR-330-5p directly inhibits Srpr expression. These data suggest that miR-330-5p-mediated regulation of the SRPR level is needed for the regulation of proliferation of epidermal keratinocytes. PMID:27768721

  11. Expression and biological significance of Ca2+-activated ion channels in human keratinocytes.

    PubMed

    Koegel, H; Alzheimer, C

    2001-01-01

    In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes.

  12. SILAC-based quantitative proteomic analysis reveals widespread molecular alterations in human skin keratinocytes upon chronic arsenic exposure.

    PubMed

    Mir, Sartaj Ahmad; Pinto, Sneha M; Paul, Somnath; Raja, Remya; Nanjappa, Vishalakshi; Syed, Nazia; Advani, Jayshree; Renuse, Santosh; Sahasrabuddhe, Nandini A; Prasad, T S Keshava; Giri, Ashok K; Gowda, Harsha; Chatterjee, Aditi

    2017-03-01

    Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin.

  13. Tacrolimus does not alter the production of several cytokines and antimicrobial peptide in Malassezia furfur-infected-keratinocytes.

    PubMed

    Balato, Anna; Paoletti, Iole; De Gregorio, Vincenza; Cantelli, Mariateresa; Ayala, Fabio; Donnarumma, Giovanna

    2014-03-01

    Topical immunosuppressant therapy is widely used in the treatment of inflammatory skin diseases, such as atopic dermatitis and psoriasis. Besides its beneficial therapeutic effects, application of topical anti-inflammatory drugs may render the epidermis more vulnerable to invading pathogens by suppressing innate immune responses in keratinocytes (KCs). Cytokines, chemokines and antimicrobial peptides (AMPs) produced by epithelial cells enable them to participate in innate and acquired immune responses. The aim of the present work was to study the influence of tacrolimus (FK506) on KCs infected with Malassezia furfur (M. furfur), evaluating the expression of pro-inflammatory cytokines IL-1α and IL-6, chemokine IL-8, anti-inflammatory cytokines transforming growth factor beta1 (TGF-β1) and IL-10 and AMP β-defensin-2. Human KCs were obtained from surgical specimens of normal adult skin. The expression of mRNAs in KCs: FK506-treated, FK506-treated and M. furfur-infected as well as only M. furfur-infected was quantified by real-time quantitative polymerase chain reaction. Next, the production of the AMP β-defensin-2 and of the above-mentioned pro-inflammatory and anti-inflammatory cytokines was evaluated using enzyme-linked immunosorbent assay. In this study, FK506 did not alter cytokine and AMP production by KCs; this led us to hypothesise that it may not enhance the risk of mycotic skin infections.

  14. Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

    PubMed

    Bisson, Francis; Paquet, Claudie; Bourget, Jean-Michel; Zaniolo, Karine; Rochette, Patrick J; Landreville, Solange; Damour, Odile; Boudreau, François; Auger, François A; Guérin, Sylvain L; Germain, Lucie

    2015-02-01

    The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

  15. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    SciTech Connect

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  16. Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes

    PubMed Central

    Oti, Martin; Kouwenhoven, Evelyn N.; Zhou, Huiqing

    2015-01-01

    The transcription factor p63 is a key regulator in epidermal keratinocyte proliferation and differentiation. However, the role of p63 in gene regulation during these processes is not well understood. To investigate this, we recently generated genome-wide profiles of gene expression, p63 binding sites and active regulatory regions with the H3K27ac histone mark (Kouwenhoven et al., 2015). We showed that only a subset of p63 binding sites are active in keratinocytes, and that differentiation-associated gene expression dynamics correlate with the activity of p63 binding sites rather than with their occurrence per se. Here we describe in detail the generation and processing of the ChIP-seq and RNA-seq datasets used in this study. These data sets are deposited in the Gene Expression Omnibus (GEO) repository under the accession number GSE59827. PMID:26484246

  17. Slug regulates integrin expression and cell proliferation in human epidermal keratinocytes.

    PubMed

    Turner, Frances E; Broad, Simon; Khanim, Farhat L; Jeanes, Alexa; Talma, Sonia; Hughes, Sharon; Tselepis, Chris; Hotchin, Neil A

    2006-07-28

    The human epidermis is a self-renewing epithelial tissue composed of several layers of keratinocytes. Within the epidermis there exists a complex array of cell adhesion structures, and many of the cellular events within the epidermis (differentiation, proliferation, and migration) require that these adhesion structures be remodeled. The link between cell adhesion, proliferation, and differentiation within the epidermis is well established, and in particular, there is strong evidence to link the process of terminal differentiation to integrin adhesion molecule expression and function. In this paper, we have analyzed the role of a transcriptional repressor called Slug in the regulation of adhesion molecule expression and function in epidermal keratinocytes. We report that activation of Slug, which is expressed predominantly in the basal layer of the epidermis, results in down-regulation of a number of cell adhesion molecules, including E-cadherin, and several integrins, including alpha3, beta1, and beta4. We demonstrate that Slug binds to the alpha3 promoter and that repression of alpha3 transcription by Slug is dependent on an E-box sequence within the promoter. This reduction in integrin expression is reflected in decreased cell adhesion to fibronectin and laminin-5. Despite the reduction in integrin expression and function, we do not observe any increase in differentiation. We do, however, find that activation of Slug results in a significant reduction in keratinocyte proliferation.

  18. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    SciTech Connect

    Shi, Ge; Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin; Ou, Bai-sheng; Kim, Sooil; Lee, Young Ho; Yoon, Tae-Jin; Kim, Seong-Jin; Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon; Kim, Chang Deok

    2010-11-15

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  19. Ectopic expression of syndecan-1 in basal epidermis affects keratinocyte proliferation and wound re-epithelialization.

    PubMed

    Ojeh, Nkemcho; Hiilesvuo, Katri; Wärri, Anni; Salmivirta, Markku; Henttinen, Tiina; Määttä, Arto

    2008-01-01

    Epidermal proliferation and differentiation can be regulated by soluble morphogens and growth factors. Heparan sulfate proteoglycans (HSPGs) modulate the action of several of these effector molecules, such as members of the fibroblast growth factor (FGF) and Wnt families. Syndecan-1 is a cell-surface proteoglycan that is expressed in differentiating keratinocytes and transiently upregulated in all layers of the epidermis upon tissue injury. To address the role of syndecan-1 in the regulation of keratinocyte proliferation and differentiation, we generated transgenic mice that overexpress syndecan-1 under K14 keratin promoter in the basal layer of the epidermis. We observed epidermal hyperproliferation in newborn transgenic mice, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers and by expression of keratin 6 in the interfollicular epidermis. Compared to both wild-type and syndecan-1-null animals, the transgene expression interfered with skin wound healing in adult mice by decreasing cell proliferation in the re-epithelialized epidermis. Thus, syndecan-1 regulates keratinocyte proliferation differently during skin development and in healing wounds.

  20. TLR2 Expression Is Increased in Rosacea and Stimulates Enhanced Serine Protease Production by Keratinocytes

    PubMed Central

    Yamasaki, Kenshi; Kanada, Kimberly; Macleod, Daniel T.; Borkowski, Andrew W.; Morizane, Shin; Nakatsuji, Teruaki; Cogen, Anna L.; Gallo, Richard L.

    2011-01-01

    A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea. PMID:21107351

  1. CXCL11 Expression by Keratinocytes Occurs Transiently Between Reaching Confluence and Cellular Compaction

    PubMed Central

    Huen, Arthur C.; Marathi, Archana; Nam, Peter K.; Wells, Alan

    2016-01-01

    Objective: To investigate whether differentiation or cellular confluence is responsible for CXCL11 expression patterns in re-epithelialization. Approach: In vitro model systems of re-epithelialization using the HaCaT keratinocyte cell line were utilized in monitoring expression of differentiation markers, including desmoplakin and various cytokeratins while evaluating for an association with chemokine CXCL11 expression. Results: CXCL11 expression was elevated in sparse culture with peak expression near the time of confluence. This somewhat followed the accumulation of desmoplakin in detergent-insoluble pool of proteins. However, in postconfluent, despite continued accumulation of desmoplakin within cells, CXCL11 expression decreased to baseline levels. This biphasic pattern was also seen in low calcium culture, an environment that inhibits keratinocyte differentiation and accumulation of desmosomal proteins. Highest CXCL11-expressing areas best correlated with newly confluent areas within culture expressing basal keratin 14, but also activated keratin 6. Innovation: Achievement of a threshold cellular density induces cell signaling cascade through CXCR3 that, in addition to other undiscovered pathways, can progress cutaneous wounds from the proliferative into the remodeling phases of cutaneous wound healing. Conclusion: These results suggest that the achievement of confluence with increased cellular density by migrating keratinocytes at the wound edge triggers expression of CXCL11. Since CXCR3 stimulation in endothelial cells results in apoptosis and causes neovascular pruning, whereas stimulation of CXCR3 in fibroblasts results decreased motility and cellular contraction, we speculate that CXCL11 expression by epidermal cells upon achieving cellular confluence could be the source of CXCR3 stimulation in the dermis ushering a transition from proliferative to remodeling phases of wound healing. PMID:28078185

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

    PubMed

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

    2012-11-01

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

  3. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    PubMed

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  4. The cytotoxicity of mercury chloride to the keratinocytes is associated with metallothionein expression.

    PubMed

    Hwang, Tsann-Long; Chen, Hsiao-Ying; Changchien, Tzu-Tsung; Wang, Chee-Chan; Wu, Chi-Ming

    2013-05-01

    There are trace amounts of heavy metals in cosmetics. Heavy metals such as mercury (Hg), which is added to skin-whitening cosmetics, may cause acute or chronic damage to human cells. The aim of this study was to investigate the cytotoxicity of mercury chloride (HgCl2) to human keratinocytes. The keratinocytes were treated with various concentrations of HgCl2 and the cell survival fractions were found to be 38.08, 17.59, 12.76, 3.29 and 0.77% when the cells were treated with 0.25, 0.5, 0.75, 1 and 1.5 μM of HgCl2, respectively. Moreover, we observed that the greatest damage was to the cell membrane. The metallothionein (MT) protein expression was also investigated. MT expression levels increased with increasing concentrations of HgCl2. The results indicated that MT protects the keratinocytes against HgCl2-induced toxicity.

  5. 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

  6. XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas.

    PubMed

    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

    2011-01-01

    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.

  7. The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling.

    PubMed

    Reinheckel, Thomas; Hagemann, Sascha; Dollwet-Mack, Susanne; Martinez, Elke; Lohmüller, Tobias; Zlatkovic, Gordana; Tobin, Desmond J; Maas-Szabowski, Nicole; Peters, Christoph

    2005-08-01

    Mice deficient for cathepsin L (CTSL) show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes. Here we show that the critical function of CTSL in the skin is keratinocyte specific. This is revealed by transgenic re-expression of CTSL in the keratinocytes of ctsl-/- mice, resulting in a rescue of the ctsl-/- skin phenotype. Cultivation of primary mouse keratinocytes with fibroblast- and keratinocyte-conditioned media, as well as heterologous organotypic co-cultures of mouse fibroblasts and human keratinocytes, showed that the altered keratinocyte proliferation is caused primarily by CTSL-deficiency in keratinocytes. In the absence of EGF, wild type and CTSL-knockout keratinocytes proliferate with the same rates, while in presence of EGF, ctsl-/- keratinocytes showed enhanced proliferation compared with controls. Internalization and degradation of radioactively labeled EGF was identical in both ctsl-/- and ctsl+/+ keratinocytes. However, ctsl-/- keratinocytes recycled more EGF to the cell surface, where it is bound to the EGF-receptor, which is also more abundant in ctsl-/- cells. We conclude that the hyperproliferation of keratinocytes in CTSL-knockout mice is caused by an enhanced recycling of growth factors and growth factor receptors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation.

  8. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    PubMed

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  9. Gene expression profiling to identify markers associated with deregulated hTERT in HPV-transformed keratinocytes and cervical cancer.

    PubMed

    de Wilde, Jillian; Wilting, Saskia M; Meijer, Chris J L M; van de Wiel, Mark A; Ylstra, Bauke; Snijders, Peter J F; Steenbergen, Renske D M

    2008-02-15

    Although high-risk human papillomavirus (HPV) infection plays a major role in the development of cervical cancer, additive oncogenic events are involved as well. One key event involves increased activity of telomerase resulting from a deregulated expression of its catalytic subunit hTERT. Our previous microcell-mediated chromosome transfer studies revealed that introduction of human chromosome 6 in the HPV16-immortalized keratinocyte cell line FK16A and in the HPV16-containing cervical cancer cell line SiHa induced growth arrest, resulting from a repression of hTERT mRNA expression and telomerase activity. Here, this model was used to analyze expression profiles associated with hTERT deregulation in HPV-transformed cells. Microarray expression analysis of 12 FK16A/chromosome 6 hybrids, 4 of which were negative for endogenous hTERT and 8 of which were positive for endogenous hTERT, resulted in the identification of 164 differentially expressed genes. Differential expression of a selection of 5 genes was verified by real-time RT-PCR. Of these 164 genes, 32 were also differentially expressed in other HPV transformed cells with deregulated hTERT. For 2 of these genes, encoding AQP3 and MGP, altered expression in hTERT positive cervical carcinomas was confirmed by real-time RT-PCR and immunohistochemistry, respectively. Moreover, increased MGP protein expression was significantly more frequent in high-grade cervical premalignant lesions with elevated hTERT mRNA expression compared to those without. In summary, we identified 32 candidate biomarkers for deregulated hTERT mRNA expression, which may enable the identification of cervical premalignant lesions that are at highest risk to progress to invasive cancer.

  10. XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.

    PubMed

    Rezvani, Hamid Reza; Rossignol, Rodrigue; Ali, Nsrein; Benard, Giovanni; Tang, Xiuwei; Yang, Hee Seung; Jouary, Thomas; de Verneuil, Hubert; Taïeb, Alain; Kim, Arianna L; Mazurier, Frédéric

    2011-06-01

    Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.

  11. XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species

    PubMed Central

    Rezvani, Hamid Reza; Rossignol, Rodrigue; Ali, Nsrein; Benard, Giovanni; Tang, Xiuwei; Yang, Hee Seung; Jouary, Thomas; de Verneuil, Hubert; Taïeb, Alain; Kim, Arianna L.; Mazurier, Frédéric

    2011-01-01

    Summary Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the interrelationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPCKD) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers. PMID:21167810

  12. Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    PubMed

    Secor, Patrick R; Jennings, Laura K; James, Garth A; Kirker, Kelly R; Pulcini, Elinor Delancey; McInnerney, Kate; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K; Bothner, Brian; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

  13. 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.

  14. Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures.

    PubMed

    Harmon, C S; Ducote, J; Xiong, Y

    1996-08-01

    Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

  15. Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

    PubMed Central

    Komoto, Tatiana Takahasi; Bitencourt, Tamires Aparecida; Silva, Gabriel; Beleboni, Rene Oliveira; Marins, Mozart; Fachin, Ana Lúcia

    2015-01-01

    Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of T. rubrum cocultured with keratinocytes and treated with the flavonoid trans-chalcone and the glycoalkaloid α-solanine. Both substances showed a marked antifungal activity against T. rubrum strain CBS (MIC = 1.15 and 17.8 µg/mL, resp.). Cytotoxicity assay against HaCaT cells produced IC50 values of 44.18 to trans-chalcone and 61.60 µM to α-solanine. The interaction of keratinocytes with T. rubrum conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter TruMDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and TruMDR2 genes. Furthermore, the trans-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets. PMID:26257814

  16. Aberrant Cytokeratin Expression During Arsenic-induced Acquired Malignant Phenotype in Human HaCaT Keratinocytes Consistent with Epidermal Carcinogenesis

    PubMed Central

    Sun, Yang; Pi, Jingbo; Wang, Xueqian; Tokar, Erik J.; Liu, Jie; Waalkes, Michael P.

    2009-01-01

    Inorganic arsenic is a known human skin carcinogen. Chronic arsenic exposure results in various human skin lesions, including hyperkeratosis and squamous cell carcinoma (SCC), both characterized by distorted cytokeratin (CK) production. Prior work shows the human skin keratinocyte HaCaT cell line, when exposed chronically for >25 weeks to a low level of inorganic arsenite (100 nM) results in cells able to produce aggressive SCC upon inoculation into nude mice. In the present study, CK expression analysis was performed in arsenic-exposed HaCaT cells during the progressive acquisition of this malignant phenotype (0 to 20 weeks) to further validate this model as relevant to epidermal carcinogenesis induced by arsenic in humans. Indeed, we observed clear evidence of acquired cancer phenotype by 20 weeks of arsenite exposure including the formation of giant cells, a >4-fold increase in colony formation in soft agar and a ∼2.5-fold increase in matrix metalloproteinase-9 secretion, an enzyme often secreted by cancer cells to help invade through the local extra-cellular matrix. During this acquired malignant phenotype, various CK genes showed markedly altered expression at the transcript and protein levels in a time-dependent manner. For example, CK1, a marker of hyperkeratosis, increased up to 34-fold during arsenic-induced transformation, while CK13, a marker for dermal cancer progression, increased up to 45-fold. The stem cell marker, CK15, increased up to 7-fold, particularly during the later stages of arsenic exposure, indicating a potential emergence of cancer stem-like cells with arsenic-induced acquired malignant phenotype. The expression of involucrin and loricrin, markers for keratinocyte differentiation, increased up to 9-fold. Thus, during arsenic-induced acquired cancer phenotype in human keratinocytes, dramatic and dynamic alterations in CK expression occur which are consistent with the process of epidermal carcinogenesis helping validate this as an

  17. Aberrant cytokeratin expression during arsenic-induced acquired malignant phenotype in human HaCaT keratinocytes consistent with epidermal carcinogenesis.

    PubMed

    Sun, Yang; Pi, Jingbo; Wang, Xueqian; Tokar, Erik J; Liu, Jie; Waalkes, Michael P

    2009-08-03

    Inorganic arsenic is a known human skin carcinogen. Chronic arsenic exposure results in various human skin lesions, including hyperkeratosis and squamous cell carcinoma (SCC), both characterized by distorted cytokeratin (CK) production. Prior work shows the human skin keratinocyte HaCaT cell line, when exposed chronically for >25 weeks to a low level of inorganic arsenite (100nM) results in cells able to produce aggressive SCC upon inoculation into nude mice. In the present study, CK expression analysis was performed in arsenic-exposed HaCaT cells during the progressive acquisition of this malignant phenotype (0-20 weeks) to further validate this model as relevant to epidermal carcinogenesis induced by arsenic in humans. Indeed, we observed clear evidence of acquired cancer phenotype by 20 weeks of arsenite exposure including the formation of giant cells, a >4-fold increase in colony formation in soft agar and a approximately 2.5-fold increase in matrix metalloproteinase-9 secretion, an enzyme often secreted by cancer cells to help invade through the local extra-cellular matrix. During this acquired malignant phenotype, various CK genes showed markedly altered expression at the transcript and protein levels in a time-dependent manner. For example, CK1, a marker of hyperkeratosis, increased up to 34-fold during arsenic-induced transformation, while CK13, a marker for dermal cancer progression, increased up to 45-fold. The stem cell marker, CK15, increased up to 7-fold, particularly during the later stages of arsenic exposure, indicating a potential emergence of cancer stem-like cells with arsenic-induced acquired malignant phenotype. The expression of involucrin and loricrin, markers for keratinocyte differentiation, increased up to 9-fold. Thus, during arsenic-induced acquired cancer phenotype in human keratinocytes, dramatic and dynamic alterations in CK expression occur which are consistent with the process of epidermal carcinogenesis helping validate this as an

  18. P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling.

    PubMed

    Bauer, Karin; Gosau, Martin; Bosserhoff, Anja; Reichert, Torsten; Bauer, Richard

    2012-12-01

    P-cadherin belongs to the family of Ca(2+)-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.

  19. Glutathione peroxidase-1 inhibits UVA-induced AP-2{alpha} expression in human keratinocytes

    SciTech Connect

    Yu Lei; Venkataraman, Sujatha; Coleman, Mitchell C.; Spitz, Douglas R.; Wertz, Philip W.; Domann, Frederick E. . E-mail: frederick-domann@uiowa.edu

    2006-12-29

    In this study, we found a role for H{sub 2}O{sub 2} in UVA-induced AP-2{alpha} expression in the HaCaT human keratinocyte cell line. UVA irradiation not only increased AP-2{alpha}, but also caused accumulation of H{sub 2}O{sub 2} in the cell culture media, and H{sub 2}O{sub 2} by itself could induce the expression of AP-2{alpha}. By catalyzing the removal of H{sub 2}O{sub 2} from cells through over-expression of GPx-1, induction of AP-2{alpha} expression by UVA was abolished. Induction of transcription factor AP-2{alpha} by UVA had been previously shown to be mediated through the second messenger ceramide. We found that not only UVA irradiation, but also H{sub 2}O{sub 2} by itself caused increases of ceramide in HaCaT cells, and C2-ceramide added to cells induced the AP-2{alpha} signaling pathway. Finally, forced expression of GPx-1 eliminated UVA-induced ceramide accumulation as well as AP-2{alpha} expression. Taken together, these findings suggest that GPx-1 inhibits UVA-induced AP-2{alpha} expression by suppressing the accumulation of H{sub 2}O{sub 2}.

  20. Meaning of relative gene expression in multilayered cultures of epidermal keratinocytes.

    PubMed

    Malaisse, Jérémy; Hermant, Maryse; Hayez, Aurélie; Poumay, Yves; Lambert de Rouvroit, Catherine

    2014-10-01

    Reconstructed human epidermis (RHE) has become an in vitro model of choice for studying cell and tissue functions. Analysis of gene expression over the course of reconstruction must take into account the heterogeneous differentiation states of keratinocytes reconstituting the typical epidermal layers. In monolayer cultures, relative mRNA expression levels of differentiation markers are usually expressed as a ratio versus a classical reference gene (also named house-keeping gene) tested to be expressed equally in certain experimental conditions. Applied to complex tissues in which the cell number increases over time together with differentiation, calculation of relative gene expression does not take enough into account a crucial phenomenon: epidermal morphogenesis results in progressive restriction of differentiation markers, such as involucrin, to a specific layer, or in the delayed onset of mRNA expression of filaggrin or TMEM45A for instance following stratification. Our study illustrates that comparing the relative expression level of mRNAs to that of a basal layer-specific gene (e.g. ITGA6) better illustrates the contribution of specific differentiation markers to the process of epidermal morphogenesis.

  1. Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

    PubMed

    Budunova, I V; Carbajal, S; Kang, H; Viaje, A; Slaga, T J

    1997-03-01

    Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

  2. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  3. MiR-26a inhibits proliferation and migration of HaCaT keratinocytes through regulating PTEN expression.

    PubMed

    Yu, Nanze; Yang, Yang; Li, Xiongwei; Zhang, Mingzi; Huang, Jiuzuo; Wang, Xiaojun; Long, Xiao

    2016-12-05

    MicroRNAs (miRNAs) have been shown to be associated with differentiation, migration and apoptosis in keratinocyte. Although it has been reported that microRNA-26a (miR-26a) plays important roles in tumor cells, its biological functions in keratinocytes are still not well elucidated. In this study, we confirmed expression of miR-26a in human keratinocytes using RT-PCR and further studied the role of miR-26a in cell proliferation and cell migration. Ectopic expression of MiR-26a mimic or inhibitor increased or decreased miR-26a expression respectively in HaCaT cells. Proliferation of HaCaT keratinocyte can be suppressed or promoted by overexpression or down-expression of miR-26a. In scratch wound-healing assay and Boyden chamber cell migration assay, upregulating miR-26a expression blocked cell migration, while downregulating miR-26a expression enhanced the migration. Using quantitative RT-PCR (qRT-PCR) and western blot, we further discovered that both mRNA and protein level of phosphatase and tensin homolog deleted from chromosome 10(PTEN) were regulated by miR-26a in HaCaT cells. Meanwhile the level of active form of AKT was also regulated by the miR-26a. In rescue experiment, knockdown of PTEN in the miR-26a mimic transduced cells recovered the migration ability of HaCaT cells. Together these results suggest that miR-26a modulates the proliferation and migration of keratinocytes via regulating PTEN/AKT signaling pathway.

  4. Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.

    PubMed

    Moutasim, Karwan A; Jenei, Veronika; Sapienza, Karen; Marsh, Daniel; Weinreb, Paul H; Violette, Shelia M; Lewis, Mark P; Marshall, John F; Fortune, Farida; Tilakaratne, Waninayaka M; Hart, Ian R; Thomas, Gareth J

    2011-02-01

    Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvβ6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvβ6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvβ6 using oral keratinocyte-derived cells genetically modified to express high αvβ6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvβ6 (OKF6/TERT-1). VB6 cells showed significant αvβ6-dependent activation of TGF-β1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-β1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvβ6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvβ6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvβ6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvβ6 integrin.

  5. Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

    PubMed

    Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

    2015-10-01

    Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

  6. Keratinocyte growth factor induces matrix metalloproteinase-9 expression and correlates with venous invasion in pancreatic cancer

    PubMed Central

    CHO, KAZUMITSU; MATSUDA, YOKO; UEDA, JUNJI; UCHIDA, EIJI; NAITO, ZENYA; ISHIWATA, TOSHIYUKI

    2012-01-01

    Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, and KGF receptor (KGFR) play important roles in the growth of epithelial cells and are overexpressed in a variety of malignant epithelial tumors, including pancreatic ductal adenocarcinoma (PDAC). We previously reported that co-expression of KGF and KGFR in PDAC is associated with venous invasion, enhanced vascular endothelial growth factor A expression and poor prognosis. Matrix metalloproteinase-9 (MMP-9) is known to participate in the degradation of type IV collagen, which is a primary component of extracellular matrices in the vascular basement membrane. In the present study, we examined the expression and roles of KGF, KGFR and MMP-9 in human PDAC cell lines and tissues. Quantitative real-time polymerase chain reaction analysis demonstrated the expression of MMP-9 mRNA in all eight PDAC cell lines. KGF, KGFR and MMP-9 were, respectively, expressed in 27 (43%), 23 (37%) and 35 (56%) of 63 patients. Each expression of KGF, KGFR or MMP-9 correlated positively with venous invasion. Furthermore, expression of KGF or MMP-9 correlated positively with liver metastasis. KGF-positive cases exhibited shorter survival than KGF-negative cases, while KGFR and MMP-9 expression were unrelated to prognosis. Administration of recombinant human KGF increased MMP-9 expression in PDAC cells, while transient transfection with short hairpin RNAs targeting KGF transcripts reduced MMP-9 expression in PDAC cells. Moreover, recombinant human KGF significantly enhanced migration and invasion of PDAC cells. These findings suggest that KGF and KGFR promote venous invasion via MMP-9 in PDAC, and closely correlate with liver metastasis. The KGF/KGFR pathway may be a critical therapeutic target for PDAC metastasis. PMID:22159401

  7. HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

    PubMed

    Kömüves, László G; Michael, Elias; Arbeit, Jeffrey M; Ma, Xiao-Kui; Kwong, Angela; Stelnicki, Eric; Rozenfeld, Sophia; Morimune, M; Yu, Qian-Chun; Largman, Corey

    2002-05-01

    The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human

  8. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  9. UVB-dependent changes in the expression of fast-responding early genes is modulated by huCOP1 in keratinocytes.

    PubMed

    Fazekas, B; Polyánka, H; Bebes, A; Tax, G; Szabó, K; Farkas, K; Kinyó, A; Nagy, F; Kemény, L; Széll, M; Ádám, É

    2014-11-01

    Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human ortholog of the Arabidopsis thaliana constitutive photomorphogenesis 1 (COP1) protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a posttranslational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. In this study, we report that stable siRNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation, indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 of the 30 examined UVB-regulated genes were organized around three central proteins. Since the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize that huCOP1 is a repressor of the identified pathway. Several members of the network have been implicated previously in the pathogenesis of non-melanoma skin cancers; therefore, clarifying the role of huCOP1 in these skin diseases may have clinical relevance in the future.

  10. Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

    SciTech Connect

    Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A. )

    1990-08-01

    Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.

  11. 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.

  12. Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture.

    PubMed

    Rennecke, J; Rehberger, P A; Fürstenberger, G; Johannes, F J; Stöhr, M; Marks, F; Richter, K H

    1999-01-05

    In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu. These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation. A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

  13. Signal transduction and nuclear responses in Staphylococcus aureus-induced expression of human beta-defensin 3 in skin keratinocytes.

    PubMed

    Menzies, Barbara E; Kenoyer, Aimee

    2006-12-01

    The human beta-defensin 3 (hBD-3) is an inducible epithelial peptide antibiotic that has potent antistaphylococcal activity. Infection of skin epithelial cells with viable Staphylococcus aureus, a common skin pathogen, induces increased gene expression of hBD-3 and other antimicrobial peptides. The aim of this study was to identify signaling pathways and nuclear responses that contribute to the gene expression of hBD-3 in primary human keratinocytes upon contact with S. aureus. Increased hBD-3 peptide was observed by immunofluorescence microscopy in keratinocytes exposed to S. aureus and to lipoteichoic acid (LTA). Both are ligands for the cell surface Toll-like receptor 2 (TLR2), and thus the contribution of TLR2 signaling in hBD-3 expression was examined. Functional inhibition of TLR2 prior to S. aureus stimulation significantly decreased hBD-3 mRNA levels by 37%, attesting to the involvement of this surface receptor in the initial recognition and downstream signaling for hBD-3 expression. Treatment of keratinocytes with a p38 mitogen-activated protein kinase (MAPK) inhibitor prior to either S. aureus or LTA stimulation was associated with reduced hBD-3 mRNA transcripts and peptide. We also propose a role for the MAPK-regulated transcriptional activating protein 1 in S. aureus-induced hBD-3 gene expression. Combined, these studies indicate a role for TLR2 signaling and MAPK activation in the upregulation of hBD-3 and demonstrate the innate immune capacity of skin keratinocytes under conditions of S. aureus challenge to enhance the local expression of this antistaphylococcal peptide antibiotic.

  14. The widely expressed extracellular matrix protein SMOC-2 promotes keratinocyte attachment and migration

    SciTech Connect

    Maier, Silke; Paulsson, Mats; Hartmann, Ursula

    2008-08-01

    SMOC-2 is a recently discovered member of the BM-40/SPARC/osteonectin family of extracellular multidomain proteins of so far unknown function. While we have shown earlier that the homologous protein SMOC-1 is associated with basement membranes, in this study we demonstrate that, in the mouse, SMOC-2 could be detected in a large number of non-basement membrane localizations, often showing a diffuse tissue distribution. A more distinct expression pattern was seen in skin where SMOC-2 is mainly present in the basal layers of the epidermis. Functionally, recombinant SMOC-2 stimulated attachment of primary epidermal cells as well as several epidermal-derived cell lines but had no effect on the attachment of non-epidermal cells. Inhibition experiments using blocking antibodies against individual integrin subunits allowed the identification of {alpha}v{beta}6 and {alpha}v{beta}1 integrins as important cellular receptors for SMOC-2. Cell attachment as well as the formation of focal adhesions could be attributed to the extracellular calcium-binding domain. The calcium-binding domain also stimulated migration, but not proliferation of keratinocyte-like HaCaT cells. We conclude that SMOC-2, like other members of the BM40/SPARC family, acts as a regulator of cell-matrix interactions.

  15. Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

    PubMed Central

    Moffett, John; Fray, Linley M; Kubat, Nicole J

    2012-01-01

    Background Pulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways. Methods and Results Using cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types. Conclusion These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting. PMID:23055776

  16. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed Central

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-01-01

    Arsenic is a carcinogen that poses a significant health risk in humans. Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis. We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes. To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate [nad(p)h] quinone oxidoreductase gene expression were measured. The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-[S,R]-sulfoximine (BSO) to culture media. Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes. Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO. These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses. They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process. PMID:12426128

  17. ΔNp63α regulates keratinocyte proliferation by controlling PTEN expression and localization.

    PubMed

    Leonard, M K; Kommagani, R; Payal, V; Mayo, L D; Shamma, H N; Kadakia, M P

    2011-12-01

    ΔNp63α, implicated as an oncogene, is upregulated by activated Akt, part of a well-known cell survival pathway. Inhibition of Akt activation by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the presence of putative p63-binding sites in the pten promoter led us to investigate whether ΔNp63α regulates PTEN expression. Knockdown of ΔNp63α led to increases in PTEN levels and loss of activated Akt, while overexpression of ΔNp63α decreased PTEN levels and elevated active Akt. The repression of PTEN by ΔNp63α occurs independently of p53 status, as loss of ΔNp63α increases PTEN expression in cell lines with and without functional p53. In addition, decreased levels of ΔNp63α resulted in an increase in nuclear PTEN. Conversely, in vivo nuclear PTEN was absent in the proliferative basal layer of the epidermis where ΔNp63α expression is highest. Additionally, we show that in keratinocytes a balance between ΔNp63α and PTEN regulates Akt activation and maintains normal proliferation rates. This balance is disrupted in non-melanoma skin cancers through increased ΔNp63α levels, and could enhance proliferation and subsequent neoplastic development. Our studies show that ΔNp63α negatively regulates PTEN, thereby providing a feedback loop between PTEN, Akt and ΔNp63α, which has an integral role in skin cancer development.

  18. MicroRNA 203 expression in keratinocytes is dependent on regulation of p53 levels by E6.

    PubMed

    McKenna, Declan J; McDade, Simon S; Patel, Daksha; McCance, Dennis J

    2010-10-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

  19. Two Mechanisms Regulate Keratin K15 Expression In Keratinocytes: Role of PKC/AP-1 and FOXM1 Mediated Signalling

    PubMed Central

    Bose, Amrita; Teh, Muy-Teck; Hutchison, Iain L.; Wan, Hong; Leigh, Irene M.; Waseem, Ahmad

    2012-01-01

    Background Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes. Methodology/Principal Findings Human keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter. Conclusions/Significance The data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving

  20. Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

    PubMed Central

    Bollag, W B; Xiong, Y; Ducote, J; Harmon, C S

    1994-01-01

    The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear. Images Figure 1 Figure 2 PMID:8198544

  1. Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

    PubMed

    Bollag, W B; Xiong, Y; Ducote, J; Harmon, C S

    1994-05-15

    The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear.

  2. Loss of flotillin expression results in weakened desmosomal adhesion and Pemphigus vulgaris-like localisation of desmoglein-3 in human keratinocytes

    PubMed Central

    Völlner, Frauke; Ali, Jawahir; Kurrle, Nina; Exner, Yvonne; Eming, Rüdiger; Hertl, Michael; Banning, Antje; Tikkanen, Ritva

    2016-01-01

    Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion. PMID:27346727

  3. Loss of flotillin expression results in weakened desmosomal adhesion and Pemphigus vulgaris-like localisation of desmoglein-3 in human keratinocytes.

    PubMed

    Völlner, Frauke; Ali, Jawahir; Kurrle, Nina; Exner, Yvonne; Eming, Rüdiger; Hertl, Michael; Banning, Antje; Tikkanen, Ritva

    2016-06-27

    Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion.

  4. Notch1 Signaling Contributes to Hypoxia-induced High Expression of Integrin β1 in Keratinocyte Migration

    PubMed Central

    Tang, Di; Yan, Tiantian; Zhang, Junhui; Jiang, Xupin; Zhang, Dongxia; Huang, Yuesheng

    2017-01-01

    Oxygen tension is an important micro-environmental factor that affects epidermal development and function. After injury, high oxygen consumption and vascular injury result in partial hypoxia. However, whether hypoxia benefits or hurts wound healing remains controversial. In this study, a tissue oxygen tension monitor was used to detect the spatial and temporal distribution of oxygen in burn wounds. In vitro, we demonstrate that hypoxia promoted the expression of integrin β1 and the migration of keratinocytes. Furthermore, hypoxia-induced migration was slowed by Notch1 ligands and a siRNA against ITGB1 (integrin β1). Our findings suggest that integrin β1 may be an oxygen-sensitive molecule that promotes keratinocyte migration during wound healing and that Notch1 signaling is involved in this process. PMID:28266574

  5. Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process.

    PubMed

    Su, Ching-Hua; Liu, Shu-Hui; Yu, Shi-Yau; Hsieh, Yi-Ling; Ho, Hsiu-O; Hu, Chung-Hong; Sheu, Ming-Thau

    2005-02-01

    SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by

  6. Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression.

    PubMed Central

    Frank, S; Kämpfer, H; Wetzler, C; Stallmeyer, B; Pfeilschifter, J

    2000-01-01

    We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1beta- and tumour-necrosis-factor-alpha-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-N(G)-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair. PMID:10727427

  7. Members of the src and ras oncogene families supplant the epidermal growth factor requirement of BALB/MK-2 keratinocytes and induce distinct alterations in their terminal differentiation program.

    PubMed Central

    Weissman, B; Aaronson, S A

    1985-01-01

    BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies. Images PMID:2427928

  8. 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

  9. Expression, subcellular localization and cytokinic modulation of Toll-like receptors (TLRs) in normal human keratinocytes: TLR2 up-regulation in psoriatic skin.

    PubMed

    Begon, Edouard; Michel, Laurence; Flageul, Béatrice; Beaudoin, Isabelle; Jean-Louis, Francette; Bachelez, Hervé; Dubertret, Louis; Musette, Philippe

    2007-01-01

    The aim of the research described here was to investigate the expression of Toll-like receptors (TLRs) in normal human keratinocytes, to study its modulation by proinflammatory cytokines, and to characterize the function of the latter within the epidermis. Our results demonstrate that normal human keratinocytes may present an intra-cytoplasmic expression of TLR2, TLR3, and TLR4. Exposure of keratinocytes to IFN-gamma and TNF-alpha increased intra-cytoplasmic expression and led to partial translocation at the cell surface. Keratinocyte activation by TLR2, TLR3, and TLR4 ligands led to the nuclear translocation of NF-kappab and the release of proinflammatory cytokines TNF-alpha and IL-8. In immunochemistry analysis, psoriatic skin showed a strong over-expression of TLR2 in the epidermis compared with normal skin. Our results thus demonstrate large TLR expression in keratinocytes and the functionality of TLRs 2, 3, and 4. TLR2 over-expression in psoriatic skin provides new insights into TLR implication in the pathogenesis of psoriasis, through inappropriate stimulation by infectious or endogen ligands.

  10. Expression of HLA-DR (Ia like) antigen on epidermal keratinocytes in human dermatoses.

    PubMed Central

    Lampert, I A

    1984-01-01

    Ia antigen (HLA-DR in man) has been demonstrated in keratinocytes in graft versus host disease. This study investigates the occurrence of HLA-DR in keratinocytes in the following dermatoses: eczematous dermatitis, discoid lupus erythematosus, with immunoglobulin and non-exposed skin from cases of systemic lupus erythematosus with immunoglobulin deposits, lichen planus, lichen simplex, bullous pemphigoid, pemphigus vulgaris, 'toxic erthema', tuberculid and chillblain. Keratinocyte staining was found in a variety of conditions. The unifying features of the instances of its occurrence was lymphoid infiltration and usually some focal evidence of keratinocyte damage. Thus in eczema the staining was mid-epidermal, while in discoid lupus erythematosus and lichen planus it was basal. HLA-DR staining was absent in bullous pemphigoid and pemphigus vulgaris, which is consistent with the hypothesis that in these conditions the damage is mediated by autoantibodies and complement in the absence of cellular immune attack. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:6204802

  11. The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

    PubMed

    Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

    2017-02-01

    Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

  12. Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

    PubMed

    Lace, Michael J; Anson, James R; Klussmann, Jens P; Wang, Dong Hong; Smith, Elaine M; Haugen, Thomas H; Turek, Lubomir P

    2011-02-01

    Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

  13. The Effect of Calcipotriol on the Expression of Human β Defensin-2 and LL-37 in Cultured Human Keratinocytes

    PubMed Central

    Kim, Beom Joon; Rho, Yong Kwan; Lee, Hye In; Jeong, Mi Sook; Li, Kapsok; Seo, Seong Jun; Kim, Myeung Nam; Hong, Chang Kwun

    2009-01-01

    Background. Vitamin D has been reported to regulate innate immunity by controlling the expression of antimicrobial peptides (AMPs). Objective. We investigated the effect of calcipotriol on the expression of AMPs in human cultured keratinocytes. Methods. Keratinocytes were treated with lipopolysaccharide (LPS), TNF-α, Calcipotriol and irradiated with UVB, cultured, and harvested. To assess the expression of human beta defensin-2 and LL-37 in the control group, not exposed to any stimulants, the experimental group was treated with LPS, TNF-α, or UVB, and another group was treated again with calcipotriol; reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical staining were performed. Results. In the experimental group treated with LPS, UVB irradiation, and TNF-α, the expression of β-defensin and LL-37 was increased more than in the control group and then decreased in the experimental group treated with calcipotriol. Conclusions. Calcipotriol suppressed HBD-2 and LL-37, which were stimulated by UVB, LPS, and TNF-α. PMID:20182640

  14. The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

    SciTech Connect

    Paes, Camila

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalian cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.

  15. Phylogenetic profiling and gene expression studies implicate a primary role of PSORS1C2 in terminal differentiation of keratinocytes.

    PubMed

    Abbas Zadeh, Salman; Mlitz, Veronika; Lachner, Julia; Golabi, Bahar; Mildner, Michael; Pammer, Johannes; Tschachler, Erwin; Eckhart, Leopold

    2017-04-01

    PSORS1C2 is a gene located between coiled-coil alpha-helical rod protein 1 (CCHCR1) and corneodesmosin (CDSN) within the psoriasis susceptibility locus 1 (PSORS1). Here, we performed a comparative genomics analysis of the as-yet incompletely characterized PSORS1C2 gene and determined its expression pattern in human tissues. In contrast to CCHCR1, which is common to all vertebrates investigated, PSORS1C2 and CDSN are present exclusively in mammals, indicating that the latter genes have originated after the evolutionary divergence of mammals and reptiles. CDSN is conserved in aquatic mammals, whereas PSORS1C2 orthologs contain gene-inactivating frame shift mutations in whales and dolphins, in which the epidermal differentiation programme has degenerated. Reverse-transcription PCR screening demonstrated that, in human tissues, PSORS1C2 is expressed principally in the epidermis and weakly in the thymus. PSORS1C2 mRNA was strongly upregulated during terminal differentiation of human keratinocytes in vitro. Immunohistochemistry revealed exclusive expression of PSORS1C2 in the granular layer of the epidermis and in cornifying epithelial cells of Hassall's corpuscles of the thymus. In summary, our results identify PSORS1C2 as a keratinocyte cornification-associated protein that has originated in evolutionarily basal mammals and has undergone gene inactivation in association with the loss of the skin barrier function in aquatic mammals.

  16. 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.

  17. The effect of two endogenous retinoids on the mRNA expression profile in human primary keratinocytes, focusing on genes causing autosomal recessive congenital ichthyosis.

    PubMed

    Törmä, H; Bergström, A; Ghiasifarahani, G; Berne, B

    2014-10-01

    Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (>350) than in proliferating keratinocytes (≈20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids.

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

    PubMed

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

    2009-03-01

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

  19. Effects of AV119, a natural sugar from avocado, on Malassezia furfur invasiveness and on the expression of HBD-2 and cytokines in human keratinocytes.

    PubMed

    Donnarumma, Giovanna; Buommino, Elisabetta; Baroni, Adone; Auricchio, Lucia; De Filippis, Anna; Cozza, Valentina; Msika, Philippe; Piccardi, Nathalie; Tufano, Maria Antonietta

    2007-11-01

    AV119 is a patented blend of two sugars from avocado that can induce human beta-defensin-2 production by normal human keratinocytes. In this study, we analysed the effect of AV119 on growth and invasiveness of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora. The ability to modulate the expression of the proinflammatory and immunomodulatory cytokines in normal human keratinocytes was also investigated. Microbiological assay demonstrated that this sugar induced the aggregation of yeast cells and inhibited the invasiveness of M. furfur, without affecting its growth. Real-time PCR analysis demonstrated that AV119 was able to modulate the HBD-2 response in treated keratinocytes, reaching a maximum after 48-h treatment, and to induce the recovery of a satisfactory proinflammatory response in human keratinocytes. As AV119 can induce aggregation of yeast cells, thus inhibiting their penetration into the keratinocytes, the sugar could be used in the preparation of cosmetics or pharmacological drugs to inhibit colonization of the skin by pathogenic strains of M. furfur.

  20. UVB and gamma-radiation induce the expression of mRNAs encoding the ribosomal subunit L13A in rat keratinocytes.

    PubMed

    Shahmolky, N; Lefebvre, D L; Poon, R; Bai, Y; Sharma, M; Rosen, C F

    1999-09-01

    Ultraviolet B radiation produces an array of cellular perturbations in the skin. We isolated a keratinocyte cDNA encoding the rat 60S ribosomal subunit protein L13a following differential cDNA library screening with UVB-enriched probes. In contrast to the reported structure of liver L13a, the keratinocyte L13a cDNA contains a longer 3'-untranslated region. Northern blot analysis detected two L13a mRNA transcripts, approximately 800 bp and approximately 1.2 kb, in keratinocytes and a variety of rat tissues. Both L13a mRNA transcripts were induced by UVB irradiation, forskolin and gamma-irradiation. In contrast, no induction of L13a mRNA transcript levels was observed following exposure of keratinocytes to 12-O-tetradecanoylphorbol-13-acetate, serum and the DNA damage-inducing agents methyl methanesulfonate or 4-nitroquinoline-N-oxide. These observations suggest that increased expression of ribosomal subunit genes may be a molecular component of the keratinocyte response to UVB in particular and not part of a nonspecific response to DNA damage.

  1. Characterization of gene expression changes associated with MNNG, arsenic, or metal mixture treatment in human keratinocytes: application of cDNA microarray technology.

    PubMed Central

    Bae, Dong-Soon; Hanneman, William H; Yang, Raymond S H; Campain, Julie A

    2002-01-01

    The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics. PMID:12634122

  2. Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

    PubMed Central

    Klymenko, T.; Hernandez-Lopez, H.; MacDonald, A. I.; Bodily, J. M.

    2016-01-01

    ABSTRACT The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCE Human papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4^L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction

  3. Characterization and functional analysis of the expression of intercellular adhesion molecule-1 in human papillomavirus-related disease of cervical keratinocytes.

    PubMed Central

    Coleman, N.; Greenfield, I. M.; Hare, J.; Kruger-Gray, H.; Chain, B. M.; Stanley, M. A.

    1993-01-01

    We have investigated the expression of intercellular adhesion molecule-1 (ICAM-1) in squamous neoplasia of the cervix and have noted a significant induction of the molecule in high-grade intra-epithelial lesions. Using monolayer and organotypic in vitro tissue culture systems, we have shown that there is no constitutive ICAM-1 expression on cervical keratinocytes immortalized but not transformed by human papillomavirus type 16, whereas two human papillomaviruses type 16 containing and fully transformed cervical keratinocyte lines do constitutively express the molecule. All cell types, including human papillomavirus-negative normal cervical keratinocytes, can be induced to up-regulate their expression of ICAM-1 by pro-inflammatory cytokines such as interferon-gamma. In addition, we have used an in vitro adhesion assay to show that ICAM-1:lymphocyte function antigen-1 interaction is functionally important in lymphocyte binding to cervical keratinocytes, suggesting a role for ICAM-1 in retaining and enabling functional activity of lymphocytes in the cervix in intraepithelial neoplasia. Images Figure 1 Figure 2 Figure 5 Figure 9 PMID:8102029

  4. Changes in localization of human discs large (hDlg) during keratinocyte differentiation is associated with expression of alternatively spliced hDlg variants

    SciTech Connect

    Roberts, S. . E-mail: s.roberts@bham.ac.uk; Calautti, E.; Vanderweil, S.; Nguyen, H.O.; Foley, A.; Baden, H.P.; Viel, A.

    2007-07-15

    Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.

  5. Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

    PubMed

    Asai, Jun; Hirakawa, Satoshi; Sakabe, Jun-ichi; Kishida, Tsunao; Wada, Makoto; Nakamura, Naomi; Takenaka, Hideya; Mazda, Osam; Urano, Tetsumei; Suzuki-Inoue, Katsue; Tokura, Yoshiki; Katoh, Norito

    2016-01-01

    Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing.

  6. Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates UV induced melanocyte homeostasis

    PubMed Central

    Hyter, Stephen; Coleman, Daniel J.; Ganguli-Indra, Gitali; Merrill, Gary F.; Ma, Steven; Yanagisawa, Masashi; Indra, Arup K.

    2013-01-01

    Summary Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of EDN1 in murine epidermis (EDN1ep−/−) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage and apoptosis after UVB irradiation. Expression of other keratinocyte derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV induced melanocyte activation and recapitulated the phenotype seen in EDN1ep−/− mice. Altogether, present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV induced melanocyte homeostasis in vivo. PMID:23279852

  7. Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes

    PubMed Central

    2010-01-01

    Background Tungsten carbide (WC) and tungsten carbide cobalt (WC-Co) nanoparticles are of occupational health relevance because of the increasing usage in hard metal industries. Earlier studies showed an enhanced toxic potential for WC-Co compared to WC or cobalt ions alone. Therefore, we investigated the impact of these particles, compared to cobalt ions applied as CoCl2, on the global gene expression level in human keratinocytes (HaCaT) in vitro. Results WC nanoparticles exerted very little effects on the transcriptomic level after 3 hours and 3 days of exposure. In contrast, WC-Co nanoparticles caused significant transcriptional changes that were similar to those provoked by CoCl2. However, CoCl2 exerted even more pronounced changes in the transcription patterns. Gene set enrichment analyses revealed that the differentially expressed genes were related to hypoxia response, carbohydrate metabolism, endocrine pathways, and targets of several transcription factors. The role of the transcription factor HIF1 (hypoxia inducible factor 1) is particularly highlighted and aspects of downstream events as well as the role of other transcription factors related to cobalt toxicity are considered. Conclusions This study provides extensive data useful for the understanding of nanoparticle and cobalt toxicity. It shows that WC nanoparticles caused low transcriptional responses while WC-Co nanoparticles are able to exert responses similar to that of free cobalt ions, particularly the induction of hypoxia-like effects via interactions with HIF1α in human keratinocytes. However, the enhanced toxicity of WC-Co particles compared to CoCl2 could not be explained by differences in gene transcription. PMID:20105288

  8. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

    PubMed

    Arredondo, J; Hall, L L; Ndoye, A; Chernyavsky, A I; Jolkovsky, D L; Grando, S A

    2003-02-01

    We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

  9. The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization

    PubMed Central

    Jinno, Chizuru; Sakamoto, Michiharu; Kakudo, Natsuko; Inoie, Masukazu; Fujisato, Toshia; Suzuki, Shigehiko; Kusumoto, Kenji; Yamaoka, Tetsuji

    2016-01-01

    We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP) higher than 200 MPa and that human cultured epidermis (hCE) engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa). Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM). hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting. PMID:27747221

  10. Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

    SciTech Connect

    Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh; Elmets, Craig A.; Athar, Mohammad

    2014-08-29

    Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34{sup +}/K15{sup +}/p63{sup +} keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in

  11. Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

    PubMed Central

    Shahbazi, Marta N.; Peña-Jimenez, Daniel; Antonucci, Francesca; Drosten, Matthias

    2017-01-01

    ABSTRACT Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian microtubule and kinetochore-associated protein Clasp2 is intimately associated with the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2-deficient keratinocytes exhibit increased centrosomal numbers and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a new role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that prevent cell cycle entry in cells that have alterations in the DNA content. PMID:28069833

  12. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  13. Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis

    PubMed Central

    Corrêa, Mara A.; Okamoto, Cinthya K.; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2016-01-01

    Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism. PMID:27078876

  14. Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis.

    PubMed

    Corrêa, Mara A; Okamoto, Cinthya K; Gonçalves-de-Andrade, Rute M; van den Berg, Carmen W; Tambourgi, Denise V

    2016-01-01

    Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.

  15. Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA.

    PubMed

    Wang, Xiao; Ramirez, Angel; Budunova, Irina

    2010-07-01

    Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co-expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co-expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co-expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43-positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down-regulation of TPA-induced activity of protein kinase C (PKC) in K5.Cx26 mice.

  16. 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.

  17. Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells

    SciTech Connect

    Du Liping; Neis, Mark M.; Ladd, Patricia A.; Yost, Garold S.; Keeney, Diane S. . E-mail: diane.keeney@vanderbilt.edu

    2006-06-01

    Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

  18. Calcitriol May Down-Regulate mRNA Over-Expression of Toll-Like Receptor-2 and -4, LL-37 and Proinflammatory Cytokines in Cultured Human Keratinocytes

    PubMed Central

    Jeong, Mi Sook; Kim, Ji-Yun; Lee, He In

    2014-01-01

    Background Although vitamin D analogs have been used in the topical treatment of psoriasis, their mechanisms of action are not well understand. Calcitriol, the hormonally active vitamin D3 metabolite, has been demonstrated to exert immunomodulatory effects in the skin by down-regulating the expression of Toll-like receptors (TLRs) and proinflammatory cytokines. Objective We investigated the effects of calcitriol on the expression of TLR2, TLR4, antimicrobial peptide LL-37, and proinflammatory cytokines in cultured human keratinocytes. Methods The mRNA expression levels of TLR2, TLR4, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and LL-37 in cultured human keratinocytes were measured by real-time polymerase chain reaction (PCR) and reverse transcription (RT). Furthermore, we measured supernatant TNF-α levels by an enzyme-linked immunosorbent assay (ELISA) to confirm the effects of calcitriol on TLR2 and TLR4. Results As measured by RT-PCR and real-time PCR, calcitriol was found to suppress the lipopolysaccharide- and ultraviolet B radiation-mediated induction of expression of TLRs, LL-37 and proinflammatory cytokines such as TNF-α and IL-1β in normal human keratinocytes. The supernatant TNF-α levels measured by ELISA were also suppressed after treatment with calcitriol. Conclusion Calcitriol may down-regulate inflammatory stated over-expression of LL-37 and proinflammatory cytokines. PMID:24966627

  19. Keratin gene expression profiles after digit amputation in C57BL/6 vs. regenerative MRL mice imply an early regenerative keratinocyte activated-like state

    PubMed Central

    Cheng, Chia-Ho; Leferovich, John; Zhang, Xiang-Ming; Bedelbaeva, Khamilia; Gourevitch, Dmitri; Hatcher, Cathy J.; Basson, Craig T.; Heber-Katz, Ellen

    2013-01-01

    Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding. PMID:23512742

  20. Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

    SciTech Connect

    Black, Adrienne T.; Joseph, Laurie B.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-06-15

    Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 {mu}M) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A{sub 4} (LTA{sub 4}) hydrolase and leukotriene C{sub 4} (LTC{sub 4}) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA{sub 4} hydrolase and LTC{sub 4} synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

  1. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

    PubMed

    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  2. Constitutive expression of MC1R in HaCaT keratinocytes inhibits basal and UVB-induced TNF-alpha production.

    PubMed

    Garcin, Geneviève; Le Gallic, Lionel; Stoebner, Pierre-Emmanuel; Guezennec, Anne; Guesnet, Joelle; Lavabre-Bertrand, Thierry; Martinez, Jean; Meunier, Laurent

    2009-01-01

    Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha). Therefore, we generated HaCaT cells that stably express human MC1R or the Arg151Cys (R151C) nonfunctional variant. We demonstrate that: (1) the constitutive activity of MC1R results in elevated intracellular cAMP level, reduced NF-kappaB activity and decreased TNF-alpha transcription; (2) binding of alpha-MSH to MC1R and the subsequent increase in cAMP production do not inhibit TNFalpha-mediated NF-kappaB activation; (3) MC1R signaling is sufficient to strongly inhibit UVB-induced TNF-alpha expression and this inhibitory effect is further enhanced by alpha-MSH stimulation. Our findings suggest that the constitutive activity of the G-protein-coupled MC1R in keratinocytes may contribute to the modulation of inflammatory events and immune response induced by UV light.

  3. Dermal fibroblast expression of stromal cell-derived factor-1 (SDF-1) promotes epidermal keratinocyte proliferation in normal and diseased skin.

    PubMed

    Quan, Chunji; Cho, Moon Kyun; Shao, Yuan; Mianecki, Laurel E; Liao, Eric; Perry, Daniel; Quan, Taihao

    2015-12-01

    Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.

  4. 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.

  5. Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

    PubMed Central

    Haertel, Beate; Straßenburg, Susanne; Wende, Kristian; von Woedtke, Thomas

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells. PMID:23936843

  6. Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes.

    PubMed

    Haertel, Beate; Straßenburg, Susanne; Oehmigen, Katrin; Wende, Kristian; von Woedtke, Thomas; Lindequist, Ulrike

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.

  7. Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

    PubMed

    Jokela, Tiina; Kärnä, Riikka; Rauhala, Leena; Bart, Genevieve; Pasonen-Seppänen, Sanna; Oikari, Sanna; Tammi, Markku I; Tammi, Raija H

    2017-03-24

    The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

  8. Induction of cytochrome P450IA1 gene expression in rat epidermis and human keratinocytes by. beta. -napthoflavone and benzanthracene

    SciTech Connect

    Khan, I.U.; Mukhtar, H.; Bickers, D.R.; Haqqi, T.M. )

    1991-03-15

    Cytochrome P450IA1 (P450IA1) plays a major role in the bioactivation of procarcinogens in various tissues including skin. However, factors controlling the expression of P450IA1 gene message in mammalian skin are unknown. In this study, the polymerase chain reaction (PCR) using specific primers was employed to study the expression of P450IA1 mRNA transcripts in rat epidermis and human keratinocytes (HK) treated with {beta}-napthoflavone ({beta}NF) and benzanthracene (BA). Total RNA was extracted from the epidermis of control and inducer-treated 4-day-old and adult Sprague Dawley rats, and from control and inducer-treated HL. cDNAs were synthesized using random primers and reverse transcriptase. PCR products were analyzed on agarose gel and quantitated by densitometry. Inducer treatment of rats and HK resulted in several-fold increases in aryl hydrocarbon hydroxylase (AHH) activity. The level of P450IA1 gene message increased 2-5-fold in treated animals as compared to controls; higher basal level and inducibility in adult than in 4-day-old rats. This induction occurred as early as 4 h after {beta}NF application, reached a maximum at 16 h and returned to basal levels by 36 h. Exposure to {beta}NF and BA resulted in 2-3-fold increase in gene message in HK. Northern blot analysis complemented PCR data. These results indicate that in mammalian skin P450IA1 gene expression is increased by the inducers of epidermal AHH activity.

  9. Downregulation of TNIP1 Expression Leads to Increased Proliferation of Human Keratinocytes and Severer Psoriasis-Like Conditions in an Imiquimod-Induced Mouse Model of Dermatitis

    PubMed Central

    Chen, Yan; Yan, Heng; Song, Zhiqiang; Chen, Fangru; Wang, Huan; Niu, Jun; Shi, Xiaowei; Zhang, Dongmei; Zhang, Na; Zhai, Zhifang; Zhong, Baiyu; Cheng, Liangjin; Qian, Tian; Hao, Fei

    2015-01-01

    Psoriasis is a chronic, inflammatory skin disease involving both environmental and genetic factors. According to genome-wide association studies (GWAS), the TNIP1 gene, which encodes the TNF-α–induced protein 3-interacting protein 1 (TNIP1), is strongly linked to the susceptibility of psoriasis. TNIP1 is a widely expressed ubiquitin sensor that binds to the ubiquitin-editing protein A20 and restricts TNF- and TLR-induced signals. In our study, TNIP1 expression decreased in specimens of epidermis affected by psoriasis. Based on previous studies suggesting a role for TNIP1 in modulating cancer cell growth, we investigated its role in keratinocyte proliferation, which is clearly abnormal in psoriasis. To mimic the downregulation or upregulation of TNIP1 in HaCaT cells and primary human keratinocytes (PHKs), we used a TNIP1 specific small interfering hairpin RNA (TNIP1 shRNA) lentiviral vector or a recombinant TNIP1 (rTNIP1) lentiviral vector, respectively. Blocking TNIP1 expression increased keratinocyte proliferation, while overexpression of TNIP1 decreased keratinocyte proliferation. Furthermore, we showed that TNIP1 signaling might involve extracellular signal-regulated kinase1/2 (Erk1/2) and CCAAT/enhancer-binding protein β (C/EBPβ) activity. Intradermal injection of TNIP1 shRNA in BALB/c mice led to exaggerated psoriatic conditions in imiquimod (IMQ)-induced psoriasis-like dermatitis. These findings indicate that TNIP1 has a protective role in psoriasis and therefore could be a promising therapeutic target. PMID:26046540

  10. 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.

  11. Fir honeydew honey flavonoids inhibit TNF-α-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing.

    PubMed

    Majtan, Juraj; Bohova, Jana; Garcia-Villalba, Rocio; Tomas-Barberan, Francisco A; Madakova, Zuzana; Majtan, Tomas; Majtan, Viktor; Klaudiny, Jaroslav

    2013-09-01

    Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.

  12. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure

    PubMed Central

    Huang, S.; Guo, S.; Guo, F.; Yang, Q.; Xiao, X.; Murata, M.; Ohnishi, S.; Kawanishi, S.; Ma, N.

    2013-01-01

    Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the human spontaneously immortalized skin keratinocytes (HaCaT) cell line to an environmentally relevant level of arsenic (0.05 ppm) in vitrofor 18 weeks. Following sodium arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem-like phenotype. Furthermore, arsenictreated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was upregulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenicinduced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway

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

    SciTech Connect

    De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.; Bolterstein, Elyse A.; Schlosser, Sandy J.; Allen-Hoffmann, B. Lynn

    2014-05-01

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

  14. Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation.

    PubMed Central

    Smith, Conrad; Zhu, Kuichun; Merritt, Anita; Picton, Rhian; Youngs, Denise; Garrod, David; Chidgey, Martyn

    2004-01-01

    Desmocollins (Dscs) are desmosomal cadherins that exhibit differentiation-specific patterns of expression in the epidermis. Dsc3 expression is strongest in basal cell layers, whereas Dsc1 is largely confined to upper, terminally differentiating strata. To understand better the processes by which Dsc expression is regulated in the epidermis, we have isolated Dsc3 and Dsc1 5'-flanking DNAs and analysed their activity in primary keratinocytes. In the present study, we found that transcription factors of the CCAAT/enhancer-binding protein family play a role in the regulation of expression of both Dscs and, in so doing, implicate this class of transcription factors in both early and late events in keratinocyte differentiation. We show that Dscs are differentially regulated by C/EBP (CCAAT/enhancer-binding protein) family members, with Dsc3 expression being activated by C/EBPbeta but not C/EBPalpha, and the reverse being the case for Dsc1. Expression of both Dscs is activated by another family member, C/EBPdelta. These results show for the first time how desmosomal cadherin gene expression is regulated and provide a mechanism for the control of other differentiation-specific genes in the epidermis. PMID:15030314

  15. Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro.

    PubMed

    Wikner, N E; Elder, J T; Persichitte, K A; Mink, P; Clark, R A

    1990-11-01

    Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

  16. The EP1 subtype of Prostaglandin E2 Receptor: Role in Keratinocyte Differentiation and Expression in Non-Melanoma Skin Cancer

    PubMed Central

    Konger, R. L.; Billings, S. D.; Prall, N. C.; Katona, T. M.; DaSilva, S. C.; Kennedy, C. R. J.; Badve, S.; Perkins, S. M.; LaCelle, P. T.

    2010-01-01

    SUMMARY We have previously demonstrated that the EP1 subtype of PGE2 receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP1 receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP1 receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP1 receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP1 receptor in non-melanoma skin cancer in humans. Well differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP1 receptor in regulating keratinocyte differentiation. PMID:19625175

  17. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation.

    PubMed

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

    2016-03-18

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

  18. The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation.

    PubMed

    Becker-Pauly, Christoph; Höwel, Markus; Walker, Tatjana; Vlad, Annica; Aufenvenne, Karin; Oji, Vinzenz; Lottaz, Daniel; Sterchi, Erwin E; Debela, Mekdes; Magdolen, Viktor; Traupe, Heiko; Stöcker, Walter

    2007-05-01

    The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

  19. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  20. [Effect of cetirizine hydrochloride on the expression of substance P receptor and cytokines production in human epidermal keratinocytes and dermal fibroblasts].

    PubMed

    Liu, Ji-Yong; Zhao, Yong-Zhe; Peng, Cheng; Li, Feng-Qian; Zhu, Quan-Gang; Hu, Jin-Hong

    2008-04-01

    To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.

  1. Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

    PubMed Central

    Bata-Csorgo, Z; Cooper, K D; Ting, K M; Voorhees, J J; Hammerberg, C

    1998-01-01

    In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis. PMID:9525994

  2. Ma Huang Tang Suppresses the Production and Expression of Inflammatory Chemokines via Downregulating STAT1 Phosphorylation in HaCaT Keratinocytes

    PubMed Central

    Jin, Seong-Eun; Lee, Mee-Young

    2016-01-01

    Ma huang tang (MHT) is a traditional herbal medicine comprising six medicinal herbs and is used to treat influenza-like illness. However, the effects of MHT on inflammatory skin diseases have not been verified scientifically. We investigated determining the inhibitory effects of MHT against inflammation responses in skin using HaCaT human keratinocyte cells. We found that MHT suppressed production of thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), regulated on activation of normal T-cell expressed and secreted (RANTES/CCL5), and interleukin-8 (IL-8) in tumor necrosis factor-α (TNF-α) and interferon-γ- (IFN-γ-) stimulated HaCaT cells. Consistently, MHT suppressed the mRNA expression of TARC, MDC, RANTES, and IL-8 in TNF-α and IFN-γ-stimulated cells. Additionally, MHT inhibited TNF-α and IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) phosphorylation in a dose-dependent manner and nuclear translocation in HaCaT cells. Our finding indicates that MHT inhibits production and expression of inflammatory chemokines in the stimulated keratinocytes by downregulating STAT1 phosphorylation, suggesting that MHT may be a possible therapeutic agent for inflammatory skin diseases. PMID:27847527

  3. Bazex Syndrome in Lung Squamous Cell Carcinoma: High Expression of Epidermal Growth Factor Receptor in Lesional Keratinocytes with Th2 Immune Shift

    PubMed Central

    Amano, Maki; Hanafusa, Takaaki; Chikazawa, Sakiko; Ueno, Makiko; Namiki, Takeshi; Igawa, Ken; Miura, Keiko; Yokozeki, Hiroo

    2016-01-01

    An 82-year-old Japanese man was referred for detailed examination of hyperkeratotic erythematous plaques on his palms and soles for 6 months. Two weeks before his first visit, he had undergone lung lobectomy for right lung squamous cell carcinoma (SCC). Laboratory findings showed elevations of eosinophil counts, serum IgE, thymus and activation-regulated chemokine, SCC antigen, and soluble interleukin-2 receptor levels. Histological results of a skin biopsy involving the left palm showed psoriasiform dermatitis. Before lung lobectomy, the hyperkeratotic erythematous plaques on the palms and soles and the erythemas on the trunk and extremities were difficult to treat with topical steroids. After lobectomy, the skin symptoms dramatically and rapidly subsided with topical steroids. Therefore, we diagnosed Bazex syndrome (BS), also known as acrokeratosis paraneoplastica, as a paraneoplastic cutaneous disease in lung SCC. The mild eosinophilia subsided and levels of SCC antigen, IgE, and soluble interleukin-2 receptor were reduced. BS is a paraneoplastic cutaneous disease characterized by acral psoriasiform lesions associated with an underlying neoplasm. In a previous report, a shift to the Th2 immune condition was found in patients with non-small cell lung cancer, as shown in our patient. Epidermal growth factor receptor (EGFR) is also known as tumor growth factor-α receptor; it is increased in psoriatic keratinocytes. In our case, EGFR expression increased in lesional keratinocytes 2 weeks after surgery and decreased 4 weeks after surgery. We speculate that a shift to Th2 immune reactions in lung SCC may be the pathogenesis of BS, whereby lesional keratinocytes highly express EGFR in parallel with disease activity. PMID:28101024

  4. A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ

    PubMed Central

    Zákány, Nóra; Tóth, Balázs I.; Bíró, Tamás

    2013-01-01

    Cannabinoid receptors (CB) are expressed throughout human skin epithelium. CB1 activation inhibits human hair growth and decreases proliferation of epidermal keratinocytes. Since psoriasis is a chronic hyperproliferative, inflammatory skin disease, it is conceivable that the therapeutic modulation of CB signaling, which can inhibit both proliferation and inflammation, could win a place in future psoriasis management. Given that psoriasis is characterized by up-regulation of keratins K6 and K16, we have investigated whether CB1 stimulation modulates their expression in human epidermis. Treatment of organ-cultured human skin with the CB1-specific agonist, arachidonoyl-chloro-ethanolamide (ACEA), decreased K6 and K16 staining intensity in situ. At the gene and protein levels, ACEA also decreased K6 expression of cultured HaCaT keratinocytes, which show some similarities to psoriatic keratinocytes. These effects were partly antagonized by the CB1-specific antagonist, AM251. While CB1-mediated signaling also significantly inhibited human epidermal keratinocyte proliferation in situ, as shown by K6/Ki-67-double immunofluorescence, the inhibitory effect of ACEA on K6 expression in situ was independent of its anti-proliferative effect. Given recent appreciation of the role of K6 as a functionally important protein that regulates epithelial wound healing in mice, it is conceivable that the novel CB1-mediated regulation of keratin 6/16 revealed here also is relevant to wound healing. Taken together, our results suggest that cannabinoids and their receptors constitute a novel, clinically relevant control element of human K6 and K16 expression. PMID:23638377

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

    SciTech Connect

    Kurita, Masakazu; Okazaki, Mutsumi; Fujino, Takashi; Takushima, Akihiko; Harii, Kiyonori

    2011-05-27

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

  6. Altered aquaporin expression in glaucoma eyes.

    PubMed

    Tran, Thuy Linh; Bek, Toke; la Cour, Morten; Nielsen, Søren; Prause, Jan Ulrik; Hamann, Steffen; Heegaard, Steffen

    2014-09-01

    Aquaporins (AQP) are channels in the cell membrane that mainly facilitate a passive transport of water. In the eye, AQPs are expressed in the ciliary body and retina and may contribute to the pathogenesis of glaucoma and optic neuropathy. We investigated the expression of AQP1, AQP3, AQP4, AQP5, AQP7 and AQP9 in human glaucoma eyes compared with normal eyes. Nine glaucoma eyes were examined. Of these, three eyes were diagnosed with primary open angle glaucoma; three eyes had neovascular glaucoma; and three eyes had chronic angle-closure glaucoma. Six eyes with normal intraocular pressure and without glaucoma were used as control. Immunohistochemistry was performed using antibodies against AQP1, AQP3, AQP4, AQP5, AQP7 and AQP9. For each specimen, optical densities of immunoprecipitates were measured using Photoshop and the staining intensities were calculated. Immunostaining showed labelling of AQP7 and AQP9 in the nonpigmented ciliary epithelium and the staining intensities were significantly decreased in glaucoma eyes (p = 0.003; p = 0.018). AQP7 expression in the Müller cell endfeet was increased (p = 0.046), and AQP9 labelling of the retinal ganglion cells (RGC) showed decreased intensity (p = 0.037). No difference in AQP1, AQP4 and AQP9 expression was found in the optic nerve fibres. This study is the first investigating AQPs in human glaucoma eyes. We found a reduced expression of AQP9 in the retinal ganglion cells of glaucoma eyes. Glaucoma also induced increased AQP7 expression in the Müller cell endfeet. In the ciliary body of glaucoma eyes, the expression of AQP7 and AQP9 was reduced. Therefore, the expression of AQPs seems to play a role in glaucoma.

  7. Ultraviolet radiation can either suppress or induce expression of intercellular adhesion molecule 1 (ICAM-1) on the surface of cultured human keratinocytes

    SciTech Connect

    Norris, D.A.; Lyons, M.B.; Middleton, M.H.; Yohn, J.J.; Kashihara-Sawami, M. )

    1990-08-01

    Interactions of the ligand/receptor pair LFA-1(CD11a/CD18) and ICAM-1(CD54) initiate and control the cell-cell interactions of leukocytes and interactions of leukocytes with parenchymal cells in all phases of the immune response. Induction of the intercellular adhesion molecule 1 (ICAM-1) on the surface of epidermal keratinocytes has been proposed as an important regulator of contact-dependent aspects of cutaneous inflammation. Ultraviolet radiation (UVR) also modifies cutaneous inflammation, producing both up- and down-regulation of contact hypersensitivity. We have found that UVR has a biphasic effect on the induction of keratinocyte CD54. Using immunofluorescence and FACS techniques to quantitate cell-surface CD54 staining, we have shown that UVR significantly (p less than 0.01) inhibits keratinocyte CD54 induction by gamma interferon 24 h after irradiation. However, at 48, 72, and 96 h after UVR, CD54 expression is significantly induced to levels even greater than are induced by gamma interferon (20 U/ml). In addition, at 48, 72, or 96 h following UVR (30-100 mJ/cm2), the gamma-interferon-induced CD54 expression on human keratinocytes is also strongly (p less than 0.05 to p less than 0.001) enhanced. In this cell-culture system, gamma interferon and TNF-alpha are both strong CD54 inducers and are synergistic, but GM-CSF, TFG-beta, and IL-1 have no direct CD54-inducing effects. Thus the effects of UVR on CD54 induction are biphasic, producing inhibition at 24 h and induction at 48, 72, and 96 h. This effect on CD54 may contribute to the biphasic effects of UVR on delayed hypersensitivity in vivo. The early inhibition of ICAM-1 by UVR may also contribute to the therapeutic effects of UVR. We also speculate that the late induction of ICAM-1 by UVR might be an important step in the induction of photosensitive diseases such as lupus erythematosus.

  8. Arsenic Induces p62 Expression to Form a Positive Feedback Loop with Nrf2 in Human Epidermal Keratinocytes: Implications for Preventing Arsenic-Induced Skin Cancer

    PubMed Central

    Shah, Palak; Trinh, Elaine; Qiang, Lei; Xie, Lishi; Hu, Wen-Yang; Prins, Gail S.; Pi, Jingbo; He, Yu-Ying

    2017-01-01

    Exposure to inorganic arsenic in contaminated drinking water poses an environmental public health threat for hundreds of millions of people in the US and around the world. Arsenic is a known carcinogen for skin cancer. However, the mechanism by which arsenic induces skin cancer remains poorly understood. Here, we have shown that arsenic induces p62 expression in an autophagy-independent manner in human HaCaT keratinocytes. In mouse skin, chronic arsenic exposure through drinking water increases p62 protein levels in the epidermis. Nrf2 is required for basal and arsenic-induced p62 up-regulation. p62 knockdown reduces arsenic-induced Nrf2 activity, and induces sustained p21 up-regulation. p62 induction is associated with increased proliferation in mouse epidermis. p62 knockdown had little effect on arsenic-induced apoptosis, while it decreased cell proliferation following arsenic treatment. Our findings indicate that arsenic induces p62 expression to regulate the Nrf2 pathway in human keratinocytes and suggest that targeting p62 may help prevent arsenic-induced skin cancer. PMID:28125038

  9. Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression

    PubMed Central

    Grossi, Maddalena; Hiou-Feige, Agnès; Di Vignano, Alice Tommasi; Calautti, Enzo; Ostano, Paola; Lee, Sam; Chiorino, Giovanna; Dotto, G. Paolo

    2005-01-01

    Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell–cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal. PMID:16061799

  10. Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells.

    PubMed

    Adase, Christopher A; Borkowski, Andrew W; Zhang, Ling-Juan; Williams, Michael R; Sato, Emi; Sanford, James A; Gallo, Richard L

    2016-05-27

    A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

  11. 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.

  12. 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

  13. RNA-binding protein CELF1 promotes tumor growth and alters gene expression in oral squamous cell carcinoma.

    PubMed

    House, Reniqua P; Talwar, Sudha; Hazard, E Starr; Hill, Elizabeth G; Palanisamy, Viswanathan

    2015-12-22

    The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention.

  14. RNA-binding protein CELF1 promotes tumor growth and alters gene expression in oral squamous cell carcinoma

    PubMed Central

    House, Reniqua P.; Talwar, Sudha; Hazard, E. Starr; Hill, Elizabeth G.; Palanisamy, Viswanathan

    2015-01-01

    The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention. PMID:26498364

  15. Malassezia furfur induces the expression of beta-defensin-2 in human keratinocytes in a protein kinase C-dependent manner.

    PubMed

    Donnarumma, Giovanna; Paoletti, Iole; Buommino, Elisabetta; Orlando, Manuela; Tufano, Maria Antonietta; Baroni, Adone

    2004-04-01

    Antimicrobial peptides of the beta-defensin family are expressed in all human epithelial tissues tested to date and have recently been the subject of vigorous investigation. Their localization and characteristics support the hypothesis that these peptides play a role in mucosal and skin defense. The lipophilic yeast Malassezia furfur is a saprophyte found in normal human cutaneous flora. Malassezia furfur is not only a saprophyte, but is also associated with several diseases such as Malassezia folliculitis, seborrheic dermatitis and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. Little is known about the mechanism by which M. furfur overcomes the natural barrier of the skin. To further define the role of the beta-defensins in the innate human skin immune response, we analyzed the mRNA expression of two human beta-defensins HBD-1 and HBD-2 in human keratinocytes treated with M. furfur. In addition, we looked into how M. furfur of TGF-beta1 and IL-10, cytokines that interfere with the development of protective cell immunity, regulate their expression. Finally, we examined the signal transduction mechanisms involved during M. furfur uptake. Cultured human keratinocytes were treated with M. furfur. The mRNA and protein expression were analyzed, respectively, by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Our data demonstrate that M. furfur does not modify HBD-1 expression, whereas it up-regulates, via protein kinase C (PKC), the expression of HBD-2, TGFbeta-1 and IL-10 48 h after treatment. Our results suggest that beta-defensins are integral components of innate host defenses. They play an essential part in the resistance of the human skin surfaces against M. furfur uptake and other microbial invasion.

  16. Modulation of constitutive cytochrome P-450 expression in vivo and in vitro in murine keratinocytes as a function of differentiation and extracellular Ca2+ concentration.

    PubMed Central

    Reiners, J J; Cantu, A R; Pavone, A

    1990-01-01

    A procedure was developed for the per cell estimation of cytochrome P-450-dependent monooxygenase activities in cultures and whole cell suspensions of murine epidermal keratinocytes (MEKs). Murine keratinocytes cultured in medium containing less than or equal to 0.04 mM Ca2+ can be induced to differentiate by raising medium Ca2+ concentrations to 1.2 mM. The per cell activities of the monooxygenases 7-ethoxyresorufin O-deethylase (7-ER) and 7-ethoxycoumarin O-deethylase (7-EC) were elevated greater than or equal to 2090% and approximately 460%, respectively, within 13-24 hr of Ca2+ shift. These increases could be completely suppressed by supplementation of culture medium with actinomycin D or cycloheximide immediately prior to Ca2+ shift. After prolonged culture in low Ca2+ medium, some MEKs detached from the monolayer. These detached cells had the characteristics of differentiating MEKs but did not have elevated 7-EC or 7-ER activities. Percoll gradient centrifugation of freshly isolated dorsal skin MEKs was used to prepare four subpopulations that differed in their stages of terminal differentiation. 7-EC and 7-ER activities varied among these subpopulations and correlated with the degree of MEK differentiation. Specifically, the lowest and highest per cell activities (greater than 7-fold difference) were in the basal and most differentiated spinous cell populations, respectively. Collectively, the current studies demonstrate that in vivo P-450 activities are markedly different in proliferating and differentiating MEKs and suggest that constitutive P-450 expression may be modulated as a function of changes in Ca2+ concentration that occur during keratinocyte terminal differentiation. PMID:2308941

  17. Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening

    PubMed Central

    Udommethaporn, Sutthirat; Tencomnao, Tewin; McGowan, Eileen M.

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine important in normal and pathological biological processes. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved to release soluble TNF-α protein: both are biologically active. Secreted TNF-α signals through TNF receptors and the membrane-bound TNF-α acts by cell contact-dependent signaling. Anti-TNF-α antibodies have been used effectively for treatment of chronic inflammation, however with adverse side effects. Thus, there is a need for new anti-TNF-α small molecule compounds. Anti-TNF-α activity assays involve treatment of keratinocytes with exogenous TNF-α before or after anti-TNF-α incubation. However, this model fails to address the dual signaling of TNF-α. Here we describe a Doxycycline (Dox)-inducible TNF-α (HaCaT-TNF-α) expression system in keratinocytes. Using this in-vitro model, we show cell inhibition and induced expression of pro-inflammatory cytokines and markers, including IL-1β, IL-6, IL-8, NF-κB1, and KRT-16, similar to cells treated with exogenous TNF-α. Sufficient secreted TNF-α produced also activated IL-1β and IL-8 expression in wt HaCaT cells. Importantly, stimulated expression of IL-1β and IL-8 in HaCaT-TNF-α were blocked by Quercetin, a flavanol shown to possess anti-TNF-α activities. This novel in vitro cell model provides an efficient tool to investigate the dual signaling of TNF-α. Importantly, this model provides an effective, fast, and simple screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies. PMID:27415000

  18. Keratinocytes contribute intrinsically to psoriasis upon loss of Tnip1 function.

    PubMed

    Ippagunta, Sirish K; Gangwar, Ruchika; Finkelstein, David; Vogel, Peter; Pelletier, Stephane; Gingras, Sebastien; Redecke, Vanessa; Häcker, Hans

    2016-10-11

    Psoriasis is a chronic inflammatory skin disease with a clear genetic contribution, characterized by keratinocyte proliferation and immune cell infiltration. Various closely interacting cell types, including innate immune cells, T cells, and keratinocytes, are known to contribute to inflammation. Innate immune cells most likely initiate the inflammatory process by secretion of IL-23. IL-23 mediates expansion of T helper 17 (Th17) cells, whose effector functions, including IL-17A, activate keratinocytes. Keratinocyte activation in turn results in cell proliferation and chemokine expression, the latter of which fuels the inflammatory process through further immune cell recruitment. One question that remains largely unanswered is how genetic susceptibility contributes to this process and, specifically, which cell type causes disease due to psoriasis-specific genetic alterations. Here we describe a mouse model based on the human psoriasis susceptibility locus TNIP1, also referred to as ABIN1, whose gene product is a negative regulator of various inflammatory signaling pathways, including the Toll-like receptor pathway in innate immune cells. We find that Tnip1-deficient mice recapitulate major features of psoriasis on pathological, genomic, and therapeutic levels. Different genetic approaches, including tissue-specific gene deletion and the use of various inflammatory triggers, reveal that Tnip1 controls not only immune cells, but also keratinocyte biology. Loss of Tnip1 in keratinocytes leads to deregulation of IL-17-induced gene expression and exaggerated chemokine production in vitro and overt psoriasis-like inflammation in vivo. Together, the data establish Tnip1 as a critical regulator of IL-17 biology and reveal a causal role of keratinocytes in the pathogenesis of psoriasis.

  19. Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing

    PubMed Central

    1995-01-01

    The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. PMID:7537276

  20. Infection of murine keratinocytes with herpes simplex virus type 1 induces the expression of interleukin-10, but not interleukin-1α or tumour necrosis factor-α

    PubMed Central

    Zak-Prelich, Malgorzata; Halliday, Katrina E; Walker, Craig; Yates, Catherine M; Norval, Mary; Mckenzie, Roddie C

    2001-01-01

    Herpes simplex virus (HSV) is known to possess several mechanisms whereby it can evade the normal host immune defences. In this study the expression of the immunosuppressive cytokine, interleukin (IL)-10, was monitored following infection of a murine keratinocyte cell line (PAM-212) and compared with the expression of two proinflammatory cytokines: IL-1α and tumour necrosis factor (TNF)-α. The PAM-212 cells were infected at a multiplicity of 0·5 with a clinical isolate of HSV type 1, and the mRNA of the three cytokines was assessed by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) over the following 24 hr. By 12 hr postinfection the amount of IL-10 mRNA had increased significantly to five-fold greater than that found in uninfected cells (P < 0·01), and this elevated level was maintained until at least 24 hr postinfection. In contrast, IL-1α and TNF-α mRNAs were not significantly up-regulated by the HSV infection. Immunostaining with an IL-10 monoclonal antibody (mAb) revealed that cytoplasmic IL-10 protein had increased by 6–12 hr postinfection. This quantity was further increased at 24 hr postinfection, when the viral cytopathic effect was apparent. Viral replication was necessary, but not sufficient on its own, for IL-10 induction. Experiments with HSV mutants lacking functional transactivating factors suggested that the viral transactivating proteins ICP-0 and VP-16 may be necessary for HSV-induced IL-10 expression. Thus, the up-regulation in the expression of IL-10 mRNA and protein induced by HSV early in the infection of keratinocytes represents a specific response and may be part of the viral strategy to avoid local immune defence mechanisms in the skin. PMID:11899434

  1. STAT3-dependent effects of IL-22 in human keratinocytes are counterregulated by sirtuin 1 through a direct inhibition of STAT3 acetylation.

    PubMed

    Sestito, Rosanna; Madonna, Stefania; Scarponi, Claudia; Cianfarani, Francesca; Failla, Cristina M; Cavani, Andrea; Girolomoni, Giampiero; Albanesi, Cristina

    2011-03-01

    IL-22 has a pathogenetic role in psoriasis, where it is responsible for the altered proliferation and differentiation of keratinocytes and induces inflammatory molecules. The IL-22-induced effects are mediated by STAT3, whose activity is proportional to acetylation in lysine (Lys)685 and phosphorylation in tyrosine (Tyr)705. Lys 685 acetylation of STAT3 is inhibited by sirtuin (SIRT)1, a class III deacetylase promoting keratinocyte differentiation. Due to the opposite effects of IL-22 and SIRT1, we investigated whether IL-22-induced effects in keratinocytes could be regulated by SIRT1 through control of STAT3. We found that SIRT1 opposes the IL-22-induced STAT3 activity by deacetylating STAT3 and reducing STAT3 Tyr705 phosphorylation. By controlling STAT3, SIRT1 also influences the IL-22-induced expression of molecules involved in proliferation and inflammation as well as proliferation and migration processes in cultured keratinocytes. Although SIRT1 levels were similar in keratinocytes of healthy individuals and patients with psoriasis, they were reduced in psoriatic skin lesions, with the lymphokine IFN-γ inhibiting SIRT1 expression. Concomitantly, IFN-γ enhanced basal acetylation of STAT3 and its phosphorylation induced by IL-22. In conclusion, STAT3-dependent IL-22 signaling and effects in keratinocytes are negatively regulated by SIRT1. In skin affected by psoriasis, SIRT1 is down-regulated by IFN-γ, which thus renders psoriatic keratinocytes more prone to respond to IL-22.

  2. Altered gene expression correlates with DNA structure.

    PubMed

    Kohwi, Y; Kohwi-Shigematsu, T

    1991-12-01

    We examined the participation of triplex DNA structure in gene regulation using a poly(dG)-poly(dC) sequence as a model. We show that a poly(dG)-poly(dC) sequence, which can adopt an intramolecular dG.dG.dC triplex under superhelical strain, strongly augments gene expression when placed 5' to a promoter. The activity of this sequence exhibits a striking length dependency: dG tracts of 27-30 bp augment the expression of a reporter gene to a level comparable to that observed with the polyoma enhancer in mouse LTK- cells, whereas tracts of 35 bp and longer have virtually no effect. A supercoiled plasmid containing a dG tract of 30 bp competes in vivo for a trans-acting factor as revealed by reduction in the reporter gene transcription driven by the (dG)29/promoter of the test plasmid, while dGs of 35 bp and longer in the competition plasmid failed to compete. In purified supercoiled plasmid DNA at a superhelical density of -0.05, dG tracts of 32 bp and longer form a triplex, whereas those of 30 bp and shorter remain double-stranded under a PBS solution. These results suggest that a localized superhelical strain can exist, at least transiently, in mouse LTK- cells, and before being relaxed by topoisomerases this rapidly induces dG tracts of 35 bp and longer to adopt a triplex preventing the factor from binding. Thus, these data suggest that a poly(dG)-poly(dC) sequence can function as a negative regulator by adopting an intramolecular triple helix structure in vivo.

  3. 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

  4. 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

    2015-06-24

    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.

  5. High Levels of EBV-Encoded RNA 1 (EBER1) Trigger Interferon and Inflammation-Related Genes in Keratinocytes Expressing HPV16 E6/E7

    PubMed Central

    Aromseree, Sirinart; Middeldorp, Jaap M.; Pientong, Chamsai; van Eijndhoven, Monique; Ramayanti, Octavia; Lougheed, Sinéad M.; Pegtel, D. Michiel; Steenbergen, Renske D. M.; Ekalaksananan, Tipaya

    2017-01-01

    Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV–modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or

  6. Functional analysis of keratinocyte and fibroblast gene expression in skin and keloid scar tissue based on deviation analysis of dynamic capabilities

    PubMed Central

    Li, Mingming; Wu, Lei

    2016-01-01

    The aim of the present study was to select key genes that are associated with fibroblasts and keratinocytes during keloid scar progression and development. The gene expression profile of GSE44270, which includes 32 samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) in case samples compared with control samples were screened using the Limma R package followed by hierarchical clustering analysis. Protein-protein interaction (PPI) networks of the total selected DEGs were constructed using Cytoscape. Moreover, the Gene Ontology biological processes and significant Kyoto Encyclopedia of Genes and Genomes pathways of the total selected DEGs were enriched using the Database for Annotation, Visualization and Integrated Discovery. Significant pathways that may be associated with keloid scar were analyzed using deviation analysis of dynamic capabilities. There were 658 DEGs in fibroblast keloid vs. normal, 112 DEGs in fibroblast non-lesion vs. normal, 439 DEGs in fibroblast keloid vs. non-lesion, 523 DEGs in keratocyte keloid vs. normal, 186 DEGs in keratocyte non-lesion vs. normal, and 963 DEGs in keratocyte keloid vs. non-lesion groups. HOXA9, BMP4, CDKN1A and SMAD2 in fibroblasts, and HOXA7, MCM8, PSMA4 and PSMB2 in keratinocytes were key genes in the PPI networks. Moreover, the amino sugar and nucleotide sugar metabolism pathway, cell cycle, and extracellular matrix (ECM)-receptor interaction pathway were significant pathways. This study suggests that several key genes (BMP4, HOXA9, SMAD2, CDKN1A, HOXA7, PSMA4 and PSMB2) that participate in some significant pathways (cell cycle and ECM-receptor interaction pathways) may be potential therapeutic targets for keloid scars. PMID:28101157

  7. Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70B′ in human keratinocytes modulating the proinflammatory response

    PubMed Central

    Buommino, Elisabetta; Schiraldi, Chiara; Baroni, Adone; Paoletti, Iole; Lamberti, Monica; De Rosa, Mario; Tufano, Maria Antonietta

    2005-01-01

    The heat shock proteins (Hsps) have an important role in the cytoprotection and repair of cells and tissues. One potential mechanism of protection is the ability of Hsp to inhibit genetic expression of proinflammatory cytokines, the transcription of which is dependent on nuclear factor–kappa B (NF-κB) activation. In this study, we evaluated the ability of ectoine, a novel natural biomolecule produced by halophilic microorganisms, to activate the hsp70 and hsp70B′. By reverse transcriptase–polymerase chain reaction and Western blot analysis, we demonstrated increased hsp70B′ gene expression in human keratinocytes treated with ectoine and heat stressed. In contrast, in the absence of heat shock, ectoine was unable to induce hsp70B′ but had the ability to induce another member of the Hsp family, the hsp70. The latter is not only elevated in response to stress but is also present at basal level in unstressed cells. In addition, ectoine had no effect on proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor–alpha and on NF-κB and IκB-α pathway, whereas it downregulated the expression of cited proinflammatory cytokines, in lipopolysaccharides-treated keratinocytes. These results highlighted the ability of ectoine to protect cells from stress conditions and to prevent cell damage by maintaining an elevated level of the Hsp70. Overall, these data might suggest the use of this compatible solute in cosmetic and even pharmaceutical preparations aiming to activate a cytoprotective heat shock response in human cells. PMID:16184764

  8. Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70B' in human keratinocytes modulating the proinflammatory response.

    PubMed

    Buommino, Elisabetta; Schiraldi, Chiara; Baroni, Adone; Paoletti, Iole; Lamberti, Monica; De Rosa, Mario; Tufano, Maria Antonietta

    2005-01-01

    The heat shock proteins (Hsps) have an important role in the cytoprotection and repair of cells and tissues. One potential mechanism of protection is the ability of Hsp to inhibit genetic expression of proinflammatory cytokines, the transcription of which is dependent on nuclear factor-kappa B (NF-kappaB) activation. In this study, we evaluated the ability of ectoine, a novel natural biomolecule produced by halophilic microorganisms, to activate the hsp70 and hsp70B'. By reverse transcriptase-polymerase chain reaction and Western blot analysis, we demonstrated increased hsp70B' gene expression in human keratinocytes treated with ectoine and heat stressed. In contrast, in the absence of heat shock, ectoine was unable to induce hsp70B' but had the ability to induce another member of the Hsp family, the hsp70. The latter is not only elevated in response to stress but is also present at basal level in unstressed cells. In addition, ectoine had no effect on proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor-alpha and on NF-kappaB and IkappaB-alpha pathway, whereas it downregulated the expression of cited proinflammatory cytokines, in lipopolysaccharides-treated keratinocytes. These results highlighted the ability of ectoine to protect cells from stress conditions and to prevent cell damage by maintaining an elevated level of the Hsp70. Overall, these data might suggest the use of this compatible solute in cosmetic and even pharmaceutical preparations aiming to activate a cytoprotective heat shock response in human cells.

  9. Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers.

    PubMed

    Lao, Victoria Valinluck; Welcsh, Piri; Luo, Yanxin; Carter, Kelly T; Dzieciatkowski, Slavomir; Dintzis, Suzanne; Meza, Jane; Sarvetnick, Nora E; Monnat, Raymond J; Loeb, Lawrence A; Grady, William M

    2013-08-01

    Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.

  10. Interferon regulatory factor 6 regulates keratinocyte migration

    PubMed Central

    Biggs, Leah C.; Naridze, Rachelle L.; DeMali, Kris A.; Lusche, Daniel F.; Kuhl, Spencer; Soll, David R.; Schutte, Brian C.; Dunnwald, Martine

    2014-01-01

    ABSTRACT Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration. PMID:24777480

  11. Rictor/mTORC2 deficiency enhances keratinocyte stress tolerance via mitohormesis.

    PubMed

    Tassone, Beatrice; Saoncella, Stefania; Neri, Francesco; Ala, Ugo; Brusa, Davide; Magnuson, Mark A; Provero, Paolo; Oliviero, Salvatore; Riganti, Chiara; Calautti, Enzo

    2017-04-01

    How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.

  12. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    PubMed

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization.

  13. Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

    SciTech Connect

    Seo, MiRan; Juhnn, Yong-Sung

    2010-03-05

    Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.

  14. 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.

  15. Caterpillar labial saliva alters tomato plant gene expression.

    PubMed

    Musser, Richard O; Hum-Musser, Sue M; Lee, Henry K; DesRochers, Brittany L; Williams, Spencer A; Vogel, Heiko

    2012-11-01

    We examined the effects of Helicoverpa zea caterpillar labial saliva on tomato plant gene expression. Caterpillars with labial salivary glands (mock-ablated) and without (ablated) were fed on tomato plants for 24 hr; then, the leaf mRNA was analyzed with tomato microarrays. Analysis of the transcript profiles revealed 384 expressed sequence tags (ESTs) that were significantly altered due to herbivory compared to the non-wounded plants. The majority of the ESTs were quantitatively altered more so by mock-ablated caterpillars with labial salivary glands than ablated caterpillars. Particularly notable, ESTs encoding acid phosphatase, arginase, acidic endochitinase, dehydrin, polyphenol oxidase, protease inhibitors, and threonine deaminase were more highly stimulated by mock-ablated caterpillars than ablated caterpillars. In addition, tomato leaves were mechanically wounded with scissors and painted with labial salivary gland extract, autoclaved salivary gland extract, or water, and compared to non-wounded tomato plants. After 4 hr, these leaves were collected and a tomato microarray analysis of the mRNA revealed correlation of the gene expression of these leaves altered by mechanical wounding and painted with salivary gland extract to the gene expression of leaves fed on by mock-ablated caterpillars. We show that caterpillar labial saliva is an important component of herbivory that can alter plant gene expression.

  16. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes.

    PubMed

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B; Rivkees, Scott A; Wendler, Christopher C

    2014-12-15

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20-60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3-65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes.

  17. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    PubMed Central

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  18. Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-γ - a method for qRT-PCR reference gene determination.

    PubMed

    Riemer, Angelika B; Keskin, Derin B; Reinherz, Ellis L

    2012-08-01

    Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-γ was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-γ-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.

  19. Temporal variations in sirtuin expression under normal and ultraviolet B-induced conditions and their correlation to energy levels in normal human epidermal keratinocytes.

    PubMed

    Pelle, Edward; Dong, Kelly; Pernodet, Nadine

    2015-01-01

    Sirtuins are post-translational modifiers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identified as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the temporal variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sirtuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. Moreover, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show significant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB-induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products.

  20. Altering sensorimotor feedback disrupts visual discrimination of facial expressions.

    PubMed

    Wood, Adrienne; Lupyan, Gary; Sherrin, Steven; Niedenthal, Paula

    2016-08-01

    Looking at another person's facial expression of emotion can trigger the same neural processes involved in producing the expression, and such responses play a functional role in emotion recognition. Disrupting individuals' facial action, for example, interferes with verbal emotion recognition tasks. We tested the hypothesis that facial responses also play a functional role in the perceptual processing of emotional expressions. We altered the facial action of participants with a gel facemask while they performed a task that involved distinguishing target expressions from highly similar distractors. Relative to control participants, participants in the facemask condition demonstrated inferior perceptual discrimination of facial expressions, but not of nonface stimuli. The findings suggest that somatosensory/motor processes involving the face contribute to the visual perceptual-and not just conceptual-processing of facial expressions. More broadly, our study contributes to growing evidence for the fundamentally interactive nature of the perceptual inputs from different sensory modalities.

  1. Candida albicans phospholipomannan triggers inflammatory responses of human keratinocytes through Toll-like receptor 2.

    PubMed

    Li, Min; Chen, Qing; Shen, Yongnian; Liu, Weida

    2009-07-01

    The Toll-like receptors (TLRs) play an important role in the recognition of Candida albicans components and activation of innate immunity. Phospholipomannan (PLM), a glycolipid, is expressed at the surface of C. albicans cell wall, which acts as a member of the pathogen-associated molecular patterns family. In this study, we sought to clarify whether C. albicans-native PLM could induce an inflammation response in human keratinocytes and to determine the underlying mechanisms. Exposure of cultured human primary keratinocytes to PLM led to the increased gene expression and secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8). PLM hydrolysed with beta-d-mannoside mannohydrolase failed to induce gene expression and secretion of IL-6 and IL-8. PLM up-regulated the mRNA and protein levels of TLR2, whereas the mRNA level of TLR4 was not altered. Keratinocytes challenged with PLM resulted in the activation of NF-kappaB and mitogen-activated protein kinase (MAPKs) including p38. Anti-TLR2 neutralizing antibody, NFkappaB and p38MAPK inhibitors blocked the PLM-induced secretion of IL-6, IL-8 in keratinocytes, but no such effect was observed in pretreatment with anti-TLR4-neutralizing antibody and lipopolysaccharide inhibitor (polymyxin B). These data suggest C. albicans-native PLM may contribute to the inflammatory responses of cutaneous candidiasis in the TLR2-NF-kappaB and p38MAPK signalling pathway dependent manner.

  2. Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes.

    PubMed

    Tucker, Dana K; Stahley, Sara N; Kowalczyk, Andrew P

    2014-04-01

    Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.

  3. Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation and epidermal organelles

    PubMed Central

    Borkowski, Andrew W.; Park, Kyungho; Uchida, Yoshikazu; Gallo, Richard L.

    2013-01-01

    Injury to the skin, and the subsequent release of non-coding double-stranded RNA from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). Since changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. Double-stranded RNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with double-stranded RNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by oil-red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that double-stranded RNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance. PMID:23353987

  4. Expression of the human papillomavirus type 16 E7 oncoprotein induces an autophagy-related process and sensitizes normal human keratinocytes to cell death in response to growth factor deprivation

    SciTech Connect

    Zhou Xiaobo; Muenger, Karl

    2009-03-01

    Expression of oncogenes, such as the human papillomavirus type 16 (HPV16) E7 oncoprotein, promotes aberrant cell proliferation. In the absence of concurrent mitogenic stimuli, this triggers a cell-intrinsic defense mechanism, the 'trophic sentinel response', which eliminates such aberrant cells. The molecular pathways that elicit this response, however, remain obscure. We set up an experimental system to investigate the trophic sentinel pathway triggered by HPV16 E7 expression in normal human keratinocytes, the natural host cells of HPVs. Keratinocytes expressing HPV16 E7 cultured in E-medium undergo cell death and show increased sub-G1 DNA content when grown to confluence or under conditions of serum deprivation. Moreover, HPV16 E7 expressing human keratinocytes express higher levels of the autophagy marker, LC3-II, which can be abrogated by 3-methyladenine, an autophagy inhibitor. These findings indicate that even under normal culture conditions, HPV16 E7 expression triggers metabolic stress that may result in autophagy, a pathway implicated in carcinogenesis.

  5. Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

    PubMed

    Wang, Yuexia; Ostlund, Cecilia; Worman, Howard J

    2010-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

  6. Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation.

    PubMed

    Monfrecola, G; Gaudiello, F; Cirillo, T; Fabbrocini, G; Balato, A; Lembo, S

    2013-03-01

    Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

  7. Expression of IL-10, TGF-β1 and TNF-α in Cultured Keratinocytes (HaCaT Cells) after IPL Treatment or ALA-IPL Photodynamic Treatment

    PubMed Central

    Byun, Ji Yeon; Choi, Hae Young; Myung, Ki Bum

    2009-01-01

    Background Depending on the light dose and concentration of photosensitizer for photodynamic treatment (PDT), a multitude of dose-related events are demonstrable in PDT-treated cells. Sublethal doses may result in the alteration of cytokine release and consequently modify immune actions, rather than cause cell death. Objective The purpose of this study was to investigate cytokine expression in cultured HaCaT cells after intense pulse light (IPL) treatment or PDT utilizing 5-aminolevulinic acid (ALA) and IPL at sublethal doses. Methods Cultured HaCaT cells were treated with either IPL only (4, 8 and 12 J/cm2) or ALA-IPL PDT (100µmol/L of ALA; 0, 4, 8, and 12 J/cm2 of IPL). The expression of IL-10, TGF-β1 and TNF-α was investigated by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Results IL-10 protein increased up to 5.95-fold after IPL treatment and up to 2.85-fold after PDT. TGF-β1 mRNA and protein showed slight increases after both IPL treatment and PDT, of which the latter induced slightly larger increases. TNF-α mRNA and protein showed no induction or reduction after PDT. Conclusion Increased expressions of IL-10 and TGF-β1 was observed after PDT. The induction of IL-10 may contribute to the anti-inflammatory effect, which explains the therapeutic benefit of PDT for inflammatory dermatoses, and that of TGF-β1 may be related to the therapeutic effect for psoriasis. The finding that IL-10 induction was more marked after IPL treatment than after PDT suggests that other mechanisms than IL-10 induction in keratinocytes after PDT may participate in the anti-inflammatory effect of PDT. PMID:20548849

  8. Inhibition of JNK promotes differentiation of epidermal keratinocytes.

    PubMed

    Gazel, Alix; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav

    2006-07-21

    In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

  9. Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

    SciTech Connect

    Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.; Speed, Terence P.; Rubin, Edward M.

    2000-05-05

    Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.

  10. Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes.

    PubMed

    Zhang, Qihao; Zhang, Lei; Xiao, Xue; Su, Zhijian; Zou, Ping; Hu, Hao; Huang, Yadong; He, Qing-Yu

    2010-06-01

    Heavy metals may exert their acute and chronic effects on the human skin through stress signals. In the present study, 2DE-based proteomics was used to analyze the protein expression in human keratinocytes exposed to heavy metals, chromium and neodymium, and 10 proteins with altered expression were identified. Among these proteins, small heat shock protein 27 (HSP27) was up-regulated significantly and the up-regulation was validated by Western blot and immunofluorescence. In addition, the mRNA expression level of HSP27 markedly increased as detected by quantitative PCR. More interestingly, the ratio of phosphorylated HSP27 and total HSP27 significantly decreased in keratinocytes treated with the heavy metals. These findings suggested that heavy metals reduced the phosphorylation level of HSP27, and that the ratio of p-HSP27 and HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

  11. Regulation of caspase 14 expression in keratinocytes by inflammatory cytokines--a possible link between reduced skin barrier function and inflammation?

    PubMed

    Hvid, Malene; Johansen, Claus; Deleuran, Bent; Kemp, Kaare; Deleuran, Mette; Vestergaard, Christian

    2011-08-01

    Caspase 14 is a unique member of the cysteinyl aspartate-specific proteinase family. Its expression is confined primarily to cornified epithelium such as the skin. Caspase 14 has been associated with the processing of filaggrin monomers and the development of natural moisturising factors of the skin, and thus, it could be speculated that caspase 14 dysregulation is implicated in the development of an impaired skin barrier function. We have investigated the regulation of caspase 14 transcription in cultured primary keratinocytes following stimulation with a number of factors present in inflamed skin, including T(H)1- and T(H)2-associated cytokines in addition to LPS and peptidoglycan. In particular, we found that T(H)2-associated cytokines reduced the caspase 14 mRNA level significantly. Furthermore, we found that the expression of caspase 14 was reduced in skin biopsies from patients with atopic dermatitis (AD), psoriasis and contact dermatitis, further supporting a role for this kinase in inflammatory skin conditions. Hence, the regulation of caspase 14 levels provides a possible link between impaired skin barrier function and inflammatory reactions in skin diseases such as AD and may offer an explanation to the skin barrier dysfunction in inflamed skin lesions.

  12. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing.

    PubMed

    Jettanacheawchankit, Suwimon; Sasithanasate, Siriruk; Sangvanich, Polkit; Banlunara, Wijit; Thunyakitpisal, Pasutha

    2009-04-01

    Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

  13. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

    PubMed

    Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

    2000-03-01

    Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

  14. Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

    PubMed Central

    Thuillier, Philippe; Brash, Alan R; Kehrer, James P; Stimmel, Julie B; Leesnitzer, Lisa M; Yang, Peiying; Newman, Robert A; Fischer, Susan M

    2002-01-01

    Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression. PMID:12069687

  15. 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

  16. Altered nestin expression in the cerebrum with periventricular leukomalacia.

    PubMed

    Okoshi, Yumi; Mizuguchi, Masashi; Itoh, Masayuki; Oka, Akira; Takashima, Sachio

    2007-03-01

    Nestin is a cytoskeletal protein expressed by neural stem cells, and by immature neurons and glial cells. In an effort to explore the potential of the infant brain for repair and plasticity, we immunohistochemically studied nestin expression in the human cerebral cortex of control subjects and of patients with periventricular leukomalacia. During normal development, nestin immunoreactivity of the cortical gray and white matter was detectable throughout the fetal period, and disappeared around birth. In brain with periventricular leukomalacia, nestin expression was altered in a time- and space-dependent manner. In the cortical gray matter, neuronal immunoreactivity was often reduced in the subacute stage, but was increased in chronic and remote stages. In the white matter near a lesion of periventricular leukomalacia, glial immunoreactivity was increased in all stages. In many cases, neurons and axons far from a lesion also showed an altered expression of nestin. These findings indicate that in brain with periventricular leukomalacia, neurons and glial cells may recapitulate nestin expression in response to ischemic brain injury, suggesting functional relevance in repair and plasticity.

  17. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  18. The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice.

    PubMed

    Tiala, Inkeri; Wakkinen, Janica; Suomela, Sari; Puolakkainen, Pauli; Tammi, Raija; Forsberg, Sofi; Rollman, Ola; Kainu, Kati; Rozell, Björn; Kere, Juha; Saarialho-Kere, Ulpu; Elomaa, Outi

    2008-04-01

    The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.

  19. Airway Epithelial miRNA Expression Is Altered in Asthma

    PubMed Central

    Solberg, Owen D.; Ostrin, Edwin J.; Love, Michael I.; Peng, Jeffrey C.; Bhakta, Nirav R.; Nguyen, Christine; Solon, Margaret; Nguyen, Cindy; Barczak, Andrea J.; Zlock, Lorna T.; Blagev, Denitza P.; Finkbeiner, Walter E.; Ansel, K. Mark; Arron, Joseph R.; Erle, David J.

    2012-01-01

    Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13–regulated miRNAs. Methods: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway

  20. 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

  1. Plasmodium infection alters Anopheles gambiae detoxification gene expression

    PubMed Central

    2010-01-01

    Background Anopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquito's innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues. Results Using a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large number of P450s were down-regulated at this stage. In the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel. While most gene expression changes were tissue-related, CYP6M2, a CYP previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion. Conclusions Most toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. Strong up-regulation of CYP6M2 in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification. PMID:20482856

  2. Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

    PubMed Central

    Walch-Rückheim, Barbara; Wickenhauser, Claudia; Doorbar, John; Pfister, Herbert; Malejczyk, Magdalena; Majewski, Sławomir; Keates, Andrew C.; Smola, Sigrun

    2012-01-01

    Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity. PMID:22911498

  3. N-Acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191): an anti-inflammatory molecule that increases the expression of the aquaglyceroporin, aquaporin-3, in human keratinocytes.

    PubMed

    Fernández, José R; Webb, Corey; Rouzard, Karl; Voronkov, Michael; Huber, Kristen L; Stock, Jeffry B; Stock, Maxwell; Gordon, Joel S; Perez, Eduardo

    2017-03-01

    Isoprenylcysteine (IPC) small molecules were discovered as signal transduction modulating compounds ~25 years ago. More recently, IPC molecules have demonstrated antioxidant and anti-inflammatory properties in a variety of dermal cells as well as antimicrobial activity, representing a novel class of compounds to ameliorate skin conditions and disease. Here, we demonstrate a new IPC compound, N-acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191), which inhibits UVB-induced inflammation blocking pro-inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) production. To investigate further the previously reported hydrating potential of IPC compounds, SIG-1191 was tested for its ability to modulate aquaporin expression. Specifically, aquaporin 3 (AQP3) the most abundant aquaporin found in skin has been reported to play a key role in skin hydration, elasticity and barrier repair. Results show here for the first time that SIG-1191 increases AQP3 expression in both cultured normal human epidermal keratinocytes as well as when applied topically in a three-dimensional (3D) reconstructed human skin equivalent. Additionally, SIG-1191 dose dependently increased AQP3 protein levels, as determined by specific antibody staining, in the epidermis of the 3D skin equivalents. To begin to elucidate which signaling pathways SIG-1191 may be modulating to increase AQP3 levels, we used several pharmacological pathway inhibitors and determined that AQP3 expression is mediated by the Mitogen-activated protein kinase/Extracellular signal-regulated kinase kinase (MEK) pathway. Altogether, these data suggest SIG-1191 represents a new IPC derivative with anti-inflammatory activity that may also promote increased skin hydration based on its ability to increase AQP3 levels.

  4. Analysis of the response of human keratinocytes to Malassezia globosa and restricta strains.

    PubMed

    Donnarumma, Giovanna; Perfetto, Brunella; Paoletti, Iole; Oliviero, Giovanni; Clavaud, Cécile; Del Bufalo, Aurelia; Guéniche, Audrey; Jourdain, Roland; Tufano, Maria Antonietta; Breton, Lionel

    2014-10-01

    Malassezia spp. are saprophyte yeasts involved in skin diseases with different degrees of severity. The aim of our study was to analyze the response of human epidermal keratinocytes to Malassezia globosa and restricta strains evaluating the host defence mechanisms induced by Malassezia spp. colonization. Our results showed a different modulation of the inflammatory and immunomodulatory cytokine pathways obtained with the different strains of Malassezia tested. In addition, this expression is altered by blocking the TLR2 receptor. In comparison with M. furfur, M. globosa and restricta displayed an unexpected and striking cytotoxicity on keratinocytes. The differences observed could be related to the different modalities of interaction between keratinocytes and Malassezia strains, but also to their growth condition. Taken together, these results indicate that M. globosa or M. restricta colonization exert a different control on the cytokine inflammatory response activated in the human keratinocyte in which TLR2 might be involved. M. globosa and M. restricta may play a synergistic role in the exacerbation of skin diseases in which both are found.

  5. Expression alterations define unique molecular characteristics of spinal ependymomas

    PubMed Central

    Lourdusamy, Anbarasu; Rahman, Ruman; Grundy, Richard G.

    2015-01-01

    Ependymomas are glial tumors that originate in either intracranial or spinal regions. Although tumors from different regions are histologically similar, they are biologically distinct. We therefore sought to identify molecular characteristics of spinal ependymomas (SEPN) in order to better understand the disease biology of these tumors. Using gene expression profiles of 256 tumor samples, we identified increased expression of 1,866 genes in SEPN when compared to intracranial ependymomas. These genes are mainly related to anterior/posterior pattern specification, response to oxidative stress, glial cell differentiation, DNA repair, and PPAR signalling, and also significantly enriched with cellular senescence genes (P = 5.5 × 10−03). In addition, a high number of significantly down-regulated genes in SEPN are localized to chromosome 22 (81 genes from chr22: 43,325,255 – 135,720,974; FDR = 1.77 × 10−23 and 22 genes from chr22: 324,739 – 32,822,302; FDR = 2.07 × 10−09) including BRD1, EP300, HDAC10, HIRA, HIC2, MKL1, and NF2. Evaluation of NF2 co-expressed genes further confirms the enrichment of chromosome 22 regions. Finally, systematic integration of chromosome 22 genes with interactome and NF2 co-expression data identifies key candidate genes. Our results reveal unique molecular characteristics of SEPN such as altered expression of cellular senescence and chromosome 22 genes. PMID:25909290

  6. Radiation Exposure Alters Expression of Metabolic Enzyme Genes In Mice

    NASA Technical Reports Server (NTRS)

    Wotring, Virginia E.; Mangala, L. S.; Zhang, Y.; Wu, H.

    2010-01-01

    Most pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Because of the importance of the liver in drug metabolism it is important to understand the effects of spaceflight on the enzymes of the liver. Exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. This study is an effort to examine the effects of adaptive mechanisms that may be triggered by early exposure to low radiation doses. Using procedures approved by the JSC Animal Care & Use Committee, C57 male mice were exposed to Cs-137 in groups: controls, low dose (50 mGy), high dose (6Gy) and a fourth group that received both radiation doses separated by 24 hours. Animals were anesthetized and sacrificed 4 hours after their last radiation exposure. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted and purified (Absolutely RNA, Agilent). Quality of RNA samples was evaluated (Agilent Bioanalyzer 2100). Complementary DNA was prepared from high-quality RNA samples, and used to run RT-qPCR screening arrays for DNA Repair and Drug Metabolism (SuperArray, SABiosciences/Qiagen; BioRad Cfx96 qPCR System). Of 91 drug metabolism genes examined, expression of 7 was altered by at least one treatment condition. Genes that had elevated expression include those that metabolize promethazine and steroids (4-8-fold), many that reduce oxidation products, and one that reduces heavy metal exposure (greater than 200-fold). Of the 91 DNA repair and general metabolism genes examined, expression of 14 was altered by at least one treatment condition. These gene expression changes are likely homeostatic and could lead to development of new radioprotective countermeasures.

  7. Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

    PubMed

    Sobel, Katrin; Tham, Marius; Stark, Hans-Jürgen; Stammer, Hermann; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-06-15

    Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

  8. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  9. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure.

    PubMed

    Huang, S; Guo, S; Guo, F; Yang, Q; Xiao, X; Murata, M; Ohnishi, S; Kawanishi, S; Ma, N

    2013-01-14

    Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm) in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

  10. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  11. Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

    PubMed

    Smulian, A George; Gibbons, Reta S; Demland, Jeffery A; Spaulding, Deborah T; Deepe, George S

    2007-11-01

    The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

  12. Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

    PubMed

    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.

  13. Activation of the δ-opioid receptor promotes cutaneous wound healing by affecting keratinocyte intercellular adhesion and migration

    PubMed Central

    Bigliardi, P L; Neumann, C; Teo, Y L; Pant, A; Bigliardi-Qi, M

    2015-01-01

    BACKGROUND AND PURPOSE In addition to its analgesic functions, the peripheral opioid receptor system affects skin homeostasis by influencing cell differentiation, migration and adhesion; also, wound healing is altered in δ-opioid receptor knockout mice (DOPr–/–). Hence, we investigated δ-opioid receptor effects on the expression of several proteins of the desmosomal junction complex and on the migratory behaviour of keratinocytes. EXPERIMENTAL APPROACH Expression levels of desmosomal cadherins in wild-type and DOPr–/– mice, and the morphology of intercellular adhesion in human keratinocytes were analysed by immunofluorescence. To investigate the δ-opioid receptor activation pathway, protein expression was studied using Western blot and its effect on cellular migration determined by in vitro live cell migration recordings from human keratinocytes. KEY RESULTS Expression of the desmosomal cadherins, desmogleins 1 and 4, was up-regulated in skin from DOPr–/– mice, and down-regulated in δ-opioid receptor-overexpressing human keratinocytes. The localization of desmoplakin expression was rearranged from linear arrays emanating from cell borders to puncta in cell periphery, resulting in less stable intercellular adhesion. Migration and wound recovery were enhanced in human keratinocyte monolayers overexpressing δ-opioid receptors in vitro. These δ-opioid receptor effects were antagonized by specific PKCα/β inhibition indicating they were mediated through the PKC signalling pathway. Finally, cells overexpressing δ-opioid receptors developed characteristically long but undirected protrusions containing filamentous actin and δ-opioid receptors, indicating an enhanced migratory phenotype. CONCLUSION AND IMPLICATIONS Opioid receptors affect intercellular adhesion and wound healing mechanisms, underlining the importance of a cutaneous neuroendocrine system in wound healing and skin homeostasis. LINKED ARTICLES This article is part of a themed section on

  14. IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes.

    PubMed

    Wang, Xiaoyong; Bi, Zhigang; Chu, Wenming; Wan, Yinsheng

    2005-12-01

    Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.

  15. Altered representation of facial expressions after early visual deprivation

    PubMed Central

    Gao, Xiaoqing; Maurer, Daphne; Nishimura, Mayu

    2013-01-01

    We investigated the effects of early visual deprivation on the underlying representation of the six basic emotions. Using multi-dimensional scaling (MDS), we compared the similarity judgments of adults who had missed early visual input because of bilateral congenital cataracts to control adults with normal vision. Participants made similarity judgments of the six basic emotional expressions, plus neutral, at three different intensities. Consistent with previous studies, the similarity judgments of typical adults could be modeled with four underlying dimensions, which can be interpreted as representing pleasure, arousal, potency and intensity of expressions. As a group, cataract-reversal patients showed a systematic structure with dimensions representing pleasure, potency, and intensity. However, an arousal dimension was not obvious in the patient group's judgments. Hierarchical clustering analysis revealed a pattern in patients seen in typical 7-year-olds but not typical 14-year-olds or adults. There was also more variability among the patients than among the controls, as evidenced by higher stress values for the MDS fit to the patients' data and more dispersed weightings on the four dimensions. The findings suggest an important role for early visual experience in shaping the later development of the representations of emotions. Since the normal underlying structure for emotion emerges postnatally and continues to be refined until late childhood, the altered representation of emotion in adult patients suggests a sleeper effect. PMID:24312071

  16. Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-β1, FGF7, VEGF-A and IL-8 genes.

    PubMed

    Pinto, Daniela; Marzani, Barbara; Minervini, Fabio; Calasso, Maria; Giuliani, Giammaria; Gobbetti, Marco; De Angelis, Maria

    2011-09-01

    This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10μg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1 gene was under expressed in the first 4h of treatments and up-regulated after 8-24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4-24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

  17. Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression

    PubMed Central

    Elizondo-Vega, Roberto; Cortés-Campos, Christian; Barahona, María José; Carril, Claudio; Ordenes, Patricio; Salgado, Magdiel; Oyarce, Karina; García-Robles, María de los Angeles

    2016-01-01

    Hypothalamic glucosensing, which involves the detection of glucose concentration changes by brain cells and subsequent release of orexigenic or anorexigenic neuropeptides, is a crucial process that regulates feeding behavior. Arcuate nucleus (AN) neurons are classically thought to be responsible for hypothalamic glucosensing through a direct sensing mechanism; however, recent data has shown a metabolic interaction between tanycytes and AN neurons through lactate that may also be contributing to this process. Monocarboxylate transporter 1 (MCT1) is the main isoform expressed by tanycytes, which could facilitate lactate release to hypothalamic AN neurons. We hypothesize that MCT1 inhibition could alter the metabolic coupling between tanycytes and AN neurons, altering feeding behavior. To test this, we inhibited MCT1 expression using adenovirus-mediated transfection of a shRNA into the third ventricle, transducing ependymal wall cells and tanycytes. Neuropeptide expression and feeding behavior were measured in MCT1-inhibited animals after intracerebroventricular glucose administration following a fasting period. Results showed a loss in glucose regulation of orexigenic neuropeptides and an abnormal expression of anorexigenic neuropeptides in response to fasting. This was accompanied by an increase in food intake and in body weight gain. Taken together, these results indicate that MCT1 expression in tanycytes plays a role in feeding behavior regulation. PMID:27677351

  18. Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

    PubMed Central

    Singhania, Akul; Rupani, Hitasha; Jayasekera, Nivenka; Lumb, Simon; Hales, Paul; Gozzard, Neil; Davies, Donna E.

    2017-01-01

    Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy. PMID:28045928

  19. Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma.

    PubMed

    Singhania, Akul; Rupani, Hitasha; Jayasekera, Nivenka; Lumb, Simon; Hales, Paul; Gozzard, Neil; Davies, Donna E; Woelk, Christopher H; Howarth, Peter H

    2017-01-01

    Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

  20. α-Synuclein Alters Toll-Like Receptor Expression

    PubMed Central

    Béraud, Dawn; Twomey, Margaret; Bloom, Benjamin; Mittereder, Andrew; Ton, Vy; Neitzke, Katherine; Chasovskikh, Sergey; Mhyre, Timothy R.; Maguire-Zeiss, Kathleen A.

    2011-01-01

    Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern. PMID:21747756

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

    PubMed Central

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

    2017-01-01

    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/cm2) 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

  2. Human hair follicle and interfollicular keratinocyte reactivity to mouse HPV16-transformed cells: an in vitro study.

    PubMed

    Smetana, Karel; Dvoránková, Barbora; Lacina, Lukás; Cada, Zdenek; Vonka, Vladimír

    2008-07-01

    The role of stem cells in cancer formation and spreading has been established. As with normal tissue, the cancer stem cells need a special microenvironment to support their growth. This microenvironment may be represented by the tumor stroma. One of the possible ways of tumor stromal formation is the epithelial-mesenchymal transition of tumor epithelium. Following this mechanism, stromal cells must share the basic genetic alterations with the tumor cells. In an attempt to create a system capable of testing some aspects of the mesenchymal cell-keratinocyte interactions, we studied the effects of the fibroblastoid mouse TC-1 cells that were prepared by the introduction of human papillomavirus type 16 (HPV16) genes E6 and E7 to lung epithelial cells on the phenotype of normal human interfollicular and hair follicle keratinocytes. From this point of view, they may resemble stromal cells formed by the epithelial-mesenchymal transition of cells from HPV-induced squamous cell carcinoma. In contrast to 3T3 murine embryonic fibroblasts which were used as control cells, TC-1 cells influenced not only the size of the keratinocytes and the shape of their colonies, but also induced the expression of keratins 8 and 19 and vimentin. In conclusion, TC-1 cells exhibited a marked biological activity by influencing the behavior of the normal human follicular and intefollicular keratinocytes. This observation is compatible with the hypothesis that stromal cells play an important role in tumor progression and spreading.

  3. 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

  4. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

    PubMed Central

    1988-01-01

    In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation- specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long- term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation. PMID:2450098

  5. Malignant progression of an SV40-transformed human epidermal keratinocyte cell line.

    PubMed Central

    Brown, K. W.; Gallimore, P. H.

    1987-01-01

    Human foetal keratinocytes were transfected with a recombinant plasmid (pSV6-1) which contained an origin defective SV40 genome. The resulting transformed cell line had many properties in common with previously described SV40-transformed keratinocytes, including expression of simple epithelial-type keratins. It was non-tumourigenic in nude mice at early passages, forming small benign cysts, however, after approximately 46 in vitro passages, these transformed keratinocytes formed invasive squamous cell carcinomas in athymic nude mice. Several in vitro changes were associated with this acquisition of tumourigenicity (a) an alteration in cellular morphology, (b) development of a cytogenetically marked clone and (c) loss of cell surface fibronectin. The loss of fibronectin was also observed in vivo; cysts formed by SV6-1 Bam/HFK produced human fibronectin whereas tumours did not, although both tumours and cysts were laminin- and keratin-positive. These results indicate that the spontaneous development of secondary events in immortalised human cells may lead to the acquisition of a malignant phenotype. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2447927

  6. Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

    PubMed

    Matus, Carola E; Ehrenfeld, Pamela; Pavicic, Francisca; González, Carlos B; Concha, Miguel; Bhoola, Kanti D; Burgos, Rafael A; Figueroa, Carlos D

    2016-09-01

    The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

  7. The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

    PubMed

    Almeida, Stéphanie; Ryser, Stephan; Obarzanek-Fojt, Magdalena; Hohl, Daniel; Huber, Marcel

    2011-02-01

    The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

  8. Altered Endoplasmic Reticulum Calcium Pump Expression during Breast Tumorigenesis

    PubMed Central

    Papp, Béla; Brouland, Jean-Philippe

    2011-01-01

    Endoplasmic reticulum calcium homeostasis is involved in several essential cell functions including cell proliferation, protein synthesis, stress responses or secretion. Calcium uptake into the endoplasmic reticulum is performed by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). In order to study endoplasmic reticulum calcium homeostasis in situ in mammary tissue, in this work SERCA3 expression was investigated in normal breast and in its benign and malignant lesions in function of the cell type, degree of malignancy, and histological and molecular parameters of the tumors. Our data indicate, that although normal breast acinar epithelial cells express SERCA3 abundantly, its expression is strongly decreased already in very early non-malignant epithelial lesions such as adenosis, and remains low in lobular carcinomas. Whereas normal duct epithelium expresses significant amounts of SERCA3, its expression is decreased in several benign ductal lesions, as well as in ductal adenocarcinoma. The loss of SERCA3 expression is correlated with Elston-Ellis grade, negative hormone receptor expression or triple negative status in ductal carcinomas. The concordance between decreased SERCA3 expression and several histological, as well as molecular markers of ductal carcinogenesis indicates that endoplasmic reticulum calcium homeostasis is remodeled during tumorigenesis in the breast epithelium. PMID:21863130

  9. Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.

    PubMed

    Pernet, I; Reymermier, C; Guezennec, A; Branka, J-E; Guesnet, J; Perrier, E; Dezutter-Dambuyant, C; Schmitt, D; Viac, J

    2003-12-01

    The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.

  10. FOXM1 allows human keratinocytes to bypass the oncogene-induced differentiation checkpoint in response to gain of MYC or loss of p53

    PubMed Central

    Molinuevo, R; Freije, A; de Pedro, I; Stoll, S W; Elder, J T; Gandarillas, A

    2017-01-01

    Tumour suppressor p53 or proto-oncogene MYC is frequently altered in squamous carcinomas, but this is insufficient to drive carcinogenesis. We have shown that overactivation of MYC or loss of p53 via DNA damage triggers an anti-oncogenic differentiation-mitosis checkpoint in human epidermal keratinocytes, resulting in impaired cell division and squamous differentiation. Forkhead box M1 (FOXM1) is a transcription factor recently proposed to govern the expression of a set of mitotic genes. Deregulation of FOXM1 occurs in a wide variety of epithelial malignancies. We have ectopically expressed FOXM1 in keratinocytes of the skin after overexpression of MYC or inactivation of endogenous p53. Ectopic FOXM1 rescues the proliferative capacity of MYC- or p53-mutant cells in spite of higher genetic damage and a larger cell size typical of differentiation. As a consequence, differentiation induced by loss of p53 or MYC is converted into increased proliferation and keratinocytes displaying genomic instability are maintained within the proliferative compartment. The results demonstrate that keratinocyte oncogene-induced differentiation is caused by mitosis control and provide new insight into the mechanisms driving malignant progression in squamous cancer. PMID:27452522

  11. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Holloway, Adele; Cook, Anthony L.; Chin, Suyin P.; Snow, Elizabeth T.

    2014-11-15

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

  12. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  13. BLT2 expression improves skin integrity and protects from alterations caused by hyperglycemia in type 2 diabetes

    PubMed Central

    2017-01-01

    ABSTRACT Type 2 diabetes (T2D) can go undiagnosed for years, leading to a stage where chronic high blood sugar produces complications such as delayed wound healing. Reports have shown that BLT2 activation improves keratinocyte migration and wound healing, as well as protecting the epidermal barrier through the promotion of actin polymerization. The goal of this study was to elucidate the role of BLT2 expression in skin epithelial integrity in T2D. For this purpose, we used both wild type (WT) and BLT2 knockout mice in a model, in which a T2D-like phenotype was induced by keeping the animals on a high fat (HF) diet over 5 weeks. In a parallel in vitro approach, we cultured BLT2-transfected HaCaT cells at both low and high glucose concentrations for 48 h. Structure, transepithelial resistance (TEER), IL-1ß, IL-8 or CXCL2, MMP9, Filaggrin, Loricrin and Keratin 10 (K10) were evaluated ex vivo and in vitro. Additionally, wound healing (WH) was studied in vitro. The skin from T2D and BLT2 knockout mice showed a reduction in TEER and the expression of IL-1ß, and in increase in CXCL2, MMP9, Filaggrin, Loricrin and K10 expression. The structure suggested an atrophic epidermis; however, the skin was dramatically affected in the BLT2 knockout mice kept on a HF diet. HaCaT-BLT2 cells presented as an organized monolayer and showed higher TEER and wound healing compared with vector only-transfected HaCaT-Mock cells. Likewise, alterations in the expression of skin inflammatory, matrix degradation and differentiation markers under low and high glucose conditions were less severe than in HaCaT-Mock cells. Our results suggest that BLT2 improves epithelial integrity and function by regulating differentiation markers, cytokines and MMP9. Furthermore, BLT2 attenuates the damaging effects of high glucose levels, thereby accelerating wound healing.

  14. A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

    PubMed Central

    Nikolovska, Katerina; Renke, Jana K.; Jungmann, Oliver; Grobe, Kay; Iozzo, Renato V.; Zamfir, Alina D.; Seidler, Daniela G.

    2016-01-01

    Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in concentration dependent manner unlike the Dcn−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of

  15. Altered expression of KLC3 may affect semen parameters

    PubMed Central

    Kargar- Dastjerdy, Pegah; Tavalaee, Marziyeh; Salehi, Mansoor; Falahati, Mojtaba; Izadi, Tayebeh; Nasr Esfahani, Mohammad Hossein

    2016-01-01

    Background: KLC3 protein as a member of the kinesin light-chain protein family plays an important role in spermatogenesis, during formation of mitochondrial sheath in the mid piece of the sperm tail. Objective: This study for the first time aims to compare the expression of the KLC3 gene between fertile and infertile individuals. Materials and Methods: Semen samples were collected from 19 fertile individuals who were selected from embryo-donor volunteers and 57 infertile individuals who had abnormal sperm parameters according to world health organization criteria. Sperm parameters using computer assisted sperm analysis and the quantitative KLC3-gene expression using the real-time PCR method were measured. Results: Our results revealed a significant correlations between sperm concentration with relative expression of KLC3 only in infertile groups (r=0.45, p=0.00). A significant correlation was not found between KLC3 expression and sperm motility; however, the relative expression of KLC3 was significantly higher in asthenozoospermic compared to non-asthenozoospermic individuals. Conclusion: Low expression of KLC3 may result in improper function of midpiece, which has important function in sperm motility. The results of this study show that aberrant expression of KLC3 might be associated with phenomena like oligozoospermia and asthenozoospermia. This article is extracted from student’s thesis. PMID:27141544

  16. 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.

  17. Responses of black and white skin to solar-simulating radiation: differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL-10 expression.

    PubMed

    Rijken, Feiko; Bruijnzeel, Piet L B; van Weelden, Huib; Kiekens, Rebecca C M

    2004-06-01

    Black skin is more resistant to the deleterious effects of ultraviolet radiation than white skin. A higher melanin content and a different melanosomal dispersion pattern in the epidermis are thought to be responsible for this. Our purpose was to compare skin responses in black and white skin following exposure to solar-simulating radiation (SSR) to further investigate the photoprotective properties of black skin. Six volunteers of skin phototype I-III (white) were exposed to (doses measured directly with a Waldmann UV detector device) 12,000-18,000 mJ per cm2 (2 MED) of SSR and compared with six volunteers of skin phototype VI (black) exposed to 18,000 mJ per cm2 (<1 MED) of SSR. The presence and distribution of skin pigment, DNA photodamage, infiltrating neutrophils, photoaging associated proteolytic enzymes, keratinocyte activation, and the source of interleukin 10 (IL-10) in skin biopsies taken before and after exposure were studied. In all white skinned subjects, 12,000-18,000 mJ per cm2 of SSR induced DNA damage in epidermal and dermal cells, an influx of neutrophils, active proteolytic enzymes, and diffuse keratinocyte activation. Additionally, in three of the white skinned volunteers IL-10 positive neutrophils were found to infiltrate the epidermis. Except for DNA damage in the supra basal epidermis, none of these changes was found in black skinned subjects. Increased skin pigmentation appears to be primarily responsible for the observed differences in skin responses. Our data could provide an explanation as to why black skin is less susceptible to sunburn, photoaging, and skin carcinogenesis.

  18. [Alteration of isozyme gene expression during cell differentiation and oncogenesis].

    PubMed

    Yamada, K; Noguchi, T

    1995-05-01

    Rat pyruvate kinase (PK) has four isozymes, called the M1-, M2-, L-, and R-types. The M1- and M2-type isozymes of PK are produced from the PKM gene by alternative splicing, whereas the L- and R-type isozymes of PK are produced from the PKL gene by use of different tissue-specific promoters. In early development, only M2-type PK expresses in all tissues. After late morphogenesis, M1-, L-, and R-type PK express tissue-specifically. In contrast, cell proliferation such as regenerating liver and oncogenesis lead to decrease or cessation of the expression of tissue-specific PK isozymes and to stimulation of the expression of M2-type PK. These phenomena from the point of view transcriptional regulatory apparatus of the PKM and PKL gene are discussed.

  19. The suppressor of cytokine signaling (SOCS)-3 determines keratinocyte proliferative and migratory potential during skin repair.

    PubMed

    Linke, Andreas; Goren, Itamar; Bösl, Michael R; Pfeilschifter, Josef; Frank, Stefan

    2010-03-01

    Recently, the suppressor of cytokine signaling (SOCS)-3 has been shown to be expressed in disturbed wound margin epithelia during diabetes-impaired wound healing in mice. To functionally connect a potential contribution of SOCS-3 expression to the control of wound keratinocyte behavior in skin repair, we created a transgenic mouse (tsgn-K5/SOCS3) overexpressing SOCS-3 in keratinocytes using the bovine keratin 5 promoter. Tsgn-K5/SOCS3 mice showed a constitutive expression of SOCS-3 in the basal layer of skin epidermis. Keratinocytes of tsgn-K5/SOCS3 mice showed full inhibition of signal transducer and activator of transcription (STAT)-3 phosphorylation. Tsgn-K5/SOCS3 keratinocytes also showed a strong inhibition of migratory and proliferative potential in vitro. In addition, tsgn-K5/SOCS3 keratinocytes co-expressed the differentiation marker loricrin in the basal layer of nonwounded skin in vivo. Upon wounding, wound tissues of tsgn-K5/SOCS3 mice showed an impairment of wound closure characterized by strongly atrophied wound margin epithelia. Atrophied epithelia of tsgn-K5/SOCS3 mice exhibited a marked reduction in proliferating cells and reduced total keratinocyte numbers. In summary, this study suggests that the presence of SOCS-3 in keratinocytes strongly disturbs epithelial repair of cutaneous wounds by interfering with keratinocyte proliferation and migration.

  20. Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour.

    PubMed

    Bondanza, Sergio; Maurelli, Riccardo; Paterna, Patrizia; Migliore, Eleonora; Giacomo, Fabio Di; Primavera, Giovanni; Paionni, Emanuel; Dellambra, Elena; Guerra, Liliana

    2007-08-01

    Vitiligo depigmentation is considered a consequence of either melanocyte disappearance or loss of functioning melanocytes in the involved areas. However, it has been reported that keratinocytes in involved vitiligo skin are damaged too. Based on this evidence, we evaluated the in vitro behaviour, in life span cultures, of involved and uninvolved vitiligo keratinocytes and their expression of proliferation, differentiation and senescence markers. An additional purpose was to investigate whether vitiligo keratinocytes from depigmented skin are able to sustain survival and growth of normal melanocytes (when added in co-culture experiments), as normal human keratinocytes manage to do. Our results demonstrate that almost all involved vitiligo keratinocytes have a shorter life span in vitro than the uninvolved cells and all of them do not maintain melanocytes in culture in a physiological ratio. Modification of proliferation and senescence marker expression also occurs. Indeed, we detected low initial expression levels of the senescence marker p16 in involved vitiligo keratinocytes, despite their shorter in vitro life span, and increased expression of proliferating cell nuclear antigen and p53. This preliminary analysis of a small number of in vitro cultured vitiligo keratinocytes suggests an impaired senescence process in lesional vitiligo keratinocytes and attempts to regulate it.

  1. Arabidopsis gene expression patterns are altered during spaceflight

    NASA Astrophysics Data System (ADS)

    Paul, Anna-Lisa; Popp, Michael P.; Gurley, William B.; Guy, Charles; Norwood, Kelly L.; Ferl, Robert J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613-621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced - but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment

  2. Non-myogenic tumors display altered expression of dystrophin (DMD) and a high frequency of genetic alterations

    PubMed Central

    Luce, Leonela N.; Abbate, Mercedes

    2017-01-01

    DMD gene mutations have been associated with the development of Dystrophinopathies. Interestingly, it has been recently reported that DMD is involved in the development and progression of myogenic tumors, assigning DMD a tumor suppressor activity in these types of cancer. However, there are only few reports that analyze DMD in non-myogenic tumors. Our study was designed to examine DMD expression and genetic alterations in non-myogenic tumors using public repositories. We also evaluated the overall survival of patients with and without DMD mutations. We studied 59 gene expression microarrays (GEO database) and RNAseq (cBioPortal) datasets that included 9817 human samples. We found reduced DMD expression in 15/27 (56%) pairwise comparisons performed (Fold-Change (FC) ≤ 0.70; p-value range = 0.04-1.5×10−20). The analysis of RNAseq studies revealed a median frequency of DMD genetic alterations of 3.4%, higher or similar to other well-known tumor suppressor genes. In addition, we observed significant poorer overall survival for patients with DMD mutations. The analyses of paired tumor/normal tissues showed that the majority of tumor specimens had lower DMD expression compared to their normal adjacent counterpart. Interestingly, statistical significant over-expression of DMD was found in 6/27 studies (FC ≥ 1.4; p-value range = 0.03-3.4×10−15). These results support that DMD expression and genetic alterations are frequent and relevant in non-myogenic tumors. The study and validation of DMD as a new player in tumor development and as a new prognostic factor for tumor progression and survival are warranted. PMID:27391342

  3. Changes in skeletal muscle gene expression consequent to altered weight bearing

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Kirby, C. R.

    1992-01-01

    Skeletal muscle is a dynamic organ that adapts to alterations in weight bearing. This brief review examines changes in muscle gene expression resulting from the removal of weight bearing by hindlimb suspension and from increased weight bearing due to eccentric exercise. Acute (less than or equal to 2 days) non-weight bearing of adult rat soleus muscle alters only the translational control of muscle gene expression, while chronic (greater than or equal to 7 days) removal of weight bearing appears to influence pretranslational, translational, and posttranslational mechanisms of control. Acute and chronic eccentric exercise are associated with alterations of translational and posttranslational control, while chronic eccentric training also alters the pretranslational control of muscle gene expression. Thus alterations in weight bearing influence multiple sites of gene regulation.

  4. Alteration in follistatin gene expression detected in prenatally androgenized rats.

    PubMed

    Salehi Jahromi, Marziyeh; Ramezani Tehrani, Fahimeh; Hill, Jennifer W; Noroozzadeh, Mahsa; Zarkesh, Maryam; Ghasemi, Asghar; Zadeh-Vakili, Azita

    2017-02-26

    Impaired ovarian follicle development, the hallmark of polycystic ovarian syndrome (PCOS), is believed to be due to the changes in expression of related genes such as follistatin (FST). Expression of FST gene and methylation level of its promoter in theca cells from adult female rats, prenatally exposed to androgen excess, during different phases of the estrus cycle was determined and compared with controls. Eight pregnant Wistar rats (experimental group) were treated by subcutaneous injection of 5 mg free testosterone on day 20 of pregnancy, while controls (n = 8) received 500 ml solvent. Based on observed vaginal smear, adult female offspring of mothers were divided into three groups. Levels of serum steroidogenic sexual hormones and gonadotropins, expression and promoter methylation of the FST gene were measured using ELISA, cyber-green real-time PCR and bisulfite sequence PCR (BSP), respectively. Compared to controls, the relative expression of FST gene in the treated group decreased overall by 0.85 fold; despite significant changes in different phases, but no significant differences in methylation of FST promoter. Our results reveal that manifestation of PCOS-like phenotype following prenatal exposure to excess androgen is associated with irregularity in expression of the FST gene during the estrus cycle.

  5. Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

    PubMed Central

    2014-01-01

    Background Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms. PMID:24885240

  6. 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.

  7. Primary structure of keratinocyte transglutaminase.

    PubMed Central

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

    1990-01-01

    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 unrelated 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. Images PMID:1979171

  8. 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

  9. 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.

  10. Altered gene expression profiles in mouse tetraploid blastocysts.

    PubMed

    Park, Mi-Ryung; Hwang, Kyu-Chan; Bui, Hong-Thuy; Cho, Ssang-Goo; Park, Chankyu; Song, Hyuk; Oh, Jae-Wook; Kim, Jin-Hoi

    2012-01-01

    In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (P<0.05). Of these 50 genes, 28 were more highly expressed in tetraploid-derived blastocysts, whereas 22 were more highly downregulated. However, some genes involved in receptor activity, cell adhesion molecule, calcium ion binding, protein biosynthesis, redox processes, transport, and transcription showed a significant decrease or increase in gene expression in the tetraploid-derived blastocyst embryos. Thus, microarray analysis can be used as a tool to screen for underlying defects responsible for the development of tetraploid-derived embryos.

  11. Transposon-induced nuclear mutations that alter chloroplast gene expression

    SciTech Connect

    Barkan, A.

    1992-01-01

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  12. Wound re-epithelialization: modulating keratinocyte migration in wound healing.

    PubMed

    Raja; Sivamani, K; Garcia, Miki Shirakawa; Isseroff, R Rivkah

    2007-05-01

    An essential feature of a healed wound is the restoration of an intact epidermal barrier through wound epithelialization, also known as re-epithelialization. The directed migration of keratinocytes is critical to wound epithelialization and defects in this function are associated with the clinical phenotype of chronic non-healing wounds. A complex balance of signaling factors and surface proteins are expressed and regulated in a temporospatial manner that promote keratinocyte motility and survival to activate wound re-epithelialization. The majority of this review focuses on the mechanisms that regulate keratinocyte migration in the re-epithelialization process. This includes a review of cell attachments via desmosomes, hemidesmosomes, and integrins, the expression of keratins, the role of growth factors, cytokines and chemokines, eicosanoids, oxygen tension, antimicrobial peptides, and matrix metalloproteinases. Also reviewed are recently emerging novel mediators of keratinocyte motility including the role of electric fields, and signaling via the acetylcholine and beta-adrenergic receptors. These multiple regulators impact the ability of keratinocytes to migrate from the wound edge or other epidermal reservoirs to efficiently re-epithelialize a breach in the integrity of the epidermis. New discoveries will continue to uncover the elegant network of events that result in restoration of epidermal integrity and complete the wound repair process.

  13. Macrophage polarization alters the expression and sulfation pattern of glycosaminoglycans.

    PubMed

    Martinez, Pierre; Denys, Agnès; Delos, Maxime; Sikora, Anne-Sophie; Carpentier, Mathieu; Julien, Sylvain; Pestel, Joël; Allain, Fabrice

    2015-05-01

    Macrophages are major cells of inflammatory process and take part in a large number of physiological and pathological processes. According to tissue environment, they can polarize into pro-inflammatory (M1) or alternative (M2) cells. Although many evidences have hinted to a potential role of cell-surface glycosaminoglycans (GAGs) in the functions of macrophages, the effect of M1 or M2 polarization on the biosynthesis of these polysaccharides has not been investigated so far. GAGs are composed of repeat sulfated disaccharide units. Heparan (HS) and chondroitin/dermatan sulfates (CS/DS) are the major GAGs expressed at the cell membrane. They are involved in numerous biological processes, which rely on their ability to selectively interact with a large panel of proteins. More than 20 genes encoding sulfotransferases have been implicated in HS and CS/DS biosynthesis, and the functional repertoire of HS and CS/DS has been related to the expression of these isoenzymes. In this study, we analyzed the expression of sulfotransferases as a response to macrophage polarization. We found that M1 and M2 activation drastically modified the profiles of expression of numerous HS and CS/DS sulfotransferases. This was accompanied by the expression of GAGs with distinct structural features. We then demonstrated that GAGs of M2 macrophages were efficient to present fibroblast growth factor-2 in an assay of tumor cell proliferation, thus indicating that changes in GAG structure may contribute to the functions of polarized macrophages. Altogether, our findings suggest a regulatory mechanism in which fine modifications in GAG biosynthesis may participate to the plasticity of macrophage functions.

  14. Reciprocal regulation of p63 by C/EBP delta in human keratinocytes

    PubMed Central

    Borrelli, Serena; Testoni, Barbara; Callari, Maurizio; Alotto, Daniela; Castagnoli, Carlotta; Romano, Rose-Anne; Sinha, Satrajit; Viganò, Alessandra M; Mantovani, Roberto

    2007-01-01

    Background Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. Results C/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. Conclusion Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. PMID:17903252

  15. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  16. 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

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

    PubMed

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

    2014-08-01

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

  18. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  19. Altered expression profile of micrornas in gastric stromal tumor.

    PubMed

    Xiao, Jun; Wang, Qi-xian; Zhu, You-qing

    2015-12-01

    MicroRNAs (miRNAs) play important roles in carcinogenesis, but the global miRNA expression profile in gastric stromal tumor tissues remains unclear. This study was to examine the miRNA expression profile in gastric stromal tumor tissues and explore the function of dysregulated miRNAs by performing gene ontology (GO) and pathway enrichment analysis. Total RNA was extracted and purified from 3 pairs of frozen gastric stromal tumor tissues and the adjacent non-tumor tissues by using mirVana™ miRNA isolation kit. The miRNA expression was analyzed with Affymetrix microarrays (version 4.0) containing 2578 human mature microRNA probes. The dysregulated microRNAs were validated by quantitative RT-PCR in 30 pairs of gastric stromal tumor tissues. The target gene of the dysregulated microRNAs was predicted by miRanda, TargetScan and PicTar. GO and pathway enrichment analysis was conducted to examine the potential function of miR-3178 and miR-193a-5p. The results showed that there were 12 differently expressed microRNAs in gastric stromal tumor tissues, among which 10 miRNAs were down-regulated, and 2 were up-regulated (P<0.05). The validation results by RT-PCR were in accordance with those by microRNA microarry. GO analysis found that the target genes of miR-3178 were involved in 5 GO terms and those of miR-193a-5p in 7 GO terms in level 2. Pathway enrichment analysis suggested that miR-3178 and miR-193a-5p were related to 57 and 122 signaling pathways, respectively. It was concluded that gastric stromal tumor displays a unique miRNA signature. This specific expression may become a new diagnostic and prognostic biomarker for gastric stromal tumor. miR-3178 and miR-193a-5p function as suppressive microRNAs, and they may also become diagnosis and treatment targets for gastric stromal tumor.

  20. 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

  1. Nickel-induced heritable alterations in retroviral transforming gene expression.

    PubMed Central

    Biggart, N W; Gallick, G E; Murphy, E C

    1987-01-01

    Determination of the mutagenic effects of carcinogenic nickel compounds has been difficult because, like many metals, nickel is poorly or nonmutagenic in procaryotic mutagenicity assays. We attempted to characterize nickel-induced genetic lesions by assessing the effect of nickel chloride on the conditionally defective expression of the v-mos transforming gene in normal rat kidney cells infected with the Murine sarcoma virus mutant ts110 (MuSVts110) retrovirus. MuSVts110 contains an out-of-frame gag gene-mos gene junction that prevents the expression of the v-mos gene at the nonpermissive temperature (39 degrees C). In MuSVts110-infected cells (6m2 cells) grown at 33 degrees C, however, this defect can be suppressed by a splicing event that restores the mos reading frame, allowing the expression of a gag-mos fusion protein which induces the transformed phenotype. The capacity to splice the viral transcript at 33 degrees C, but not at 39 degrees C, is an intrinsic property of the viral RNA. This property allowed us to target the MuSVts110 genome using a positive selection scheme whereby nickel was used to induce genetic changes which resulted in expression of the transformed phenotype at 39 degrees C. We treated 6m2 cells with NiCl2 and isolated foci consisting of cells which had reverted to the transformed phenotype at 39 degrees C. We found that brief nickel treatment increased the reversion frequency of 6m2 cells grown at 39 degrees C sevenfold over the spontaneous reversion frequency. The nickel-induced revertants displayed the following heritable characteristics: They stably maintained the transformed phenotype at 39 degrees C; unlike the MuSVts110 RNA in 6m2 cells, the nickel-induced revertant viral RNA could be spliced efficiently at 39 degrees C; as a consequence of the enhanced accumulation of spliced viral RNA, the nickel-induced revertants produced substantial amounts of the transforming v-mos protein P85gag-mos at 39 degrees C; the nickel

  2. Knockdown of Leptin A Expression Dramatically Alters Zebrafish Development

    PubMed Central

    Liu, Qin; Dalman, Mark; Chen, Yun; Akhter, Mashal; Brahmandam, Sravya; Patel, Yesha; Lowe, Josef; Thakkar, Mitesh; Gregory, Akil-Vuai; Phelps, Daryllanae; Riley, Caitlin; Londraville, Richard L.

    2012-01-01

    Using morpholino antisense oligonucleotide (MO) technology, we blocked leptin A or leptin receptor expression in embryonic zebrafish, and analyzed consequences of leptin knock-down on fish development. Embryos injected with leptin A or leptin receptor MOs (leptin A or leptin receptor morphants) had smaller bodies and eyes, undeveloped inner ear, enlarged pericardial cavity, curved body and/or tail and larger yolk compared to control embryos of the same stages. The defects persisted in 6-9 day old larvae. We found that blocking leptin A function had little effect on the development of early brain (1 day old), but differentiation of both the morphant dorsal brain and retinal cells was severely disrupted in older (2 day old) embryos. Despite the enlarged pericardial cavity, differentiation of cardiac cells appeared to be similar to control embryos. Formation of the morphants’ inner ear is also severely disrupted, which corroborates existing reports of leptin receptor expression in inner ear of both zebrafish and mammals. Co-injection of leptin A MO and recombinant leptin results in partial rescue of the wild-type phenotype. Our results suggest that leptin A plays distinct roles in zebrafish development. PMID:22841760

  3. Characterization of primary human keratinocytes transformed by human papillomavirus type 18

    SciTech Connect

    Kaur, P.; McDougall, J.K. )

    1988-06-01

    Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.

  4. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    PubMed

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  5. Nursing frequency alters circadian patterns of mammary gene expression in lactating mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Milking frequency impacts lactation in dairy cattle and in rodent models of lactation. The role of circadian gene expression in this process is unknown. The hypothesis tested was that changing nursing frequency alters the circadian patterns of mammary gene expression. Mid-lactation CD1 mice were stu...

  6. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

    PubMed Central

    Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

  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. Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization.

    PubMed

    Usui, Marcia L; Underwood, Robert A; Mansbridge, Jonathan N; Muffley, Lara A; Carter, William G; Olerud, John E

    2005-01-01

    The process by which wounds reepithelialize remains controversial. Two models have been proposed to describe reepithelialization: the "sliding" model and the "rolling" model. In the "sliding" model, basal keratinocytes are the principal cells responsible for migration and wound closure. In this model, basal and suprabasal keratinocytes remain strongly attached to leading edge basal keratinocytes and are then passively dragged along as a sheet. The "rolling" model postulates that basal keratinocytes remain strongly attached to the basement membrane zone while suprabasal keratinocytes at the wound margin are activated to roll into the wound site. The purpose of this study was to determine which populations of keratinocytes are actively involved in reepithelialization. We evaluated expression of keratins K14, K15, K10, K2e, and K16 as well as the proliferation marker Ki67 in the migrating tongue of normal human incisional 1-hour to 28-day wounds and normal human 3 mm diameter excisional 1- to 7-day wounds. Our results show dramatic changes in phenotype and protein expression of keratins K10, K2e, K14, K15, and K16 in suprabasal keratinocytes in response to injury. We conclude that this large population of suprabasal keratinocytes actively participates in wound closure.

  9. Effects of transforming growth factor beta-1 on growth-regulatory genes in tumour-derived human oral keratinocytes.

    PubMed Central

    Paterson, I. C.; Patel, V.; Sandy, J. R.; Prime, S. S.; Yeudall, W. A.

    1995-01-01

    This study examined the effect of transforming growth factor beta-1 (TGF-beta 1) on c-myc, RB1, junB and p53 expression together with pRb phosphorylation, in carcinoma-derived and normal human oral keratinocytes with a range of inhibitory responses to this ligand. Amplification of c-myc was observed in eight of eight tumour-derived cell lines and resulted in corresponding mRNA expression. The down-regulation of c-myc expression by TGF-beta 1 predominantly reflected growth inhibition by TGF-beta 1, but in two of eight tumour-derived cell lines which were partially responsive to TGF-beta 1 c-myc expression was unaltered by this ligand. While RB1 mRNA levels were unaltered by TGF-beta 1, the ligand caused the accumulation of the underphosphorylated form of the Rb protein in all cells irrespective of TGF-beta 1-induced growth arrest. junB expression was up-regulated by TGF-beta 1 in cells with a range of growth inhibitory responses. All cells contained mutant p53. TGF-beta 1 did not affect p53 mRNA expression in both tumour-derived and normal keratinocytes and there was no alteration in p53 protein levels in keratinocytes expressing stable p53 protein following TGF-beta 1 treatment. The data indicate that TGF-beta-induced growth control can exist independently of the presence of mutant p53 and the control of Rb phosphorylation and c-myc down-regulation. It may be that TGF-beta growth inhibition occurs via multiple mechanisms and that the loss of one pathway during tumour progression does not necessarily result in the abrogation of TGF-beta-induced growth control. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7547241

  10. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

  11. IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression.

    PubMed

    Johnston, Andrew; Xing, Xianying; Guzman, Andrew M; Riblett, MaryBeth; Loyd, Candace M; Ward, Nicole L; Wohn, Christian; Prens, Errol P; Wang, Frank; Maier, Lisa E; Kang, Sewon; Voorhees, John J; Elder, James T; Gudjonsson, Johann E

    2011-02-15

    IL-1F6, IL-1F8, and IL-1F9 and the IL-1R6(RP2) receptor antagonist IL-1F5 constitute a novel IL-1 signaling system that is poorly characterized in skin. To further characterize these cytokines in healthy and inflamed skin, we studied their expression in healthy control, uninvolved psoriasis, and psoriasis plaque skin using quantitative RT-PCR and immunohistochemistry. Expression of IL-1F5, -1F6, -1F8, and -1F9 were increased 2 to 3 orders of magnitude in psoriasis plaque versus uninvolved psoriasis skin, which was supported immunohistologically. Moreover, treatment of psoriasis with etanercept led to significantly decreased IL-1F5, -1F6, -1F8, and -1F9 mRNAs, concomitant with clinical improvement. Similarly increased expression of IL-1F5, -1F6, -1F8, and -1F9 was seen in the involved skin of two mouse models of psoriasis. Suggestive of their importance in inflamed epithelia, IL-1α and TNF-α induced IL-1F5, -1F6, -1F8, and -1F9 transcript expression by normal human keratinocytes. Microarray analysis revealed that these cytokines induce the expression of antimicrobial peptides and matrix metalloproteinases by reconstituted human epidermis. In particular, IL-1F8 increased mRNA expression of human β-defensin (HBD)-2, HBD-3, and CAMP and protein secretion of HBD-2 and HBD-3. Collectively, our data suggest important roles for these novel cytokines in inflammatory skin diseases and identify these peptides as potential targets for antipsoriatic therapies.

  12. Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer

    PubMed Central

    Perez, Itxaro; Blanco, Lorena; Sanz, Begoña; Errarte, Peio; Ariz, Usue; Beitia, Maider; Fernández, Ainhoa; Loizate, Alberto; Candenas, M Luz; Pinto, Francisco M; Gil, Javier; López, José I.; Larrinaga, Gorka

    2015-01-01

    Background and Objective: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. Methods: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). Results: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall suvival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. Conclusions: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis. PMID:26078706

  13. Chemical derivatization combined with capillary LC or MALDI-TOF MS for trace determination of lipoic acid in cosmetics and integrated protein expression profiling in human keratinocytes.

    PubMed

    Tsai, Chia-Ju; Lin, Ying-Chi; Chen, Yen-Ling; Feng, Chia-Hsien

    2014-12-01

    Lipoic acid (LA) is an essential cofactor in mitochondrial enzymes and an ideal antioxidant in prokaryotic and eukaryotic cells. Capillary liquid chromatography coupled with ultraviolet detection (CapLC-UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are two environmentally friendly methods for determining LA. In this study, a pre-column microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin enhanced the UV absorbance of LA and was monitored at 345 nm by CapLC-UV. Gradient separation was performed using a reversed-phase C18 column with a mobile phase consisting of acetonitrile-0.1% formic acid solution. The ionization of LA was increased, and the LA derivative was detected by MALDI-TOF MS at m/z 683 with an α-cyano-4-hydroxycinnamic acid matrix. The linear response ranged from 0.1 to 40 μM with a correlation coefficient of 0.999. The CapLC-UV and MALDI-TOF MS had detection limits of 5 and 4 fmol, respectively. These methods effectively detected LA in dietary supplements and cosmetics. Cellular proteomes of a human keratinocyte cell line (HaCaT) irradiated with UV radiation were also compared with and without LA treatment. The cellular proteomes were identified by nanoultra performance LC with LTQ Orbitrap system after trypsin digestion. Protein identification was performed by simultaneous peptide sequencing and MASCOT search. The analysis revealed changes in several proteins, including CDC42, TPI1, HNRPA2B1, PRDX1, PTGES3 and MYL6.

  14. Epidermal differentiation and dermal changes in healing following treatment of surgical wounds with sheets of cultured allogeneic keratinocytes.

    PubMed Central

    Myers, S R; Navsaria, H A; Brain, A N; Purkis, P E; Leigh, I M

    1995-01-01

    AIMS--To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation. METHODS--Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components. RESULTS--The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds. CONCLUSIONS--Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding. Images PMID:8567992

  15. Increased Skin Tumor Incidence and Keratinocyte Hyper-Proliferation in a Mouse Model of Down Syndrome

    PubMed Central

    Yang, Annan; Currier, Duane; Poitras, Jennifer L.; Reeves, Roger H.

    2016-01-01

    Down syndrome (DS) is a genetic disorder caused by the presence of an extra copy of human chromosome 21 (Hsa21). People with DS display multiple clinical traits as a result of the dosage imbalance of several hundred genes. While many outcomes of trisomy are deleterious, epidemiological studies have shown a significant risk reduction for most solid tumors in DS. Reduced tumor incidence has also been demonstrated in functional studies using trisomic DS mouse models. Therefore, it was interesting to find that Ts1Rhr trisomic mice developed more papillomas than did their euploid littermates in a DMBA-TPA chemical carcinogenesis paradigm. Papillomas in Ts1Rhr mice also proliferated faster. The increased proliferation was likely caused by a stronger response of trisomy to TPA induction. Treatment with TPA caused hyperkeratosis to a greater degree in Ts1Rhr mice than in euploid, reminiscent of hyperkeratosis seen in people with DS. Cultured trisomic keratinocytes also showed increased TPA-induced proliferation compared to euploid controls. These outcomes suggest that altered gene expression in trisomy could elevate a proliferation signalling pathway. Gene expression analysis of cultured keratinocytes revealed upregulation of several trisomic and disomic genes may contribute to this hyperproliferation. The contributions of these genes to hyper-proliferation were further validated in a siRNA knockdown experiment. The unexpected findings reported here add a new aspect to our understanding of tumorigenesis with clinical implications for DS and demonstrates the complexity of the tumor repression phenotype in this frequent condition. PMID:26752700

  16. HPV-18 confers resistance to TNF-{alpha} in organotypic cultures of human keratinocytes

    SciTech Connect

    Boccardo, Enrique . E-mail: eboccardo@ludwig.org.br; Noya, Francisco; Broker, Thomas R.; Chow, Louise T.; Villa, Luisa L.

    2004-10-25

    The proinflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-{alpha} antiproliferative effect. The present study analyzes the effects of TNF-{alpha} on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-{alpha}. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-{alpha} cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-{alpha}. Besides, TNF-{alpha} treatment did not alter p16{sup ink4a}, p21{sup cip1}, p27{sup kip1}, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.

  17. Computational identification of altered metabolism using gene expression and metabolic pathways.

    PubMed

    Nam, Hojung; Lee, Jinwon; Lee, Doheon

    2009-07-01

    Understanding altered metabolism is an important issue because altered metabolism is often revealed as a cause or an effect in pathogenesis. It has also been shown to be an important factor in the manipulation of an organism's metabolism in metabolic engineering. Unfortunately, it is not yet possible to measure the concentration levels of all metabolites in the genome-wide scale of a metabolic network; consequently, a method that infers the alteration of metabolism is beneficial. The present study proposes a computational method that identifies genome-wide altered metabolism by analyzing functional units of KEGG pathways. As control of a metabolic pathway is accomplished by altering the activity of at least one rate-determining step enzyme, not all gene expressions of enzymes in the pathway demonstrate significant changes even if the pathway is altered. Therefore, we measure the alteration levels of a metabolic pathway by selectively observing expression levels of significantly changed genes in a pathway. The proposed method was applied to two strains of Saccharomyces cerevisiae gene expression profiles measured in very high-gravity (VHG) fermentation. The method identified altered metabolic pathways whose properties are related to ethanol and osmotic stress responses which had been known to be observed in VHG fermentation because of the high sugar concentration in growth media and high ethanol concentration in fermentation products. With the identified altered pathways, the proposed method achieved best accuracy and sensitivity rates for the Red Star (RS) strain compared to other three related studies (gene-set enrichment analysis (GSEA), significance analysis of microarray to gene set (SAM-GS), reporter metabolite), and for the CEN.PK 113-7D (CEN) strain, the proposed method and the GSEA method showed comparably similar performances.

  18. Melatonin promotes diabetic wound healing in vitro by regulating keratinocyte activity

    PubMed Central

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

    2016-01-01

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

  19. miR-21 promotes keratinocyte migration and re-epithelialization during wound healing.

    PubMed

    Yang, Xue; Wang, Jun; Guo, Shui-Long; Fan, Kai-Ji; Li, Jun; Wang, You-Liang; Teng, Yan; Yang, Xiao

    2011-01-01

    MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.

  20. Cigarette smoke alters IL-33 expression and release in airway epithelial cells.

    PubMed

    Pace, Elisabetta; Di Sano, Caterina; Sciarrino, Serafina; Scafidi, Valeria; Ferraro, Maria; Chiappara, Giuseppina; Siena, Liboria; Gangemi, Sebastiano; Vitulo, Patrizio; Giarratano, Antonino; Gjomarkaj, Mark

    2014-09-01

    Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored. Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.

  1. Prediction of drug-induced liver injury using keratinocytes.

    PubMed

    Hirashima, Rika; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2017-01-31

    Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1β are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1β basal expression. They also showed a higher inducibility of IL-1β when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    PubMed

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

  3. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  4. Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

    PubMed

    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

  5. 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.

  6. Expression of liver fatty acid binding protein alters growth and differentiation of embryonic stem cells.

    PubMed

    Schroeder, F; Atshaves, B P; Starodub, O; Boedeker, A L; Smith, R R; Roths, J B; Foxworth, W B; Kier, A B

    2001-03-01

    Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higher levels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.

  7. Keratinocyte stem cells and the targets for nonmelanoma skin cancer.

    PubMed

    Singh, Ashok; Park, Heuijoon; Kangsamaksin, Thaned; Singh, Anupama; Readio, Nyssa; Morris, Rebecca J

    2012-01-01

    The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.

  8. Formaldehyde exposure alters miRNA expression profiles in the olfactory bulb.

    PubMed

    Li, Guifa; Yang, Jing; Ling, Shucai

    2015-01-01

    It has been reported that inhaling formaldehyde (FA) causes damage to the central nervous system. However, it is unclear whether FA can disturb the function of the olfactory bulb. Using a microarray, we found that FA inhalation altered the miRNA expression profile. Functional enrichment analysis of the predicted targets of the changed miRNA showed that the enrichment canonical pathways and networks associated with cancer and transcriptional regulation. FA exposure disrupts miRNA expression profiles within the olfactory bulb.

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

    PubMed

    Uchida, Ryo; Aoki, Reiji; Aoki-Yoshida, Ayako; Tajima, Atsushi; Takayama, Yoshiharu

    2017-02-01

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

  10. Cell shape controls terminal differentiation of human epidermal keratinocytes.

    PubMed Central

    Watt, F M; Jordan, P W; O'Neill, C H

    1988-01-01

    Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture. Images PMID:2456572

  11. 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

  12. 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

  13. Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

    PubMed Central

    Shirai, Cara Lunn; Ley, James N.; White, Brian S.; Kim, Sanghyun; Tibbitts, Justin; Shao, Jin; Ndonwi, Matthew; Wadugu, Brian; Duncavage, Eric J.; Okeyo-Owuor, Theresa; Liu, Tuoen; Griffith, Malachi; McGrath, Sean; Magrini, Vincent; Fulton, Robert S.; Fronick, Catrina; O’Laughlin, Michelle; Graubert, Timothy A.; Walter, Matthew J.

    2015-01-01

    SUMMARY Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ~11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently-mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in MDS patients. PMID:25965570

  14. POTENTIAL ALTERATIONS IN GENE EXPRESSION ASSOCIATED WITH CARCINOGEN EXPOSURE IN MYA ARENARIA

    EPA Science Inventory

    Gonadal cancers in soft-shell clams (Mya arenaria) have been found at high prevalences (20-40%) in populations in eastern Maine. The aetiology of these tumours is unknown. We hypothesized that gene expression would be altered in gonadal tumours and that examination of gene expres...

  15. Methyl-ß-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was designed to determine if metabolic stress as induced by methyl-ß-cyclodextrin (MCD) can alter cytokine expression in neonatal swine adipose tissue explants. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21 day old pigs. Explants were incubated in medium 199 s...

  16. Induced keratinocyte hyper-proliferation in alpha2beta1 integrin transgenic mice results in systemic immune cell activation.

    PubMed

    Teige, Ingrid; Bäcklund, Alexandra; Svensson, Lars; Kvist, Peter Helding; Petersen, Thomas Kongstad; Kemp, Kåre

    2010-01-01

    alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.

  17. 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

    2015-11-05

    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.

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

    PubMed

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

    2003-12-16

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

  19. HuCOP1 contributes to the regulation of DNA repair in keratinocytes.

    PubMed

    Fazekas, B; Carty, M P; Németh, I; Kemény, L; Széll, M; Ádám, É

    2017-03-01

    We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

  20. Atypical Diabetic Foot Ulcer Keratinocyte Protein Signaling Correlates with Impaired Wound Healing

    PubMed Central

    Hoke, Glenn D.; Ramos, Corrine; Hoke, Nicholas N.; Crossland, Mary C.; Shawler, Lisa G.

    2016-01-01

    Diabetes mellitus is associated with chronic diabetic foot ulcers (DFUs) and wound infections often resulting in lower extremity amputations. The protein signaling architecture of the mechanisms responsible for impaired DFU healing has not been characterized. In this preliminary clinical study, the intracellular levels of proteins involved in signal transduction networks relevant to wound healing were non-biasedly measured using reverse-phase protein arrays (RPPA) in keratinocytes isolated from DFU wound biopsies. RPPA allows for the simultaneous documentation and assessment of the signaling pathways active in each DFU. Thus, RPPA provides for the accurate mapping of wound healing pathways associated with apoptosis, proliferation, senescence, survival, and angiogenesis. From the study data, we have identified potential diagnostic, or predictive, biomarkers for DFU wound healing derived from the ratios of quantified signaling protein expressions within interconnected pathways. These biomarkers may allow physicians to personalize therapeutic strategies for DFU management on an individual basis based upon the signaling architecture present in each wound. Additionally, we have identified altered, interconnected signaling pathways within DFU keratinocytes that may help guide the development of therapeutics to modulate these dysregulated pathways, many of which parallel the therapeutic targets which are the hallmarks of molecular therapies for treating cancer. PMID:27840833

  1. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice

    PubMed Central

    Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

    2014-01-01

    The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

  2. Low Density Lipoprotein Receptor-Related Protein and Apolipoprotein E Expression is Altered in Schizophrenia

    PubMed Central

    Gibbons, Andrew Stuart; Thomas, Elizabeth A.; Scarr, Elizabeth; Dean, Brian

    2010-01-01

    Our recent microarray study reported altered mRNA expression of several low density lipoprotein receptor-related proteins (LRP) associated with the first 4 years following diagnosis with schizophrenia. Whilst this finding is novel, apolipoprotein E (APOE), which mediates its activity through LRPs, has been reported by several studies to be altered in brains of subjects with schizophrenia. We used qPCR to measure the expression of LRP2, LRP4, LRP6, LRP8, LRP10 and LRP12 mRNA in Brodmann's area (BA) 46 of the dorsolateral prefrontal cortex in 15 subjects with short duration of illness schizophrenia (SDS) and 15 pair matched controls. We also used Western blotting to measure APOE protein expression in BA46 from these subjects. Amongst the LRPs examined, LRP10 expression was significantly increased (P = 0.03) and LRP12 was significantly decreased (P < 0.01) in SDS. APOE protein expression was also increased in SDS (P = 0.01). No other marker examined in this study was altered with diagnosis. Our data supports a role for distinct members of the LRP family in the pathology of schizophrenia and adds weight to the hypothesis that aberrant apolipoprotein signaling is involved in the early stages of schizophrenia. PMID:21423430

  3. Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

    PubMed

    Kato, Hiroko; Izumi, Kenji; Uenoyama, Atsushi; Shiomi, Aki; Kuo, Shiuhyang; Feinberg, Stephen E

    2014-01-01

    The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

  4. Fisetin inhibits TNF-α-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression.

    PubMed

    Seo, Seung-Hee; Jeong, Gil-Saeng

    2015-12-01

    Oxidative skin damage and skin inflammation play key roles in the pathogenesis of skin-related diseases. Fisetin is a naturally occurring flavonoid abundantly found in several vegetables and fruits. Fisetin has been shown to exert various positive biological effects, such as anti-cancer, anti-proliferative, neuroprotective and anti-oxidative effects. In this study, we investigate the skin protective effects and anti-inflammatory properties of fisetin in hydrogen peroxide- and TNF-α-challenged human keratinocyte HaCaT cells. When HaCaT cells were treated with non-cytotoxic concentrations of fisetin (1-20μM), heme oxygenase (HO)-1 mRNA and protein expression increased in a dose-dependent manner. Furthermore, fisetin dose-dependently increased cell viability and reduced ROS production in hydrogen peroxide-treated HaCaT cells. Fisetin also inhibited the production of NO, PGE2 IL-1β, IL-6, expression of iNOS and COX-2, and activation of NF-κB in HaCaT cells treated with TNF-α. Fisetin induced Nrf2 translocation to the nuclei. HO-1 siRNA transient transfection reversed the effects of fisetin on cytoprotection, ROS reduction, NO, PGE2, IL-1β, IL-6, and TNF-α production, and NF-κB DNA-binding activity. Moreover, fisetin increased Akt phosphorylation and a PI3K pathway inhibitor (LY294002) abolished fisetin-induced cytoprotection and NO inhibition. Taken together, these results provide evidence for a beneficial role of fisetin in skin therapy.

  5. Alcohol consumption modulates host defense in rhesus macaques by altering gene expression in circulating leukocytes

    PubMed Central

    Barr, Tasha; Girke, Thomas; Sureshchandra, Suhas; Nguyen, Christina; Grant, Kathleen; Messaoudi, Ilhem

    2015-01-01

    Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections whereas moderate alcohol consumption decreases incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations (BECs) >80 mg/dl) suppressed, whereas moderate alcohol consumption (BEC <50 mg/dl) enhanced T and B-cell responses to Modified Vaccinia Ankara (MVA) vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-MVA vaccination, the earliest time point at which we detected differences in T-cell and antibody responses. Overall, chronic heavy alcohol consumption reduced expression of immune genes involved in response to infection and wound healing, and increased expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated expression of genes involved in immune response and reduced expression of genes involved in cancer. In order to uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate expression of mRNAs differentially expressed in our dataset. PMID:26621857

  6. Altered expression patterns of syndecan-1 and -2 predict biochemical recurrence in prostate cancer.

    PubMed

    Ledezma, Rodrigo; Cifuentes, Federico; Gallegos, Iván; Fullá, Juan; Ossandon, Enrique; Castellon, Enrique A; Contreras, Héctor R

    2011-05-01

    The clinical features of prostate cancer do not provide an accurate determination of patients undergoing biochemical relapse and are therefore not suitable as indicators of prognosis for recurrence. New molecular markers are needed for proper pre-treatment risk stratification of patients. Our aim was to assess the value of altered expression of syndecan-1 and -2 as a marker for predicting biochemical relapse in patients with clinically localized prostate cancer treated by radical prostatectomy. The expression of syndecan-1 and -2 was examined by immunohistochemical staining in a series of 60 paraffin-embedded tissue samples from patients with localized prostate cancer. Ten specimens from patients with benign prostatic hyperplasia were used as non-malignant controls. Semiquantitative analysis was performed to evaluate the staining patterns. To investigate the prognostic value, Kaplan-Meier survival curves were performed and compared by a log-rank test. In benign samples, syndecan-1 was expressed in basal and secretory epithelial cells with basolateral membrane localisation, whereas syndecan-2 was expressed preferentially in basal cells. In prostate cancer samples, the expression patterns of both syndecans shifted to granular-cytoplasmic localisation. Survival analysis showed a significant difference (P < 0.05) between normal and altered expression of syndecan-1 and -2 in free prostate-specific antigen recurrence survival curves. These data suggest that the expression of syndecan-1 and -2 can be used as a prognostic marker for patients with clinically localized prostate cancer, improving the prostate-specific antigen recurrence risk stratification.

  7. Altered expression patterns of syndecan-1 and -2 predict biochemical recurrence in prostate cancer

    PubMed Central

    Ledezma, Rodrigo; Cifuentes, Federico; Gallegos, Iván; Fullá, Juan; Ossandon, Enrique; Castellon, Enrique A; Contreras, Héctor R

    2011-01-01

    The clinical features of prostate cancer do not provide an accurate determination of patients undergoing biochemical relapse and are therefore not suitable as indicators of prognosis for recurrence. New molecular markers are needed for proper pre-treatment risk stratification of patients. Our aim was to assess the value of altered expression of syndecan-1 and -2 as a marker for predicting biochemical relapse in patients with clinically localized prostate cancer treated by radical prostatectomy. The expression of syndecan-1 and -2 was examined by immunohistochemical staining in a series of 60 paraffin-embedded tissue samples from patients with localized prostate cancer. Ten specimens from patients with benign prostatic hyperplasia were used as non-malignant controls. Semiquantitative analysis was performed to evaluate the staining patterns. To investigate the prognostic value, Kaplan–Meier survival curves were performed and compared by a log-rank test. In benign samples, syndecan-1 was expressed in basal and secretory epithelial cells with basolateral membrane localisation, whereas syndecan-2 was expressed preferentially in basal cells. In prostate cancer samples, the expression patterns of both syndecans shifted to granular-cytoplasmic localisation. Survival analysis showed a significant difference (P<0.05) between normal and altered expression of syndecan-1 and -2 in free prostate-specific antigen recurrence survival curves. These data suggest that the expression of syndecan-1 and -2 can be used as a prognostic marker for patients with clinically localized prostate cancer, improving the prostate-specific antigen recurrence risk stratification. PMID:21317913

  8. Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Gene Expression in Circulating Leukocytes.

    PubMed

    Barr, Tasha; Girke, Thomas; Sureshchandra, Suhas; Nguyen, Christina; Grant, Kathleen; Messaoudi, Ilhem

    2016-01-01

    Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations < 50 mg/dl) enhanced, T and B cell responses to modified vaccinia Ankara vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in T cell and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune genes involved in response to infection and wound healing and increased the expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set.

  9. Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells.

    PubMed

    Takeshige, Keiko; Sekido, Takashi; Kitahara, Jun-ichirou; Ohkubo, Yousuke; Hiwatashi, Dai; Ishii, Hiroaki; Nishio, Shin-ichi; Takeda, Teiji; Komatsu, Mitsuhisa; Suzuki, Satoru

    2014-01-01

    μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

  10. High-resolution analysis of copy number alterations and associated expression changes in ovarian tumors

    PubMed Central

    Haverty, Peter M; Hon, Lawrence S; Kaminker, Joshua S; Chant, John; Zhang, Zemin

    2009-01-01

    Background DNA copy number alterations are frequently observed in ovarian cancer, but it remains a challenge to identify the most relevant alterations and the specific causal genes in those regions. Methods We obtained high-resolution 500K SNP array data for 52 ovarian tumors and identified the most statistically significant minimal genomic regions with the most prevalent and highest-level copy number alterations (recurrent CNAs). Within a region of recurrent CNA, comparison of expression levels in tumors with a given CNA to tumors lacking that CNA and to whole normal ovary samples was used to select genes with CNA-specific expression patterns. A public expression array data set of laser capture micro-dissected (LCM) non-malignant fallopian tube epithelia and LCM ovarian serous adenocarcinoma was used to evaluate the effect of cell-type mixture biases. Results Fourteen recurrent deletions were detected on chromosomes 4, 6, 9, 12, 13, 15, 16, 17, 18, 22 and most prevalently on X and 8. Copy number and expression data suggest several apoptosis mediators as candidate drivers of the 8p deletions. Sixteen recurrent gains were identified on chromosomes 1, 2, 3, 5, 8, 10, 12, 15, 17, 19, and 20, with the most prevalent gains localized to 8q and 3q. Within the 8q amplicon, PVT1, but not MYC, was strongly over-expressed relative to tumors lacking this CNA and showed over-expression relative to normal ovary. Likewise, the cell polarity regulators PRKCI and ECT2 were identified as putative drivers of two distinct amplicons on 3q. Co-occurrence analyses suggested potential synergistic or antagonistic relationships between recurrent CNAs. Genes within regions of recurrent CNA showed an enrichment of Cancer Census genes, particularly when filtered for CNA-specific expression. Conclusion These analyses provide detailed views of ovarian cancer genomic changes and highlight the benefits of using multiple reference sample types for the evaluation of CNA-specific expression changes

  11. 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.

  12. Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

    PubMed Central

    Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; Harding, Scott A.

    2016-01-01

    Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism in root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability. PMID:27641356

  13. Vimentin is necessary for colony growth of human diploid keratinocytes.

    PubMed

    Castro-Muñozledo, Federico; Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Hernández-Quintero, Miriam; Kuri-Harcuch, Walid

    2015-01-01

    The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

  14. C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation.

    PubMed

    Lopez, Rodolphe G; Garcia-Silva, Susana; Moore, Susan J; Bereshchenko, Oksana; Martinez-Cruz, Ana B; Ermakova, Olga; Kurz, Elke; Paramio, Jesus M; Nerlov, Claus

    2009-10-01

    The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

  15. Notch Cooperates with Survivin to Maintain Stemness and to Stimulate Proliferation in Human Keratinocytes during Ageing

    PubMed Central

    Palazzo, Elisabetta; Morandi, Paolo; Lotti, Roberta; Saltari, Annalisa; Truzzi, Francesca; Schnebert, Sylvianne; Dumas, Marc; Marconi, Alessandra; Pincelli, Carlo

    2015-01-01

    The Notch signaling pathway orchestrates cell fate by either inducing cell differentiation or maintaining cells in an undifferentiated state. This study aims to evaluate Notch expression and function in normal human keratinocytes. Notch1 is expressed in all epidermal layers, though to a different degree of intensity, with a dramatic decrease during ageing. Notch1 intracellular domain (N1ICD) levels are decreased during transit from keratinocyte stem cells (KSC) to transit amplifying (TA) cells, mimicking survivin expression in samples from donors of all ages. Calcium markedly reduces N1ICD levels in keratinocytes. N1ICD overexpression induces the up-regulation of survivin and the down-regulation of keratin 10 and involucrin, while increasing the S phase of the cell cycle. On the other hand, Notch1 inhibition (DAPT) dose-dependently decreases survivin, stimulates differentiation, and reduces keratinocyte proliferation in samples from donors of all ages. Silencing Notch downgrades survivin and increases keratin 10. In addition, Notch1 inhibition decreases survivin levels and proliferation both in KSC and TA cells. Finally, while survivin overexpression decreases keratinocyte differentiation and increases N1ICD expression both in KSC and TA cells, silencing survivin results in N1ICD down-regulation and an increase in differentiation markers. These results suggest that the Notch1/survivin crosstalk contributes to the maintenance of stemness in human keratinocytes. PMID:26540052

  16. Restricted maternal nutrition alters myogenic regulatory factor expression in satellite cells of ovine offspring.

    PubMed

    Raja, J S; Hoffman, M L; Govoni, K E; Zinn, S A; Reed, S A

    2016-07-01

    Poor maternal nutrition inhibits muscle development and postnatal muscle growth. Satellite cells are myogenic precursor cells that contribute to postnatal muscle growth, and their activity can be evaluated by the expression of several transcription factors. Paired-box (Pax)7 is expressed in quiescent and active satellite cells. MyoD is expressed in activated and proliferating satellite cells and myogenin is expressed in terminally differentiating cells. Disruption in the expression pattern or timing of expression of myogenic regulatory factors negatively affects muscle development and growth. We hypothesized that poor maternal nutrition during gestation would alter the in vitro temporal expression of MyoD and myogenin in satellite cells from offspring at birth and 3 months of age. Ewes were fed 100% or 60% of NRC requirements from day 31±1.3 of gestation. Lambs from control-fed (CON) or restricted-fed (RES) ewes were euthanized within 24 h of birth (birth; n=5) or were fed a control diet until 3 months of age (n=5). Satellite cells isolated from the semitendinosus muscle were used for gene expression analysis or cultured for 24, 48 or 72 h and immunostained for Pax7, MyoD or myogenin. Fusion index was calculated from a subset of cells allowed to differentiate. Compared with CON, temporal expression of MyoD and myogenin was altered in cultured satellite cells isolated from RES lambs at birth. The percent of cells expressing MyoD was greater in RES than CON (P=0.03) after 24 h in culture. After 48 h of culture, there was a greater percent of cells expressing myogenin in RES compared with CON (P0.05). In satellite cells from RES lambs at 3 months of age, the percent of cells expressing MyoD and myogenin were greater than CON after 72 h in culture (P<0.05). Fusion index was reduced in RES lambs at 3 months of age compared with CON (P<0.001). Restricted nutrition during gestation alters the temporal expression of myogenic regulatory factors in satellite cells of the

  17. Role of keratin 24 in human epidermal keratinocytes

    PubMed Central

    Min, Min; Chen, Xi-Bei; Wang, Ping; Landeck, Lilla; Chen, Jia-Qi; Li, Wei; Cai, Sui-Qing; Zheng, Min; Man, Xiao-Yong

    2017-01-01

    Keratin 24 (K24) is a new kind of keratin genes, which encodes a novel keratin protein, K24 that bears high similarity to the type I keratins and displays a unique expression profile. However, the role of K24 is incompletely understood. In our study, we investigated the localization of K24 within the epidermis and possible functions. Keratin 24 was found to be modestly overexpressed in senescent keratinocytes and was mainly restricted to the upper stratum spinosum of epidermis. The protein was required for terminal differentiation upon CaCl2-induced differentiation. In vitro results showed that increased K24 in keratinocytes dramatically changed the differentiation of primary keratinocytes. It also inhibited cell survival by G1/S phase cell cycle arrest and induced senescence, autophagy and apoptosis of keratinocytes. In addition, K24 activated PKCδ signal pathway involving in cellular survival. In summary, K24 may be suggested as a potential differentiation marker and anti-proliferative factor in the epidermis. PMID:28362807

  18. Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin.

    PubMed

    Yang, Hsin-Ya; La, Thi Dinh; Gurenko, Zhanna; Steenhuis, Pieter; Liu, Wei; Isseroff, R Rivkah

    2015-02-01

    Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.

  19. The expression of p73 is increased in lung cancer, independent of p53 gene alteration

    PubMed Central

    Tokuchi, Y; Hashimoto, T; Kobayashi, Y; Hayashi, M; Nishida, K; Hayashi, S; Imai, K; Nakachi, K; Ishikawa, Y; Nakagawa, K; Kawakami, Y; Tsuchiya, E

    1999-01-01

    p73 gene, a new p53 homologue, has been identified: it supposedly acts as tumour suppressor gene in neuroblastoma. To clarify whether p73 might be involved in lung carcinogenesis, we examined p73 expression in resected lung cancer and paired normal lung in 60 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We also examined p73 gene status in three representative cases using Southern blot, and p53 gene alteration in 49 cases using PCR-single-strand conformation polymorphism (PCR-SSCP) and direct sequence. In 87% of the cases (52/60) p73 expression in tumour was more than twice as high as that in paired normal lung tissues, and the difference between p73 expression in tumour and normal lung tissue was significant (P < 0.0001). However, Southern blot analysis revealed that none of the cases showed p73 gene amplification. Compared with clinicopathological characteristics, p73 expression correlates significantly with histological differences and age of patient, independently (P < 0.05). Concerning p53 gene status, 43% (21/49) showed p53 gene alteration, but there was no correlation between p73 overexpression and p53 gene alteration. Our results suggest that need for further functional analysis of the role of p73 in lung carcinogenesis. © 1999 Cancer Research Campaign PMID:10408409

  20. HER2 expression and PI3K-Akt pathway alterations in gastric cancer.

    PubMed

    Sukawa, Yasutaka; Yamamoto, Hiroyuki; Nosho, Katsuhiko; Ito, Miki; Igarashi, Hisayoshi; Naito, Takafumi; Mitsuhashi, Kei; Matsunaga, Yasutaka; Takahashi, Taiga; Mikami, Masashi; Adachi, Yasushi; Suzuki, Hiromu; Shinomura, Yasuhisa

    2014-01-01

    The anti-HER2 antibody trastuzumab has led to an era of personalized therapy in gastric cancer (GC). As a result, HER2 expression has become a major concern in GC. HER2 overexpression is seen in 7-34% of GC cases. Trastuzumab is an antibody that targets the HER2 extracellular domain and induces antibody-dependent cellular cytotoxicity and inhibition of the HER2 downstream signals. Mechanisms of resistance to trastuzumab have been reported in breast cancer. There are various mechanisms underlying trastuzumab resistance, such as alterations of HER2 structure or surroundings, dysregulation of HER2 downstream signal effectors and interaction of HER2 with other membrane receptors. The PI3K-Akt pathway is one of the main downstream signaling pathways of HER2. It is well known that PIK3CA mutations and phosphate and tensin homolog (PTEN) inactivation cause over-activation of the downstream signal without an upstream signal activation. Frequencies of PIK3CA mutations and PTEN inactivation have been reported to be 4-25 and 16-77%, respectively. However, little is known about the association between HER2 expression and PI3K-Akt pathway alterations in GC. We have found that HER2 over-expression was significantly correlated with pAkt expression in GC tissues. Furthermore, pAkt expression was correlated with poor prognosis. These results suggest that the PI3K-Akt pathway plays an important role in HER2-positive GC. Moreover, PIK3CA mutations and/or PTEN inactivation might affect the effectiveness of HER2-targeting therapy. Hence, it is necessary to clarify not only HER2 alterations but also PI3K-Akt pathway alterations for HER2-targeting therapy in GC. This review will introduce recent investigations and consider the current status of HER2-targeted therapy for treatment of GC.

  1. Chronic ethanol feeding alters miRNA expression dynamics during liver regeneration

    PubMed Central

    Dippold, Rachael P.; Vadigepalli, Rajanikanth; Gonye, Gregory E.; Patra, Biswanath; Hoek, Jan B.

    2012-01-01

    Background Adaptation to chronic ethanol treatment of rats results in a changed functional state of the liver and greatly inhibits its regenerative ability, which may contribute to the progression of alcoholic liver disease. Methods In this study we investigated the effect of chronic ethanol intake on hepatic miRNA expression in male Sprague-Dawley rats during the initial 24 hrs of liver regeneration following 70% partial hepatectomy (PHx) using microRNA (miRNA) microarrays. miRNA expression during adaptation to ethanol was investigated using RT-qPCR. Nuclear Factor kappa B (NFκB) binding at target miRNA promoters was investigated with chromatin immunoprecipitation. Results Unsupervised clustering of miRNA expression profiles suggested that miRNA expression was more affected by chronic ethanol feeding than by the acute challenge of liver regeneration after PHx. Several miRNAs that were significantly altered by chronic ethanol feeding, including miRs-34a, -103, -107, and -122 have been reported to play a role in regulating hepatic metabolism and the onset of these miRNA changes occurred gradually during the time course of ethanol feeding. Chronic ethanol feeding also altered the dynamic miRNA profile during liver regeneration. Promoter analysis predicted a role for Nuclear Factor kappa B (NFκB) in the immediate early miRNA response to PHx. NFκB binding at target miRNA promoters in the chronic ethanol-fed group was significantly altered and these changes directly correlated with the observed expression dynamics of the target miRNA. Conclusions Chronic ethanol consumption alters the hepatic miRNA expression profile such that the response of the metabolism-associated miRNAs occurs during long-term adaptation to ethanol rather than as an acute transient response to ethanol metabolism. Additionally, the dynamic miRNA program during liver regeneration in response to PHx is altered in the chronically ethanol-fed liver and these differences reflect, in part, differences

  2. Altered JS-2 expression in colorectal cancers and its clinical pathological relevance.

    PubMed

    Lam, Alfred King-Yin; Gopalan, Vinod; Nassiri, Mohammad Reza; Kasim, Kais; Dissanayake, Jayampathy; Tang, Johnny Chuek-On; Smith, Robert Anthony

    2011-10-01

    JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer.

  3. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    SciTech Connect

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  4. MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

    PubMed

    Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

    2017-01-01

    The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

  5. Maternal immune activation alters glutamic acid decarboxylase-67 expression in the brains of adult rat offspring

    PubMed Central

    Cassella, Sarah N.; Hemmerle, Ann M.; Lundgren, Kerstin H.; Kyser, Tara L.; Ahlbrand, Rebecca; Bronson, Stefanie L.; Richtand, Neil M.; Seroogy, Kim B.

    2016-01-01

    Activation of the maternal innate immune system, termed “maternal immune activation” (MIA), represents a common environmental risk factor for schizophrenia. Whereas evidence suggests dysregulation of GABA systems may underlie the pathophysiology of schizophrenia, a role for MIA in alteration of GABAergic systems is less clear. Here, pregnant rats received either the viral mimetic polyriboinosinic-polyribocytidilic acid or vehicle injection on gestational day 14. Glutamic acid decarboxylase-67 (GAD67) mRNA expression was examined in male offspring at postnatal day (P)14, P30 and P60. At P60, GAD67 mRNA was elevated in hippocampus and thalamus and decreased in prefrontal cortex of MIA offspring. MIA-induced alterations in GAD expression could contribute to the pathophysiology of schizophrenia. PMID:26830319

  6. Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion.

    PubMed

    Juengel, Eva; Krueger, Geraldine; Rutz, Jochen; Nelson, Karen; Werner, Isabella; Relja, Borna; Seliger, Barbara; Fisslthaler, Beate; Fleming, Ingrid; Tsaur, Igor; Haferkamp, Axel; Blaheta, Roman A

    2016-04-12

    Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.

  7. Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation.

    PubMed

    Díaz-Delfín, Julieta; Domingo, Pere; Giralt, Marta; Villarroya, Francesc

    2013-03-01

    Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patients with CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequently appear in HIV-infected patients under HAART, there is no information available on the effects of MVC on adipose tissue. Here we studied the effects of MVC during and after the differentiation of human adipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured the acquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity, adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally, we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed no alterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistent with this, there were no changes in the mRNA levels of PPARγ or SREBP-1c, two master regulators of adipogenesis. In addition, MVC caused a significant decrease in the gene expression and release of pro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflammation-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes in differentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rather shows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatory cytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissue development, metabolism, and inflammation, and thus could be a potentially beneficial component of antiretroviral therapy.

  8. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid.

    PubMed

    Tang, Xiao-Han; 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 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) 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.

  9. 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.

  10. Role for WNT16B in human epidermal keratinocyte proliferation and differentiation.

    PubMed

    Teh, Muy-Teck; Blaydon, Diana; Ghali, Lucy R; Briggs, Victoria; Edmunds, Scott; Pantazi, Eleni; Barnes, Michael R; Leigh, Irene M; Kelsell, David P; Philpott, Michael P

    2007-01-15

    WNT signalling regulates a variety of cell functions including cell fate, polarity, and differentiation via the canonical or beta-catenin stabilisation pathway and/or the planar cell polarity or non-canonical pathway. We have previously demonstrated that two isoforms (A and B) from the WNT16 locus have differential expression in various adult human tissues. In this study we show that WNT16B but not WNT16A isoform was upregulated in basal cell carcinomas compared with normal skin. We further investigated the cellular and molecular functions of WNT16B in primary human epidermal keratinocytes and a keratinocyte cell line. Cellular expression of WNT16B neither stabilised beta-catenin nor activated the lymphoid enhancer factor or T-cell factor transcriptional reporter in primary keratinocytes. WNT16B activated the Jun-N-terminal kinase cascade suggesting the activation of a non-canonical WNT signalling pathway. Constitutive expression of WNT16B significantly enhanced the rate of cell proliferation and prolonged clonogenicity in primary keratinocytes. Silencing WNT16B by RNA interference reduced keratinocyte proliferation. Furthermore, overexpression of WNT16B induced a hyperproliferation phenotype in an organotypical culture system. This work presents the first evidence that WNT16B activates human keratinocyte proliferation possibly via a beta-catenin-independent non-canonical WNT transduction pathway.

  11. Sex differences in learned fear expression and extinction involve altered gamma oscillations in medial prefrontal cortex.

    PubMed

    Fenton, Georgina E; Halliday, David M; Mason, Rob; Bredy, Timothy W; Stevenson, Carl W

    2016-11-01

    Sex differences in learned fear expression and extinction involve the medial prefrontal cortex (mPFC). We recently demonstrated that enhanced learned fear expression during auditory fear extinction and its recall is linked to persistent theta activation in the prelimbic (PL) but not infralimbic (IL) cortex of female rats. Emerging evidence indicates that gamma oscillations in mPFC are also implicated in the expression and extinction of learned fear. Therefore we re-examined our in vivo electrophysiology data and found that females showed persistent PL gamma activation during extinction and a failure of IL gamma activation during extinction recall. Altered prefrontal gamma oscillations thus accompany sex differences in learned fear expression and its extinction. These findings are relevant for understanding the neural basis of post-traumatic stress disorder, which is more prevalent in women and involves impaired extinction and mPFC dysfunction.

  12. Heart failure alters matrix metalloproteinase gene expression and activity in rat skeletal muscle.

    PubMed

    Carvalho, Robson Francisco; Dariolli, Rafael; Justulin Junior, Luis Antonio; Sugizaki, Mário Mateus; Politi Okoshi, Marina; Cicogna, Antonio Carlos; Felisbino, Sérgio Luis; Dal Pai-Silva, Maeli

    2006-12-01

    Heart failure is associated with a skeletal muscle myopathy with cellular and extracellular alterations. The hypothesis of this investigation is that extracellular changes may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP). We examined MMP mRNA expression and MMP activity in Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) muscles of young Wistar rat with monocrotaline-induced heart failure. Rats injected with saline served as age-matched controls. MMP2 and MMP9 mRNA contents were determined by RT-PCR and MMP activity by electrophoresis in gelatin-containing polyacrylamide gels in the presence of SDS under non-reducing conditions. Heart failure increased MMP9 mRNA expression and activity in SOL, EDL and DIA and MMP2 mRNA expression in DIA. These results suggest that MMP changes may contribute to the skeletal muscle myopathy during heart failure.

  13. Epstein-Barr virus growth/latency III program alters cellular microRNA expression

    SciTech Connect

    Cameron, Jennifer E. Fewell, Claire Yin, Qinyan McBride, Jane Wang Xia Lin Zhen

    2008-12-20

    The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

  14. Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts

    PubMed Central

    Cross, Courtney E.; Tolba, Mai F.; Rondelli, Catherine M.; Xu, Meixiang; Abdel-Rahman, Sherif Z.

    2015-01-01

    The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development. PMID:26339600

  15. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

    NASA Astrophysics Data System (ADS)

    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A.; Woodman, Scott E.; Kwong, Lawrence N.

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy.

  16. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

    PubMed Central

    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A; Woodman, Scott E; Kwong, Lawrence N

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy. PMID:26787600

  17. Altered glial gene expression, density, and architecture in the visual cortex upon retinal degeneration.

    PubMed

    Cornett, Ashley; Sucic, Joseph F; Hillsburg, Dylan; Cyr, Lindsay; Johnson, Catherine; Polanco, Anthony; Figuereo, Joe; Cabine, Kenneth; Russo, Nickole; Sturtevant, Ann; Jarvinen, Michael K

    2011-11-08

    Genes encoding the proteins of cytoskeletal intermediate filaments (IF) are tightly regulated, and they are important for establishing neural connections. However, it remains uncertain to what extent neurological disease alters IF gene expression or impacts cells that express IFs. In this study, we determined the onset of visual deficits in a mouse model of progressive retinal degeneration (Pde6b(-) mice; Pde6b(+) mice have normal vision) by observing murine responses to a visual task throughout development, from postnatal day (PND) 21 to adult (N=174 reliable observations). Using Q-PCR, we evaluated whether expression of the genes encoding two Type III IF proteins, glial fibrillary acidic protein (GFAP) and vimentin was altered in the visual cortex before, during, and after the onset of visual deficits. Using immunohistochemical techniques, we investigated the impact of vision loss on the density and morphology of astrocytes that expressed GFAP and vimentin in the visual cortex. We found that Pde6b(-) mice displayed 1) evidence of blindness at PND 49, with visual deficits detected at PND 35, 2) reduced GFAP mRNA expression in the visual cortex between PND 28 and PND 49, and 3) an increased ratio of vimentin:GFAP-labeled astrocytes at PND 49 with reduced GFAP cell body area. Together, these findings demonstrate that retinal degeneration modifies cellular and molecular indices of glial plasticity in a visual system with drastically reduced visual input. The functional consequences of these structural changes remain uncertain.

  18. Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin.

    PubMed

    Hu, Wei; Chen, Songyu; Zhang, Ran; Lin, Yushuang

    2013-06-01

    The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.

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

    PubMed

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

    2015-03-01

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

  20. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer.

    PubMed

    Chen, Yi; Pirisi, Lucia; Creek, Kim E

    2013-09-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.

  1. Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning.

    PubMed

    Xue, Kai; Xie, Jianping; Zhou, Aifen; Liu, Feifei; Li, Dejun; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy D; Luo, Yiqi; Zhou, Jizhong

    2016-01-01

    Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming.

  2. Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning

    PubMed Central

    Xue, Kai; Xie, Jianping; Zhou, Aifen; Liu, Feifei; Li, Dejun; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Luo, Yiqi; Zhou, Jizhong

    2016-01-01

    Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming. PMID:27199978

  3. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection. PMID:27698715

  4. Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew

    PubMed Central

    Chowdhury, Jamil; Lück, Stefanie; Rajaraman, Jeyaraman; Douchkov, Dimitar; Shirley, Neil J.; Schwerdt, Julian G.; Schweizer, Patrick; Fincher, Geoffrey B.; Burton, Rachel A.; Little, Alan

    2017-01-01

    Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

  5. Specificity in stress response: epidermal keratinocytes exhibit specialized UV-responsive signal transduction pathways.

    PubMed

    Adachi, Makoto; Gazel, Alix; Pintucci, Giuseppe; Shuck, Alyssa; Shifteh, Shiva; Ginsburg, Dov; Rao, Laxmi S; Kaneko, Takehiko; Freedberg, Irwin M; Tamaki, Kunihiko; Blumenberg, Miroslav

    2003-10-01

    UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses.

  6. Comparison of gene expression profiles altered by comfrey and riddelliine in rat liver

    PubMed Central

    Guo, Lei; Mei, Nan; Dial, Stacey; Fuscoe, James; Chen, Tao

    2007-01-01

    Background Comfrey (Symphytum officinale) is a perennial plant and has been consumed by humans as a vegetable, a tea and an herbal medicine for more than 2000 years. It, however, is hepatotoxic and carcinogenic in experimental animals and hepatotoxic in humans. Pyrrolizidine alkaloids (PAs) exist in many plants and many of them cause liver toxicity and/or cancer in humans and experimental animals. In our previous study, we found that the mutagenicity of comfrey was associated with the PAs contained in the plant. Therefore, we suggest that carcinogenicity of comfrey result from those PAs. To confirm our hypothesis, we compared the expression of genes and processes of biological functions that were altered by comfrey (mixture of the plant with PAs) and riddelliine (a prototype of carcinogenic PA) in rat liver for carcinogenesis in this study. Results Groups of 6 Big Blue Fisher 344 rats were treated with riddelliine at 1 mg/kg body weight by gavage five times a week for 12 weeks or fed a diet containing 8% comfrey root for 12 weeks. Animals were sacrificed one day after the last treatment and the livers were isolated for gene expression analysis. The gene expressions were investigated using Applied Biosystems Rat Whole Genome Survey Microarrays and the biological functions were analyzed with Ingenuity Analysis Pathway software. Although there were large differences between the significant genes and between the biological processes that were altered by comfrey and riddelliine, there were a number of common genes and function processes that were related to carcinogenesis. There was a strong correlation between the two treatments for fold-change alterations in expression of drug metabolizing and cancer-related genes. Conclusion Our results suggest that the carcinogenesis-related gene expression patterns resulting from the treatments of comfrey and riddelliine are very similar, and PAs contained in comfrey are the main active components responsible for carcinogenicity of

  7. Global Loss of Bmal1 Expression Alters Adipose Tissue Hormones, Gene Expression and Glucose Metabolism

    PubMed Central

    Kennaway, David John; Varcoe, Tamara Jayne; Voultsios, Athena; Boden, Michael James

    2013-01-01

    The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism. PMID:23750248

  8. The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells.

    PubMed

    Lin, Yi-Han; Fu, Keng-Yen; Hong, Po-Da; Ma, Hsu; Liou, Nien-Hsien; Ma, Kuo-Hsing; Liu, Jiang-Chuan; Huang, Kun-Lun; Dai, Lien-Guo; Chang, Shun-Cheng; Yi-Hsin Chan, James; Chen, Shyi-Gen; Chen, Tim-Mo; Dai, Niann-Tzyy

    2013-12-01

    Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable microniches. In this study, we aimed to investigate the effects of culture media, cell coculture, and a tissue-engineering biocomposite on the differentiation of mouse ESCs (MESCs) into keratinocyte-like cells and applied these cells to a surgical skin wound model. MESCs from BALB/c mice (ESC26GJ), which were transfected using pCX-EGFP expressing green fluorescence, were used to track MESC-derived keratinocytes. Weak expression of the keratinocyte early marker Cytokeratin 14 (CK-14) was observed up to 12 days when MESCs were cultured in a keratinocyte culture medium on tissue culture plastic and on a gelatin/collagen/polycaprolactone (GCP) biocomposite. MESCs cocultured with human keratinocyte cells (HKCs) also expressed CK-14, but did not express CK-14 when cocultured with human fibroblast cells (HFCs). Furthermore, CK-14 expression was observed when MESCs were cocultured by seeding HKCs or HFCs on the same or opposite side of the GCP biocomposite. The highest CK-14 expression was observed by seeding MESCs and HKCs on the same side of the GCP composite and with HFCs on the opposite side. To verify the effectiveness of wound healing in vivo, adipose-derived stem cells were applied to treat surgical wounds in nude mice. An obvious epidermis multilayer and better collagen deposition during wound healing were observed, as assessed by Masson staining. This study demonstrated the potential of keratinocyte-like differentiation from mesenchymal stem cells for use in promoting wound closure and skin regeneration.

  9. Tissue-specific alterations of PRL-1 and PRL-2 expression in cancer.

    PubMed

    Dumaual, Carmen M; Sandusky, George E; Soo, Han Weng; Werner, Sean R; Crowell, Pamela L; Randall, Stephen K

    2012-01-01

    The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of these molecules in human neoplasms is not well understood. To increase understanding of the role PRL-1 and PRL-2 play in the neoplastic process, in situ hybridization was used to examine PRL-1 and PRL-2 mRNA expression in 285 normal, benign, and malignant human tissues of diverse origin. Immunohistochemical analysis was performed on a subset of these. PRL-1 and PRL-2 mRNA expression was also assessed in a small set of samples from a variety of diseases other than cancer. Where possible, associations with clinicopathological characteristics were evaluated. Alterations in PRL-1 or -2 expression were a frequent event, but the nature of those alterations was highly tumor type specific. PRL-1 was significantly overexpressed in 100% of hepatocellular and gastric carcinomas, but significantly under-expressed in 100% of ovarian, 80% of breast, and 75% of lung tumors. PRL-2 expression was significantly increased in 100% of hepatocellular carcinomas, yet significantly downregulated in 54% of kidney carcinomas. PRL-1 expression was correlated to patient gender in the bladder and to patient age in the brain and skeletal muscle. PRL-1 expression was also associated with tumor grade in the prostate, ovary, and uterus. These results suggest a pleiotropic role for PRL-1 and PRL-2 in the neoplastic process. These molecules may associate with tumor progression and serve as clinical markers of tumor aggressiveness in some tissues, but be involved in inhibition of tumor formation or growth in others.

  10. Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging.

    PubMed

    Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

    2016-05-18

    Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

  11. 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

  12. Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients.

    PubMed

    Wölfler, Monika Martina; Küppers, Mareike; Rath, Werner; Buck, Volker Uwe; Meinhold-Heerlein, Ivo; Classen-Linke, Irmgard

    2016-07-01

    Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle. All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies. In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected. These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis.

  13. Altered expression of the Smad signalling pathway: implications for COPD pathogenesis.

    PubMed

    Zandvoort, A; Postma, D S; Jonker, M R; Noordhoek, J A; Vos, J T W M; van der Geld, Y M; Timens, W

    2006-09-01

    Pulmonary emphysema, as a feature of chronic obstructive pulmonary disease (COPD), is characterised by destruction of alveolar tissue. The present authors previously demonstrated reduced decorin expression in the peribronchial area of COPD patients, reflecting an altered extracellular matrix (ECM) modulation. Decorin transcription is regulated by the transforming growth factor (TGF)-beta-Smad pathway, the key intracellular signal route for initiation of ECM component gene transcription. Whether this pathway is aberrantly expressed in COPD is not known. An immunohistochemical study was performed to compare protein expression of TGF-beta1 and TGF-beta receptors, Smad 2, 3, 4 and 7, and decorin in lung tissue of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages II and IV COPD patients and controls. Epithelial expression of the inhibitory Smad 7 was significantly lower in patients with GOLD stages II and IV than in controls, with other Smad protein expressions being similar in the groups. The expression of TGF-beta1 and TGF-beta receptor type I was significantly lower in stage II patients. Decorin staining of the adventitia and alveolar walls was significantly reduced in COPD stage IV. In conclusion, the transforming growth factor-beta-Smad pathway is aberrantly expressed in chronic obstructive pulmonary disease patients, implying an abnormal tissue repair ultimately resulting in reduced decorin production. The results of the present study contribute to better understanding of the pathogenesis of emphysema and the airway fibrosis observed in chronic obstructive pulmonary disease patients.

  14. Heparanase Overexpression Reduces Hepcidin Expression, Affects Iron Homeostasis and Alters the Response to Inflammation

    PubMed Central

    Asperti, Michela; Stuemler, Tanja; Poli, Maura; Gryzik, Magdalena; Lifshitz, Lena; Meyron-Holtz, Esther G.; Vlodavsky, Israel

    2016-01-01

    Hepcidin is the key regulator of systemic iron availability that acts by controlling the degradation of the iron exporter ferroportin. It is expressed mainly in the liver and regulated by iron, inflammation, erythropoiesis and hypoxia. The various agents that control its expression act mainly via the BMP6/SMAD signaling pathway. Among them are exogenous heparins, which are strong hepcidin repressors with a mechanism of action not fully understood but that may involve the competition with the structurally similar endogenous Heparan Sulfates (HS). To verify this hypothesis, we analyzed how the overexpression of heparanase, the HS degrading enzyme, modified hepcidin expression and iron homeostasis in hepatic cell lines and in transgenic mice. The results showed that transient and stable overexpression of heparanase in HepG2 cells caused a reduction of hepcidin expression and of SMAD5 phosphorylation. Interestingly, the clones showed also altered level of TfR1 and ferritin, indices of a modified iron homeostasis. The heparanase transgenic mice showed a low level of liver hepcidin, an increase of serum and liver iron with a decrease in spleen iron content. The hepcidin expression remained surprisingly low even after treatment with the inflammatory LPS. The finding that modification of HS structure mediated by heparanase overexpression affects hepcidin expression and iron homeostasis supports the hypothesis that HS participate in the mechanisms controlling hepcidin expression. PMID:27711215

  15. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  16. Primary Biliary Cirrhosis is Associated With Altered Hepatic microRNA Expression

    PubMed Central

    Padgett, Kerstien A.; Lan, Ruth Y.; Leung, Patrick C.; Lleo, Ana; Dawson, Kevin; Pfeiff, Janice; Mao, Tin K.; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

    2009-01-01

    MicroRNAs (miRNAs) are small RNA molecules that negatively regulate protein coding gene expression and are thought to play a critical role in many biological processes. Aberrant levels of miRNAs have been associated with numerous diseases and cancers, and as such, miRNAs have gain much interests as diagnostic biomarkers, and as therapeutic targets. However, their role in autoimmunity is largely unknown. The aims of this study are to: (1) identify differentially expressed miRNAs in human primary biliary cirrhosis (PBC); (2) validate these independently; and (3) indentify potential targets of differentially expressed miRNAs. We compared the expression of 377 miRNAs in explanted livers form subjects with PBC versus controls with normal liver histology. A total of 35 independent miRNAs were found to be differentially expressed in PBC (p< 0.001). Quantitative PCR was employed to validate down-regulation of microRNA-122a (miR-122a) and miR-26a and the increased expression of miR-328 and miR-299-5p. The predicted targets of these miRNAs are known to affect cell proliferation, apoptosis, inflammation, oxidative stress, and metabolism. Our data are the first to demonstrate that PBC is characterized by altered expression of hepatic miRNA; however additional studies are required to demonstrate a causal link between those miRNA and the development of PBC. PMID:19345069

  17. Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease

    PubMed Central

    Soderquest, Katrina; Hertweck, Arnulf; Mohamed, Rami; Goldberg, Rimma; Perucha, Esperanza; Franke, Lude; Herrero, Javier; Lord, Graham M.

    2017-01-01

    The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21) specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn’s disease, ulcerative colitis (UC) and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL) for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner. PMID:28187197

  18. p53 expression and its relationship to DNA alterations in bone and soft tissue sarcomas.

    PubMed Central

    Wadayama, B.; Toguchida, J.; Yamaguchi, T.; Sasaki, M. S.; Yamamuro, T.

    1993-01-01

    The p53 gene is one of the best studied tumour suppressor genes. Recently we performed mutation analysis on the p53 gene in a large number of bone and soft tissue sarcomas, and found that approximately one-third of the sarcomas have some type of DNA alteration at the p53 locus (Toguchida et al., 1992). However, the expression of the p53 protein resulting from these alterations still remains to be clarified. In this study, p53 expression in the sarcoma tissues was analysed immunohistochemically using antibody PAb421 (Oncogene Science) and its relationship to DNA alterations was examined. Of 113 tumours, 29 (25.7%) showed positive staining for the p53 protein. These included 19 of 67 osteosarcomas, five of 20 chondrosarcomas, four of 11 malignant fibrous histiocytomas (MFHs) and one Ewing's sarcoma. In chondrosarcomas, most of the p53-positive tumours belonged to highly malignant and atypical tumour types (dedifferentiated or mesenchymal type), suggesting a role for p53 mutation in the progression of cartilaginous tumours. All the cases with a missense mutation showed strongly positive staining, while no immunoreactivity was observed in the remaining three-quarters with DNA alterations including gross rearrangement, frame-shift mutation, nonsense mutation or mutation at splicing site except in one case. These results demonstrated the dominance of the p53 mutations with null protein expression in bone and soft tissue sarcomas, showing a unique characteristic of these types of tumours compared with other malignancies such as colon carcinomas. Images Figure 1 Figure 2 Figure 3 PMID:8260365

  19. Altered Gene Expression in Cerulein-Stimulated Pancreatic Acinar Cells: Pathologic Mechanism of Acute Pancreatitis

    PubMed Central

    Yu, Ji Hoon; Lim, Joo Weon

    2009-01-01

    Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with 10-8 M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis. PMID:20054485

  20. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    PubMed

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  1. A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells.

    PubMed

    Ceci, Claudia; Barbaccia, Maria Luisa; Pistritto, Giuseppa

    2015-03-01

    Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10μM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10μM) increased the expression of specific neuronal differentiation markers such as neural cell adhesion molecule (NCAM) and microtubule associated protein 2 (MAP2). Furthermore, nickel exposure increased the expression of hypoxia-inducible-factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life.

  2. Respiratory function decline and DNA mutation in mitochondria, oxidative stress and altered gene expression during aging.

    PubMed

    Wei, Yau-Huei; Wu, Shi-Bei; Ma, Yi-Shing; Lee, Hsin-Chen

    2009-01-01

    Aging is a biological process that is characterized by the gradual loss of physiological function and increases in the susceptibility to disease of an individual. During the aging process, a wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) has been observed in somatic tissues of humans and animals. This is associated with the decline in mitochondrial respiratory function; excess production of the reactive oxygen species (ROS); increase in the oxidative damage to mtDNA, lipids and proteins in mitochondria; accumulation of point mutations and large-scale deletions of mtDNA; and altered expression of genes involved in intermediary metabolism. It has been demonstrated that the ROS may cause oxidative damage and mutations of mtDNA and alterations of the expression of several clusters of genes in aging tissues and senescent cells. We found that intracellular levels of hydrogen peroxide (H2O2) and oxidative damage to DNA in the tissue cells and skin fibroblasts of old donors were higher than those of young donors. In H2O2-induced senescent skin fibroblasts, we observed an increase in the protein expression and activity levels of manganese-dependent superoxide dismutase and a concurrent decrease in the activity of cytochrome c oxidase and the rate of oxygen consumption. Moreover, the mRNA and protein expression levels of pyruvate dehydrogenase (PDH) were decreased but those of PDH kinase and lactate dehydrogenase were increased in senescent skin fibroblasts. The changes in the expression of these enzymes suggest a metabolic shift from mitochondrial respiration to glycolysis as a major supply of ATP in aging human cells. On the other hand, recent studies on mitochondrial mutant mice, which carry a proofreading deficient subunit of DNA polymerase gamma, revealed that mtDNA mutations accumulated in somatic tissues in the mice that displayed prominent features of aging. Taken together, we suggest that the respiratory function decline and increase in

  3. The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

    PubMed

    Lai, Yuping; Li, Dongqing; Li, Changwei; Muehleisen, Beda; Radek, Katherine A; Park, Hyun Jeong; Jiang, Ziwei; Li, Zhiheng; Lei, Hu; Quan, Yanchun; Zhang, Tian; Wu, Yelin; Kotol, Paul; Morizane, Shin; Hata, Tissa R; Iwatsuki, Keiji; Tang, Ce; Gallo, Richard L

    2012-07-27

    Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

  4. In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation.

    PubMed

    Fernandez, Tara L; Van Lonkhuyzen, Derek R; Dawson, Rebecca A; Kimlin, Michael G; Upton, Zee

    2014-01-01

    Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

  5. Gene expression in developing fibres of Upland cotton (Gossypium hirsutum L.) was massively altered by domestication

    PubMed Central

    2010-01-01

    Background Understanding the evolutionary genetics of modern crop phenotypes has a dual relevance to evolutionary biology and crop improvement. Modern upland cotton (Gossypium hirsutum L.) was developed following thousands of years of artificial selection from a wild form, G. hirsutum var. yucatanense, which bears a shorter, sparser, layer of single-celled, ovular trichomes ('fibre'). In order to gain an insight into the nature of the developmental genetic transformations that accompanied domestication and crop improvement, we studied the transcriptomes of cotton fibres from wild and domesticated accessions over a developmental time course. Results Fibre cells were harvested between 2 and 25 days post-anthesis and encompassed the primary and secondary wall synthesis stages. Using amplified messenger RNA and a custom microarray platform designed to interrogate expression for 40,430 genes, we determined global patterns of expression during fibre development. The fibre transcriptome of domesticated cotton is far more dynamic than that of wild cotton, with over twice as many genes being differentially expressed during development (12,626 versus 5273). Remarkably, a total of 9465 genes were diagnosed as differentially expressed between wild and domesticated fibres when summed across five key developmental time points. Human selection during the initial domestication and subsequent crop improvement has resulted in a biased upregulation of components of the transcriptional network that are important for agronomically advanced fibre, especially in the early stages of development. About 15% of the differentially expressed genes in wild versus domesticated cotton fibre have no homology to the genes in databases. Conclusions We show that artificial selection during crop domestication can radically alter the transcriptional developmental network of even a single-celled structure, affecting nearly a quarter of the genes in the genome. Gene expression during fibre development

  6. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice.

    PubMed

    Weindruch, R; Kayo, T; Lee, C K; Prolla, T A

    2001-03-01

    An active research area in biological gerontology concerns the mechanisms by which caloric restriction (CR) retards the aging process in laboratory rodents. We used high density oligonucleotide arrays representing 6347 genes to determine the gene expression profile of the aging process in gastrocnemius muscle of male C57BL/6 mice. Aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were completely or partially prevented by CR. Transcriptional patterns of muscle from calorie-restricted animals suggest that CR retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. The use of high density oligonucleotide microarrays provides a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of nutritional interventions designed to retard the aging process.

  7. Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

    PubMed Central

    Liu, Xiaochen; Lu, Deyi; Bowser, Robert; Liu, Jian

    2016-01-01

    Carbonic anhydrase I (CA1) is the cytosolic isoform of mammalian α-CA family members which are responsible for maintaining pH homeostasis in the physiology and pathology of organisms. A subset of CA isoforms are known to be expressed and function in the central nervous system (CNS). CA1 has not been extensively characterized in the CNS. In this study, we demonstrate that CA1 is expressed in the motor neurons in human spinal cord. Unexpectedly, a subpopulation of CA1 appears to be associated with endoplasmic reticulum (ER) membranes. In addition, the membrane-associated CA1s are preferentially upregulated in amyotrophic lateral sclerosis (ALS) and exhibit altered distribution in motor neurons. Furthermore, long-term expression of CA1 in mammalian cells activates apoptosis. Our results suggest a previously unknown role for CA1 function in the CNS and its potential involvement in motor neuron degeneration in ALS. PMID:27809276

  8. Persistent alterations of gene expression profiling of human peripheral blood mononuclear cells from smokers.

    PubMed

    Weng, Daniel Y; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P; Loffredo, Christopher A; Shields, Peter G

    2016-10-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers' peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers' blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. © 2015 Wiley Periodicals, Inc.

  9. Persistent Alterations of Gene Expression Profiling of Human Peripheral Blood Mononuclear Cells From Smokers

    PubMed Central

    Weng, Daniel Y.; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P.; Loffredo, Christopher A.; Shields, Peter G.

    2016-01-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. PMID:26294040

  10. Ethanol-related alterations in gene expression patterns in the developing murine hippocampus.

    PubMed

    Mandal, Chanchal; Park, Kyoung Sun; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-08-01

    It is well known that consuming alcohol prior to and during pregnancy can cause harm to the developing fetus. Fetal alcohol spectrum disorder is a term commonly used to describe a range of disabilities that may arise from prenatal alcohol exposure such as fetal alcohol syndrome, partial fetal alcohol syndrome, alcohol-related neurodevelopmental disorders, and alcohol-related birth defects. Here, we report that maternal binge alcohol consumption alters several important genes that are involved in nervous system development in the mouse hippocampus at embryonic day 18. Microarray analysis revealed that Nova1, Ntng1, Gal, Neurog2, Neurod2, and Fezf2 gene expressions are altered in the fetal hippocampus. Pathway analysis also revealed the association of the calcium signaling pathway in addition to other pathways with the differentially expressed genes during early brain development. Alteration of such important genes and dynamics of the signaling pathways may cause neurodevelopmental disorders. Our findings offer insight into the molecular mechanism involved in neurodevelopmental disorders associated with alcohol-related defects.

  11. 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

  12. Bisphenol A Exposure Alters Developmental Gene Expression in the Fetal Rhesus Macaque Uterus

    PubMed Central

    Calhoun, Kathryn C.; Padilla-Banks, Elizabeth; Jefferson, Wendy N.; Liu, Liwen; Gerrish, Kevin E.; Young, Steven L.; Wood, Charles E.; Hunt, Patricia A.; VandeVoort, Catherine A.; Williams, Carmen J.

    2014-01-01

    Bisphenol A (BPA) exposure results in numerous developmental and functional abnormalities in reproductive organs in rodent models, but limited data are available regarding BPA effects in the primate uterus. To determine if maternal oral BPA exposure affects fetal uterine development in a non-human primate model, pregnant rhesus macaques carrying female fetuses were exposed orally to 400 µg/kg BPA or vehicle control daily from gestation day (GD) 50–100 or GD100–165. Fetal uteri were collected at the completion of treatment (GD100 or GD165); tissue histology, cell proliferation, and expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) were compared to that of controls. Gene expression analysis was conducted using rhesus macaque microarrays. There were no significant differences in histology or in the percentage of cells expressing the proliferation marker Ki-67, ERα, or PR in BPA-exposed uteri compared to controls at GD100 or GD165. Minimal differences in gene expression were observed between BPA-exposed and control GD100 uteri. However, at GD165, BPA-exposed uteri had significant differences in gene expression compared to controls. Several of the altered genes, including HOXA13, WNT4, and WNT5A, are critical for reproductive organ development and/or adult function. We conclude that second or third trimester BPA exposure does not significantly affect fetal uterus development based on morphological, proliferation, and steroid hormone receptor assessments. However, differences in expression of key developmental genes after third trimester exposure suggest that BPA could alter transcriptional signals influencing uterine function later in life. PMID:24465770

  13. Altered Protein Composition and Gene Expression in Strabismic Human Extraocular Muscles and Tendons

    PubMed Central

    Agarwal, Andrea B.; Feng, Cheng-Yuan; Altick, Amy L.; Quilici, David R.; Wen, Dan; Johnson, L. Alan; von Bartheld, Christopher S.

    2016-01-01

    Purpose To determine whether structural protein composition and expression of key regulatory genes are altered in strabismic human extraocular muscles. Methods Samples from strabismic horizontal extraocular muscles were obtained during strabismus surgery and compared with normal muscles from organ donors. We used proteomics, standard and customized PCR arrays, and microarrays to identify changes in major structural proteins and changes in gene expression. We focused on muscle and connective tissue and its control by enzymes, growth factors, and cytokines. Results Strabismic muscles showed downregulation of myosins, tropomyosins, troponins, and titin. Expression of collagens and regulators of collagen synthesis and degradation, the collagenase matrix metalloproteinase (MMP)2 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2, was upregulated, along with tumor necrosis factor (TNF), TNF receptors, and connective tissue growth factor (CTGF), as well as proteoglycans. Growth factors controlling extracellular matrix (ECM) were also upregulated. Among 410 signaling genes examined by PCR arrays, molecules with downregulation in the strabismic phenotype included GDNF, NRG1, and PAX7; CTGF, CXCR4, NPY1R, TNF, NTRK1, and NTRK2 were upregulated. Signaling molecules known to control extraocular muscle plasticity were predominantly expressed in the tendon rather than the muscle component. The two horizontal muscles, medial and lateral rectus, displayed similar changes in protein and gene expression, and no obvious effect of age. Conclusions Quantification of proteins and gene expression showed significant differences in the composition of extraocular muscles of strabismic patients with respect to important motor proteins, elements of the ECM, and connective tissue. Therefore, our study supports the emerging view that the molecular composition of strabismic muscles is substantially altered. PMID:27768799

  14. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy.

    PubMed

    Tsai, Chieh-Chih; Wu, Shi-Bei; Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO.

  15. Inhalation of ultrafine particles alters blood leukocyte expression of adhesion molecules in humans.

    PubMed

    Frampton, Mark W; Stewart, Judith C; Oberdörster, Günter; Morrow, Paul E; Chalupa, David; Pietropaoli, Anthony P; Frasier, Lauren M; Speers, Donna M; Cox, Christopher; Huang, Li-Shan; Utell, Mark J

    2006-01-01

    Ultrafine particles (UFPs; aerodynamic diameter < 100 nm) may contribute to the respiratory and cardiovascular morbidity and mortality associated with particulate air pollution. We tested the hypothesis that inhalation of carbon UFPs has vascular effects in healthy and asthmatic subjects, detectable as alterations in blood leukocyte expression of adhesion molecules. Healthy subjects inhaled filtered air and freshly generated elemental carbon particles (count median diameter approximately 25nm, geometric standard deviation approximately 1.6), for 2 hr, in three separate protocols: 10 microg/m3 at rest, 10 and 25 microg/m3 with exercise, and 50 microg/m3 with exercise. In a fourth protocol, subjects with asthma inhaled air and 10 microg/m3 UFPs with exercise. Peripheral venous blood was obtained before and at intervals after exposure, and leukocyte expression of surface markers was quantitated using multiparameter flow cytometry. In healthy subjects, particle exposure with exercise reduced expression of adhesion molecules CD54 and CD18 on monocytes and CD18 and CD49d on granulocytes. There were also concentration-related reductions in blood monocytes, basophils, and eosinophils and increased lymphocyte expression of the activation marker CD25. In subjects with asthma, exposure with exercise to 10 microg/m3 UFPs reduced expression of CD11b on monocytes and eosinophils and CD54 on granulocytes. Particle exposure also reduced the percentage of CD4+ T cells, basophils, and eosinophils. Inhalation of elemental carbon UFPs alters peripheral blood leukocyte distribution and expression of adhesion molecules, in a pattern consistent with increased retention of leukocytes in the pulmonary vascular bed.

  16. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves’ Ophthalmopathy

    PubMed Central

    Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO. PMID:26599235

  17. Alterations in gene expression in Caenorhabditis elegans associated with organophosphate pesticide intoxication and recovery

    PubMed Central

    2013-01-01

    Background The principal toxicity of acute organophosphate (OP) pesticide poisoning is the disruption of neurotransmission through inhibition of acetylcholinesterase (AChE). However, other mechanisms leading to persistent effects and neurodegeneration remain controversial and difficult to detect. Because Caenorhabditis elegans is relatively resistant to OP lethality—particularly through the inhibition of AChE—studies in this nematode provide an opportunity to observe alterations in global gene expression following OP exposure that cannot be readily observed in less resistant organisms. Results We exposed cultures of worms in axenic, defined medium to dichlorvos under three exposure protocols. In the first, worms were exposed continuously throughout the experiment. In the second and third, the worms were exposed for either 2 or 8 h, the dichlorvos was washed out of the culture, and the worms were allowed to recover. We then analyzed gene expression using whole genome microarrays from RNA obtained from worms sampled at multiple time points throughout the exposure. The worms showed a time-dependent increase in the expression of genes involved in stress responses. Early in the exposure, the predominant effect was on metabolic processes, while at later times, an immune-like response and cellular repair mechanisms dominated the expression pattern. Following removal of dichlorvos, the gene expression in the worms appeared to relatively rapidly return to steady-state levels. Conclusion The changes in gene expression observed in the worms following exposure to dichlorvos point towards two potential mechanisms of toxicity: inhibition of AChE and mitochondrial disruption. PMID:23631360

  18. Genetic association and altered gene expression of osteoprotegerin in otosclerosis patients.

    PubMed

    Priyadarshi, Saurabh; Ray, Chinmay Sundar; Biswal, Narayan Chandra; Nayak, Soumya Ranjan; Panda, Khirod Chandra; Desai, Ashim; Ramchander, Puppala Venkat

    2015-07-01

    Otosclerosis (OTSC) is a late-onset hearing disorder characterized by increased bone turnover in the otic capsule. Disturbed osteoprotegerin expression has been found in the otosclerotic foci which may have an important role in the pathogenesis of OTSC. To identify the genetic risk factors, we sequenced the coding region and exon-intron boundaries of the OPG gene in 254 OTSC patients and 262 controls. Sequence analysis identified five known polymorphisms c.9C>G, c.30+15C>T, c.400+4C>T, c.768A>G, and c.817+8A>C. Testing of these SNPs revealed sex specific association with c.9C>G in males and c.30+15C>T in females after multiple correction. Furthermore, meta-analysis provided evidence of association of the c.9C>G polymorphism with OTSC. In secondary analysis, we investigated the mRNA expression of OPG and associated genes RANK and RANKL in otosclerotic tissues compared to controls. Expression analysis revealed significantly missing/reduced OPG expression only in otosclerotic tissues. However, the signal sequence polymorphism c.9C>G has shown no effect on OPG mRNA expression. In conclusion, our results suggest that the risk of OTSC is influenced by variations in the OPG gene along with other factors which might regulate its altered expression in otosclerotic tissues. Further research is warranted to elucidate the mechanisms underlying these observations.

  19. Consistently altered expression of gene sets in postmortem brains of individuals with major psychiatric disorders

    PubMed Central

    Darby, M M; Yolken, R H; Sabunciyan, S

    2016-01-01

    The measurement of gene expression in postmortem brain is an important tool for understanding the pathogenesis of serious psychiatric disorders. We hypothesized that major molecular deficits associated with psychiatric disease would affect the entire brain, and such deficits may be shared across disorders. We performed RNA sequencing and quantified gene expression in the hippocampus of 100 brains in the Stanley Array Collection followed by replication in the orbitofrontal cortex of 57 brains in the Stanley Neuropathology Consortium. We then identified genes and canonical pathway gene sets with significantly altered expression in schizophrenia and bipolar disorder in the hippocampus and in schizophrenia, bipolar disorder and major depression in the orbitofrontal cortex. Although expression of individual genes varied, gene sets were significantly enriched in both of the brain regions, and many of these were consistent across diagnostic groups. Further examination of core gene sets with consistently increased or decreased expression in both of the brain regions and across target disorders revealed that ribosomal genes are overexpressed while genes involved in neuronal processes, GABAergic signaling, endocytosis and antigen processing have predominantly decreased expression in affected individuals compared to controls without a psychiatric disorder. Our results highlight pathways of central importance to psychiatric health and emphasize messenger RNA processing and protein synthesis as potential therapeutic targets for all three of the disorders. PMID:27622934

  20. Manipulation of BK channel expression is sufficient to alter auditory hair cell thresholds in larval zebrafish

    PubMed Central

    Rohmann, Kevin N.; Tripp, Joel A.; Genova, Rachel M.; Bass, Andrew H.

    2014-01-01

    Non-mammalian vertebrates rely on electrical resonance for frequency tuning in auditory hair cells. A key component of the resonance exhibited by these cells is an outward calcium-activated potassium current that flows through large-conductance calcium-activated potassium (BK) channels. Previous work in midshipman fish (Porichthys notatus) has shown that BK expression correlates with seasonal changes in hearing sensitivity and that pharmacologically blocking these channels replicates the natural decreases in sensitivity during the winter non-reproductive season. To test the hypothesis that reducing BK channel function is sufficient to change auditory thresholds in fish, morpholino oligonucleotides (MOs) were used in larval zebrafish (Danio rerio) to alter expression of slo1a and slo1b, duplicate genes coding for the pore-forming α-subunits of BK channels. Following MO injection, microphonic potentials were recorded from the inner ear of larvae. Quantitative real-time PCR was then used to determine the MO effect on slo1a and slo1b expression in these same fish. Knockdown of either slo1a or slo1b resulted in disrupted gene expression and increased auditory thresholds across the same range of frequencies of natural auditory plasticity observed in midshipman. We conclude that interference with the normal expression of individual slo1 genes is sufficient to increase auditory thresholds in zebrafish larvae and that changes in BK channel expression are a direct mechanism for regulation of peripheral hearing sensitivity among fishes. PMID:24803460

  1. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

  2. Altered Purkinje cell responses and calmodulin expression in the spontaneously ataxic mouse, Pogo.

    PubMed

    Lee, Kwan Young; Kim, Jin Seong; Kim, Se Hoon; Park, Hyung Seo; Jeong, Young-Gil; Lee, Nam-Seob; Kim, Dong Kwan

    2011-04-01

    Ataxia is often associated with altered cerebellar motor control, a process in which Purkinje cells (PCs) play a principal role. Pogo mice display severe motor deficits characterized by an ataxic gait accompanying hindlimb hyperextension. Here, using whole-cell patch-clamp recordings, we show that parallel fiber (PF)-excitatory post-synaptic currents (PF-EPSCs) are reduced, paired-pulse facilitation (PPF) is increased and PF-PC long-term depression (LTD) is impaired in Pogo mice; in contrast, climbing-fiber EPSCs are preserved. In control mice, treatment with the calmodulin antagonist calmidazolium (5 μm) impaired PPF and LTD. Notably, cerebellar calmodulin expression was significantly reduced in Pogo mice compared with control mice. Control PCs predominantly exhibited a tonic firing pattern, whereas the firing pattern in Pogo PCs was mainly a complex burst type. These results implicate alterations in PC responses and calmodulin content in the abnormal cerebellar function of Pogo mice.

  3. Dendritic remodeling of hippocampal neurons is associated with altered NMDA receptor expression in alcohol dependent rats

    PubMed Central

    Staples, Miranda C.; Kim, Airee; Mandyam, Chitra D.

    2015-01-01

    Prolonged alcohol exposure has been previously shown to impair the structure and function of the hippocampus, although the underlying structural and biochemical alterations contributing to these deleterious effects are unclear. Also unclear is whether these changes persist into prolonged periods of abstinence. Previous work from our lab utilizing a clinically relevant rodent model of alcohol consumption demonstrated that alcohol dependence (induced by chronic intermittent ethanol vapor exposure or CIE) decreases proliferation and survival of neural stem cells in the hippocampal subgranular zone and hippocampal neurogenesis in the dentate gyrus, implicating this region of the cortex as particularly sensitive to the toxic effects of prolonged ethanol exposure. For this study, we investigated seven weeks of CIE-induced morphological changes (dendritic complexity and dendritic spine density) of dentate gyrus (DG) granule cell neurons, CA3, and CA1 pyramidal neurons and the associated alterations in biochemical markers of synaptic plasticity and toxicity (NMDA receptors and PSD-95) in the hippocampus in ethanol-experienced Wistar rats 3h (CIE) and 21 days (protracted abstinence) after the last ethanol vapor exposure. CIE reduced dendritic arborization of DG neurons and this effect persisted into protracted abstinence. CIE enhanced dendritic arborization of pyramidal neurons and this effect did not persist into protracted abstinence. The architectural changes in dendrites did not correlate with alterations in dendritic spine density, however, they were associated with increases in the expression of pNR2B, total NR2B, and total NR2A immediately following CIE with expression levels returning to control levels in prolonged abstinence. Overall, these data provide the evidence that CIE produces profound changes in hippocampal structural plasticity and in molecular tools that maintain hippocampal structural plasticity, and these alterations may underlie cognitive dysfunction

  4. Rat embryo fibroblast cells expressing human papillomavirus 1a genes exhibit altered growth properties and tumorigenicity.

    PubMed Central

    Green, M; Brackmann, K H; Loewenstein, P M

    1986-01-01

    Human papillomavirus 1a (HPV1a) induces benign tumors (papillomas or warts) in humans under natural conditions of infection but has not been found to replicate significantly in cell culture or in experimental animals. To establish model systems to study the oncogenic properties and expression of HPV genes, we established cell lines by cotransfecting the 3Y1 rat fibroblast cell line with HPV1a DNA constructs containing an intact early gene region and the Tn5 neomycin resistance gene. Most cell lines selected for expression of the neomycin resistance gene by treatment with the antibiotic G-418 contained viral DNA in a high-molecular-weight form. The growth characteristics of several cell lines containing high copy numbers of HPV1a DNA were studied further. They were shown to differ from the parental cell line and from G-418-resistant cell lines that did not incorporate viral DNA in the following properties: morphological alteration, increased cell density at confluence, growth in 0.5% serum, efficient anchorage-independent growth in soft agar, and rapid formation of tumors in nude mice. Those cell lines that possessed altered growth properties and tumorigenicity were found to express abundant quantities of polyadenylated virus-specific RNA species in the cytoplasm. Images PMID:3023676

  5. Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

    PubMed Central

    Darbro, Benjamin W.; Schneider, Galen B.; Klingelhutz, Aloysius J.

    2007-01-01

    Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system. PMID:16117791

  6. Control of keratinocyte proliferation and differentiation by p63.

    PubMed

    Truong, Amy B; Khavari, Paul A

    2007-02-01

    The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKalpha and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.

  7. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    PubMed

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  8. Adrenergic stimulation alters the expression of inflammasome components and interleukins in primary human monocytes

    PubMed Central

    HORSTMANN, JOHANN-PHILIPP; MARZI, INGO; RELJA, BORNA

    2016-01-01

    Prior to their release, interleukin (IL)-1β and IL-18 are cleaved to their bioactive forms by a multiprotein complex known as an inflammasome, which is comprised of a number of elements that are subject to nuclear factor-κB-dependent transcription. Catecholamines have been indicated to exert an enhancing effect on the IL-1β release. The aim of the present study was to determine whether alterations in inflammasome gene expression may be responsible for the modified IL-1β and IL-18 secretion following lipopolysaccharide (LPS) and catecholamine co-stimulation. Monocytes were isolated from the peripheral blood of 21 healthy volunteers using CD14+ microbeads. Following stimulation with LPS (2 µg/ml) and/or phenylephrine (PE; 10 µM) for 24 h, the supernatants were subjected to ELISA to evaluate the ex vivo protein expression levels of IL-1β and IL-18. In addition, the gene expression levels of inflammasome components associated with the cleavage of IL-1β and IL-18, including NLRP1, NLRP3, caspase-1 and PYCARD were determined using polymerase chain reaction. The results indicated that LPS significantly increased IL-1β expression compared with the unstimulated control samples. Co-stimulation with LPS + PE significantly enhanced IL-1β expression compared with LPS alone. Furthermore, IL-18 expression was significantly reduced by LPS and LPS + PE co-stimulation. The gene expression levels of IL-18, NLRP1, caspase-1 and PYCARD were comparable in the LPS- and LPS + PE-stimulated cells. LPS significantly induced the expression levels of IL-1β and NLRP3, and to a lesser degree, the expression of NLRP1, compared with the control. By contrast, PE markedly induced the expression levels of IL-18 and NLRP1, while LPS reduced the gene expression of IL-18. In conclusion, adrenergic stimulation suppressed NLRP3 expression and enhanced NLRP1 expression, indicating that NLRP3 may regulate IL-1β secretion and NLRP1 may regulate the release of IL-18. PMID:26889257

  9. Ectopic expression of SOX9 in osteoblasts alters bone mechanical propertie

    PubMed Central

    Liang, Bojian; Cotter, Meghan M.; Chen, Dongxing; Hernandez, Christopher J.; Zhou, Guang

    2011-01-01

    Osteoporosis is a common skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. We previously demonstrated that Col1a1-SOX9 transgenic mice, in which SOX9 specifically expresses in osteoblasts driven by a 2.3kb Col1a1 promoter, display osteopenia during the early postnatal stage. In this study, to further analyze the osteopenia phenotype and especially the effect of the osteoblast-specific expression of SOX9 on bone mechanical properties, we performed bone geometry and mechanical property analysis of long bones from Col1a1-SOX9 transgenic mice (TG) and wild type littermates (WT) at different time points. Interestingly, after body weight adjustment, TG mice have similar whole-bone strength as WT mice, but exhibit significantly thinner cortical bone, lower elastic modulus, and higher moment of inertia. Thus, osteoblast-specific SOX9 expression results in altered bone structure and material properties. Furthermore, the expression levels of Pcna, Col1a1, Osteocalcin, and the Opg/Rankl ratio in TG mice are significantly lower until 4 months of age compared with WT mice, suggesting that TG mice have dysregulated bone homeostasis. Finally, bone marrow stromal cells (MSCs) isolated from TG mice display enhanced adipocyte differentiation and decreased osteoblast differentiation in vitro, suggesting that osteoblast-specific expression of SOX9 can lead to altered mesenchymal stem cell differentiation potentials. In conclusion, our study implies that SOX9 activity has to be tightly regulated in adult skeleton to ensure optimal bone quality. PMID:22143895

  10. Spaceflight alters expression of microRNA during T-cell activation

    PubMed Central

    Hughes-Fulford, Millie; Chang, Tammy T.; Martinez, Emily M.; Li, Chai-Fei

    2015-01-01

    Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.—Hughes-Fulford, M., Chang, T. T., Martinez, E. M., Li, C.-F. Spaceflight alters expression of microRNA during T-cell activation. PMID:26276131

  11. Haem oxygenase 1 expression is altered in monocytes from patients with systemic lupus erythematosus

    PubMed Central

    Herrada, Andrés A; Llanos, Carolina; Mackern-Oberti, Juan P; Carreño, Leandro J; Henriquez, Carla; Gómez, Roberto S; Gutierrez, Miguel A; Anegon, Ignacio; Jacobelli, Sergio H; Kalergis, Alexis M

    2012-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple functional alterations affecting immune cells, such as B cells, T cells, dendritic cells (DCs) and monocytes. During SLE, the immunogenicity of monocytes and DCs is significantly up-regulated, promoting the activation of self-reactive T cells. Accordingly, it is important to understand the contribution of these cells to the pathogenesis of SLE and the mechanisms responsible for their altered functionality during disease. One of the key enzymes that control monocyte and DC function is haem oxygenase-1 (HO-1), which catalyses the degradation of the haem group into biliverdin, carbon monoxide and free iron. These products possess immunosuppressive and anti-inflammatory capacities. The main goal of this work was to determine HO-1 expression in monocytes and DCs from patients with SLE and healthy controls. Hence, peripheral blood mononuclear cells were obtained from 43 patients with SLE and 30 healthy controls. CD14+ monocytes and CD4+ T cells were sorted by FACS and HO-1 expression was measured by RT-PCR. In addition, HO-1 protein expression was determined by FACS. HO-1 levels in monocytes were significantly reduced in patients with SLE compared with healthy controls. These results were confirmed by flow cytometry. No differences were observed in other cell types, such as DCs or CD4+ T cells, although decreased MHC-II levels were observed in DCs from patients with SLE. In conclusion, we found a significant decrease in HO-1 expression, specifically in monocytes from patients with SLE, suggesting that an imbalance of monocyte function could be partly the result of a decrease in HO-1 expression. PMID:22587389

  12. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit

    PubMed Central

    Ho, Emily V.; Klenotich, Stephanie J.; McMurray, Matthew S.; Dulawa, Stephanie C

    2016-01-01

    Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology. PMID:27861553

  13. Inferring causal genomic alterations in breast cancer using gene expression data

    PubMed Central

    2011-01-01

    Background One of the primary objectives in cancer research is to identify causal genomic alterations, such as somatic copy number variation (CNV) and somatic mutations, during tumor development. Many valuable studies lack genomic data to detect CNV; therefore, methods that are able to infer CNVs from gene expression data would help maximize the value of these studies. Results We developed a framework for identifying recurrent regions of CNV and distinguishing the cancer driver genes from the passenger genes in the regions. By inferring CNV regions across many datasets we were able to identify 109 recurrent amplified/deleted CNV regions. Many of these regions are enriched for genes involved in many important processes associated with tumorigenesis and cancer progression. Genes in these recurrent CNV regions were then examined in the context of gene regulatory networks to prioritize putative cancer driver genes. The cancer driver genes uncovered by the framework include not only well-known oncogenes but also a number of novel cancer susceptibility genes validated via siRNA experiments. Conclusions To our knowledge, this is the first effort to systematically identify and validate drivers for expression based CNV regions in breast cancer. The framework where the wavelet analysis of copy number alteration based on expression coupled with the gene regulatory network analysis, provides a blueprint for leveraging genomic data to identify key regulatory components and gene targets. This integrative approach can be applied to many other large-scale gene expression studies and other novel types of cancer data such as next-generation sequencing based expression (RNA-Seq) as well as CNV data. PMID:21806811

  14. Modulation of NFAT-5, an outlying member of the NFAT family, in human keratinocytes and skin

    PubMed Central

    Al-Daraji, Wael I; Afolayan, John; Zelger, Bettina G; Abdellaoui, Adel; Zelger, Bernhard

    2009-01-01

    Background Cyclosporin A (CsA) and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). NFAT compose a family of transcription factors that are turned on during T cell activation. Aims To study the expression of NFAT-5 mRNA and protein in normal human keratinocytes and to investigate the cellular and subcellular pattern of expression of NFAT-5 in normal human skin and psoriasis, and analyze effects of different agonists and ultraviolet radiation on NFAT-5 in normal human skin. Methods Tissue cultures, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Western analysis, immunostaining, confocal microscopy. Results Sequencing of RT-PCR products confirmed the identity of the product that showed 100 % homology with the predicted NFAT-5 sequence. anti-NFAT-5 mainly detected a single band in cultured keratinocytes and dermal fibroblasts using Western analysis. Immunohistochemistry showed that epidermal keratinocytes and dermal fibroblasts in normal human and psoriatic skin express NFAT-5. NFAT-5 showed predominantly nuclear localization in epidermal keratinocytes and dermal fibroblasts within five normal adult skin biopsies. Our data also suggest that UV irradiation reduces NFAT-5 nuclear localization within the epidermis. Unlike NFAT 1-4, NFAT-5/TonEBP was localized to both nucleus and cytoplasm of cultured keratinocytes. Cyclosporin A induces nuclear membrane translocation of NFAT-5 in cultured keratinocytes and raffinose (a hypertonicity inducing agent) induces more nuclear localization of NFAT-5 compared to untreated cells. In addition, differentiation-promoting agonists that induce sustained rise in intracellular calcium did not result in changes in NFAT-5 localization in cultured keratinocytes. Conclusion These studies provide the first observation of expression of NFAT-5/TonEBP mRNA protein in

  15. ALTERED EXPRESSION OF NEUROPLASTICITY-RELATED GENES IN THE BRAIN OF DEPRESSED SUICIDES

    PubMed Central

    FUCHSOVA, B.; ALVAREZ JULIÁ, A.; RIZAVI, H. S.; FRASCH, A. C.; PANDEY, G. N.

    2015-01-01

    Background Expression of the neuronal membrane glycoprotein M6a (GPM6A), the proteolipid protein (PLP/DM20) family member, is downregulated in the hippocampus of chronically stressed animals. Its neuroplastic function involves a role in neurite formation, filopodium outgrowth and synaptogenesis through an unknown mechanism. Disruptions in neuroplasticity mechanisms have been shown to play a significant part in the etiology of depression. Thus, the current investigation examined whether GPM6A expression is also altered in human depressed brain. Methods Expression levels and coexpression patterns of GPM6A, GPM6B, and PLP1 (two other members of PLP/DM20 family) as well as of the neuroplasticity-related genes identified to associate with GPM6A were determined using quantitative polymerase chain reaction (qPCR) in postmortem samples from the hippocampus (n =18) and the prefrontal cortex (PFC) (n= 25) of depressed suicide victims and compared with control subjects (hippocampus n= 18; PFC n =25). Neuroplasticity-related proteins that form complexes with GPM6A were identified by coimmunoprecipitation technique followed by mass spectrometry. Results Results indicated transcriptional downregulation of GPM6A and GPM6B in the hippocampus of depressed suicides. The expression level of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) and coronin1A (CORO1A) was also significantly decreased. Subsequent analysis of coexpression patterns demonstrated coordinated gene expression in the hippocampus and in the PFC indicating that the function of these genes might be coregulated in the human brain. However, in the brain of depressed suicides this coordinated response was disrupted. Conclusions Disruption of coordinated gene expression as well as abnormalities in GPM6A and GPM6B expression and expression of the components of GPM6A complexes were detected in the brain of depressed suicides. PMID:25934039

  16. Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors

    PubMed Central

    2010-01-01

    Background Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established - namely, the Swarm Rat Chondrosarcoma (SRC) - and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy. Methods To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC. Results The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression. Conclusion This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-β4 may have a role

  17. DNA copy-number alterations underlie gene expression differences between microsatellite stable and unstable colorectal cancers

    PubMed Central

    Jorissen, Robert N.; Lipton, Lara; Gibbs, Peter; Chapman, Matthew; Desai, Jayesh; Jones, Ian T.; Yeatman, Timothy J.; East, Philip; Tomlinson, Ian P.M.; Verspaget, Hein W.; Aaltonen, Lauri A.; Kruhøffer, Mogens; Ørntoft, Torben F.; Andersen, Claus Lindbjerg; Sieber, Oliver M.

    2008-01-01

    Purpose About 15% of colorectal cancers (CRCs) harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in CRCs, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression. Experimental Design DNA microarray data on 89 MSI and 140 MSS CRCs from this study, and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples, and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data. Results MSI-associated gene expression changes in CRCs were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P<0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion-binding and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P<0.001). Conclusions Our results demonstrate cross-study consistency of MSI-associated gene expression changes in CRCs. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers. PMID:19088021

  18. Oxaliplatin Alters Expression of T1R2 Receptor and Sensitivity to Sweet Taste in Rats.

    PubMed

    Ohishi, Akihiro; Nishida, Kentaro; Yamanaka, Yuri; Miyata, Ai; Ikukawa, Akiko; Yabu, Miharu; Miyamoto, Karin; Bansho, Saho; Nagasawa, Kazuki

    2016-01-01

    As one of the adverse effects of oxaliplatin, a key agent in colon cancer chemotherapy, a taste disorder is a severe issue in a clinical situation because it decreases the quality of life of patients. However, there is little information on the mechanism underlying the oxaliplatin-induced taste disorder. Here, we examined the molecular and behavioral characteristics of the oxaliplatin-induced taste disorder in rats. Oxaliplatin (4-16 mg/kg) was administered to Sprague-Dawley (SD) rats intraperitoneally for 2 d. Expression levels of mRNA and protein of taste receptors in circumvallate papillae (CP) were measured by real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. Taste sensitivity was assessed by their behavioral change using a brief-access test. Morphological change of the taste buds in CP was evaluated by hematoxyline-eosin (HE) staining, and the number of taste cells in taste buds was counted by immunohistochemical analysis. Among taste receptors, the expression levels of mRNA and protein of T1R2, a sweet taste receptor subunit, were increased transiently in CP of oxaliplatin-administered rats on day 7. In a brief-access test, the lick ratio was decreased in oxaliplatin-administered rats on day 7 and the alteration was recovered to the control level on day 14. There was no detectable alteration in the morphology of taste buds, number of taste cells or plasma zinc level in oxaliplatin-administered rats. These results suggest that decreased sensitivity to sweet taste in oxaliplatin-administered rats is due, at least in part, to increased expression of T1R2, while these alterations are reversible.

  19. Altered expression of glycosaminoglycans in metastatic 13762NF rat mammary adenocarcinoma cells

    SciTech Connect

    Steck, P.A.; Cheong, P.H.; Nakajima, M.; Yung, W.K.A.; Moser, R.P.; Nicolson, G.L.

    1987-02-24

    A difference in the expression and metabolism of (/sup 35/S)sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate. These results suggested that altered glycosaminoglycan expression and metabolism may be associated with the metastatic process in 13762NF rat mammary tumor cells.

  20. Chronic maternal morphine alters calbindin D-28k expression pattern in postnatal mouse brain.

    PubMed

    Mithbaokar, Pratibha; Fiorito, Filomena; Della Morte, Rossella; Maharajan, Veeramani; Costagliola, Anna

    2016-01-01

    The distribution pattern of calbindin (CB)-D28k-expressing neurons results to be altered in several brain regions of chronic morphine exposed adult mice. In this study, the influence of chronic maternal exposure to morphine on the distribution pattern of CB-D28k-expressing neurons in the brain of mouse offspring was investigated. Females of CD-1 mice were daily administered with saline or morphine for 7 days before mating, during the whole gestation period, and until 21 day post-partum. Their offspring were sacrificed on postnatal day 18, and the brains were examined by histology using cresyl violet and by immunohistochemistry using a rabbit polyclonal anti-CB-D28k antibody. Histology revealed no significant differences in the distribution pattern and the number of neurons between the offspring forebrain of the control group of mice and the two groups of mice treated with different doses of morphine. However, immunohistochemical analysis revealed that the number of CB-D28k-immunoreactive neurons remarkably decreased in the cingulate cortex, in the layers II-IV of the parietal cortex and in all regions of the hippocampus, while it increased in the layers V-VI of the parietal cortex and in the subicular region of the offspring brain of morphine treated mice. Overall, our findings demonstrate that maternal exposure to morphine alters the pattern of CB-D28k-expressing neuron pattern in specific regions of murine developing brain, in a layer- and dose-dependent way, thus suggesting that these alterations might represent a mechanism by which morphine modifies the functional aspects of developing brain.

  1. Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

    PubMed

    Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

    2015-02-01

    We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.

  2. Mice lacking integrin β3 expression exhibit altered response to chronic stress

    PubMed Central

    Varney, Seth; Polston, Keith F.; Jessen, Tammy; Carneiro, Ana M.D.

    2015-01-01

    Recent studies indicate multiple roles for integrin αvβ3 in adult neurons, including response to pharmacological agents such as cocaine and selective serotonin reuptake inhibitors. In this study, we examined the role of the integrin β3 gene (Itgb3) in the response to environmental stimuli by subjecting Itgb3+/+ and Itgb3−/− mice to unpredictable chronic mild stressors. We found that genetic abrogation of integrin β3 expression elicits an exaggerated vulnerability to chronic unpredictable stress in the open field test. In this test, chronic stress elicited significant decreases in stereotypic behavior and horizontal locomotor activity, including increases in anxiety behaviors. Mild chronic stress led to reductions in dopamine turnover in midbrains of Itgb3+/+, but not Itgb3−/− mice, suggesting a disruption of stress-dependent regulation of DA homeostasis. Chronic stress elicited altered synaptic expression of syntaxin and synaptophysin in midbrains of Itgb3−/− mice, when compared to Itgb3+/+. Semi-quantitative Western blot studies revealed that the synaptic expression, but not total tissue expression, of multiple signaling proteins is correlated with integrin αv levels in the midbrain. Moreover, loss of integrin β3 expression modifies this correlation network. Together, these findings demonstrate that Itgb3−/− mice display a pattern of changes indicating disrupted regulation of midbrain synaptic systems involved in conferring resilience to mild stressors. PMID:26634222

  3. Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

    PubMed

    Bonaccorsi, Lorella; Luciani, Paola; Nesi, Gabriella; Mannucci, Edoardo; Deledda, Cristiana; Dichiara, Francesca; Paglierani, Milena; Rosati, Fabiana; Masieri, Lorenzo; Serni, Sergio; Carini, Marco; Proietti-Pannunzi, Laura; Monti, Salvatore; Forti, Gianni; Danza, Giovanna; Serio, Mario; Peri, Alessandro

    2008-10-01

    Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

  4. A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology

    PubMed Central

    Mullen, Brian R.; Ross, Brennan; Chou, Joan Wang; Khankan, Rana; Khialeeva, Elvira; Bui, Kimberly

    2016-01-01

    Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors—reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon—gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology. PMID:27364165

  5. West Nile Virus Infection Alters Midgut Gene Expression in Culex pipiens quinquefasciatus Say (Diptera: Culicidae)

    PubMed Central

    Smartt, Chelsea T.; Richards, Stephanie L.; Anderson, Sheri L.; Erickson, Jennifer S.

    2009-01-01

    Alterations in gene expression in the midgut of female Culex pipiens quinquefasciatus exposed to blood meals containing 6.8 logs plaque-forming units/mL of West Nile virus (WNV) were studied by fluorescent differential display. Twenty-six different cDNAs exhibited reproducible differences after feeding on infected blood. Of these, 21 cDNAs showed an increase in expression, and 5 showed a decrease in expression as a result of WNV presence in the blood meal. GenBank database searches showed that one clone with increased expression, CQ G12A2, shares 94% identity with a leucine-rich repeat-containing protein from Cx. p. quinquefasciatus and 32% identity to Toll-like receptors from Aedes aegypti. We present the first cDNA clone isolated from female Cx. p. quinquefasciatus midgut tissue whose expression changes on exposure to WNV. This cDNA represents a mosquito gene that is an excellent candidate for interacting with WNV in Cx. p. quinquefasciatus and may play a role in disease transmission. PMID:19635880

  6. Localisation of Neuregulin 1-{beta}3 to different sub-nuclear structures alters gene expression

    SciTech Connect

    Wang, Ming; Trim, Carol M.; Gullick, William J.

    2011-02-15

    Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-{beta}3 (NRG1-{beta}3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-{beta}3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-{beta}3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-{beta}3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B'. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.

  7. Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster

    SciTech Connect

    Slusarski, D.C.; Motzny, C.K.; Holmgren, R.

    1995-01-01

    The cubitus interruptus (ci) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode tra-1 gene. Three classes of existing mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. The first class of ci mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci product in the posterior compartment of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks the formation of the ventral naked cuticle. The loss of ci expression at these two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique ci{sup Ce} mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In ci{sup Ce}/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development. 47 refs., 9 figs., 1 tab.

  8. Absence of caveolin-1 alters heat shock protein expression in spontaneous mammary tumors driven by Her-2/neu expression.

    PubMed

    Ciocca, Daniel R; Cuello-Carrión, F Darío; Natoli, Anthony L; Restall, Christina; Anderson, Robin L

    2012-02-01

    In a previous study, we measured caveolin-1 protein levels, both in the normal breast and in breast cancer. The study revealed no association between caveolin-1 expression in the epithelial compartment and clinical disease outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor associated strongly with reduced metastasis and improved survival. Using an animal model, we found that the onset of mammary tumors driven by Her-2/neu expression was accelerated in mice lacking caveolin-1. We have analysed the heat shock protein (Hsp) response in the tumors of mice lacking caveolin-1. In all cases, the mammary tumors were estrogen and progesterone receptor negative, and the levels of Her-2/neu (evaluated by immunohistochemistry) were not different between the caveolin-1 +/+ (n = 8) and the caveolin-1 -/- (n = 7) tumors. However, a significant reduction in the extent of apoptosis was observed in mammary tumors from animals lacking caveolin-1. While Bcl-2, Bax, and survivin levels in the tumors were not different, the amount of HSPA (Hsp70) was almost double in the caveolin-1 -/- tumors. In contrast, HSPB1 (Hsp27/Hsp25) levels were significantly lower in the caveolin-1 -/- tumors. The mammary tumors from caveolin-1 null mice expressed more HSPC4 (gp96 or grp94), but HSPC1 (Hsp90), HSPA5 (grp78), HSPD1 (Hsp60), and CHOP were not altered. No significant changes in these proteins were found in the stroma surrounding these tumors. These results demonstrate that the disruption of the Cav-1 gene can cause alterations of specific Hsps as well as tumor development.

  9. ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes

    PubMed Central

    Dakic, Aleksandra; DiVito, Kyle; Fang, Shuang; Suprynowicz, Frank; Gaur, Anirudh; Li, Xin; Palechor-Ceron, Nancy; Simic, Vera; Choudhury, Sujata; Yu, Songtao; Simbulan-Rosenthal, Cynthia M.; Rosenthal, Dean; Schlegel, Richard; Liu, Xuefeng

    2016-01-01

    The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death. PMID:27556514

  10. Genes and small RNA transcripts exhibit dosage-dependent expression pattern in maize copy-number alterations