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1.
Exp Dermatol ; 27(10): 1142-1151, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30033522

RESUMO

We have reported recently that inactivation of the essential autophagy-related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP-LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age-matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5-fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58-60 carbon atoms) were increased, while shorter species (54-55 carbon atoms) were under-represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Autofagia/genética , Glândulas Sebáceas/patologia , Glândulas Sebáceas/fisiopatologia , Sebo/química , Animais , Autofagossomos , Proliferação de Células/genética , Colesterol/análise , Ácidos Graxos não Esterificados/análise , Cabelo , Masculino , Camundongos , Fenótipo , Ceras/análise
3.
Parasit Vectors ; 10(1): 205, 2017 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-28441957

RESUMO

BACKGROUND: Vector-pathogen dynamics are controlled by fluctuations of potential vector communities, such as the Culicidae. Assessment of mosquito community diversity and, in particular, identification of environmental parameters shaping these communities is therefore of key importance for the design of adequate surveillance approaches. In this study, we assess effects of climatic parameters and habitat structure on mosquito communities in eastern Austria to deliver these highly relevant baseline data. METHODS: Female mosquitoes were sampled twice a month from April to October 2014 and 2015 at 35 permanent and 23 non-permanent trapping sites using carbon dioxide-baited traps. Differences in spatial and seasonal abundance patterns of Culicidae taxa were identified using likelihood ratio tests; possible effects of environmental parameters on seasonal and spatial mosquito distribution were analysed using multivariate statistical methods. We assessed community responses to environmental parameters based on 14-day-average values that affect ontogenesis. RESULTS: Altogether 29,734 female mosquitoes were collected, and 21 of 42 native as well as two of four non-native mosquito species were reconfirmed in eastern Austria. Statistical analyses revealed significant differences in mosquito abundance between sampling years and provinces. Incidence and abundance patterns were found to be linked to 14-day mean sunshine duration, humidity, water-level maxima and the amount of precipitation. However, land cover classes were found to be the most important factor, effectively assigning both indigenous and non-native mosquito species to various communities, which responded differentially to environmental variables. CONCLUSIONS: These findings thus underline the significance of non-climatic variables for future mosquito prediction models and the necessity to consider these in mosquito surveillance programmes.


Assuntos
Culicidae/crescimento & desenvolvimento , Ecossistema , Meio Ambiente , Insetos Vetores , Animais , Áustria , Clima , Culicidae/classificação , Densidade Demográfica , Estações do Ano , Análise Espaço-Temporal
4.
J Invest Dermatol ; 137(3): 587-594, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27771328

RESUMO

Sebaceous glands produce sebum via holocrine secretion, a largely uncharacterized mode of programmed cell death that contributes to the homeostasis and barrier function of the skin. To determine the mechanism of DNA degradation during sebocyte cell death, we have inactivated candidate DNA-degrading enzymes by targeted gene deletions in mice. DNase1 and DNase1-like 2 were dispensable for nuclear DNA degradation in sebocytes. By contrast, epithelial cell-specific deletion of lysosomal DNase2 blocked DNA degradation in these cells. DNA breakdown during sebocyte differentiation coincided with the loss of LAMP1 and was accelerated by the abrogation of autophagy, the central cellular program of lysosome-dependent catabolism. Suppression of DNA degradation by the deletion of DNase2 resulted in aberrantly increased concentrations of residual DNA and decreased amounts of the DNA metabolite uric acid in secreted sebum. These results define holocrine secretion as a DNase2-mediated form of programmed cell death and suggest that autophagy-dependent metabolism, DNA degradation, and the molecular composition of sebum are mechanistically linked.


Assuntos
Apoptose , Endodesoxirribonucleases/metabolismo , Glândulas Sebáceas/metabolismo , Sebo/citologia , Animais , DNA , DNA Mitocondrial/metabolismo , Endodesoxirribonucleases/genética , Células Epiteliais/citologia , Histonas/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/metabolismo , Ácido Úrico/metabolismo
5.
Int J Biochem Cell Biol ; 81(Pt B): 375-382, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27732890

RESUMO

Autophagy is a recycling program which allows cells to adapt to metabolic needs and to stress. Defects in autophagy can affect metabolism, aging, proteostasis and inflammation. Autophagy pathway genes, including autophagy related 7 (Atg7), have been associated with the regulation of skin pigmentation, and autophagy defects disturb the biogenesis and transport of melanosomes in melanocytes as well as transfer and processing of melanin into keratinocytes. We have previously shown that mice whose melanocytes or keratinocytes lack Atg7 (and thus autophagy) as a result of specific gene knockout still retained functioning melanosome synthesis and transfer, and displayed only moderate reduction of pigmentation. In cell culture the Atg7 deficient melanocytes were prone to premature senescence and dysregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling. To elucidate the biochemical basis of this phenotype, we performed a study on global gene expression, protein secretion and phospholipid composition in Atg7 deficient versus Atg7 expressing melanocytes. In cell culture Atg7 deficient melanocytes showed a pro-inflammatory gene expression signature and secreted higher levels of C-X-C motif chemokine ligand -1,-2,-10 and -12 (Cxcl1, Cxcl2, Cxcl10, Cxcl12), which are implicated in the pathogenesis of pigmentary disorders and expressed higher amounts of matrix metalloproteinases -3 and -13 (Mmp3, Mmp13). The analysis of membrane phospholipid composition identified an increase in the arachidonic- to linoleic acid ratio in the autophagy deficient cells, as well as an increase in oxidized phospholipid species that act as danger associated molecular patterns (DAMPs). The secretion of inflammation related factors suggests that autophagy deficient melanocytes display a senescence associated secretory phenotype (SASP), and we propose oxidized lipid mediators as novel components of this SASP.


Assuntos
Envelhecimento , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Metabolismo dos Lipídeos , Melanócitos/citologia , Melanócitos/metabolismo , Animais , Autofagia/genética , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Lipídeos/química , Camundongos , Fenótipo , Reação em Cadeia da Polimerase
6.
PLoS One ; 11(8): e0161640, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27537685

RESUMO

Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.


Assuntos
Proteína 7 Relacionada à Autofagia/fisiologia , Epitélio Pigmentado da Retina/metabolismo , cis-trans-Isomerases/metabolismo , Animais , Autofagia/genética , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Western Blotting , Feminino , Imunofluorescência , Deleção de Genes , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monofenol Mono-Oxigenase/metabolismo
7.
Parasit Vectors ; 9: 197, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27067139

RESUMO

BACKGROUND: Culex pipiens complex taxa differ in behaviour, ecophysiology and epidemiologic importance. Despite their epidemiologic significance, information on genetic diversity, occurrence and seasonal and spatial distribution patterns of the Cx. pipiens complex is still insufficient. Assessment of seasonal and spatial distribution patterns of Culex pipiens forms and their congener Cx. torrentium is crucial for the understanding of their vector-pathogen dynamics. METHODS: Female mosquitoes were trapped from April-October 2014 twice a month for a 24-h time period with BG-sentinel traps at 24 sampling sites in eastern Austria, using carbon dioxide as attractant. Ecological forms of Cx. pipiens s.l. and their hybrids were differentiated using the CQ11 locus, and Cx. pipiens forms and their congener Cx. torrentium using the ACE-2 gene. Differential exploitation of ecological niches by Cx. pipiens forms and Cx. torrentium was analysed using likelihood ratio tests. Possible effects of environmental parameters on these taxa were tested using PERMANOVA based on distance matrices and, if significant, were modelled in nMDS ordination space to estimate non-linear relationships. RESULTS: For this study, 1476 Culex spp. were sampled. Culex pipiens f. pipiens representing 87.33 % of the total catch was most abundant, followed by hybrids of both forms (5.62 %), Cx. torrentium (3.79 %) and Cx. pipiens f. molestus (3.25 %). Differences in proportional abundances were found between land cover classes. Ecological parameters affecting seasonal and spatial distribution of these taxa in eastern Austria are precipitation duration, air temperature, sunlight and the interaction term of precipitation amount and the Danube water level, which can be interpreted as a proxy for breeding habitat availability. CONCLUSIONS: The Cx. pipiens complex of eastern Austria comprises both ecologically different forms, the mainly ornithophilic form pipiens and the mainly mammalophilic and anthropophilic form molestus. Heterogeneous agricultural areas as areas of coexistence may serve as hybridization zones, resulting in potential bridge vectors between birds and humans. Occurrence, seasonal and spatial distribution patterns of the Cx. pipiens complex and Cx. torrentium and the presence of hybrids between both forms were quantified for the first time in Austria. These findings will improve the knowledge of their vector-pathogen dynamics in this country.


Assuntos
Biota , Culex/classificação , Culex/crescimento & desenvolvimento , Variação Genética , Animais , Áustria , Culex/genética , Feminino , Estações do Ano , Análise de Sequência de DNA , Análise Espaço-Temporal
8.
J Invest Dermatol ; 135(5): 1348-1357, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25290687

RESUMO

Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.


Assuntos
Senilidade Prematura/fisiopatologia , Antioxidantes/metabolismo , Autofagia/fisiologia , Senescência Celular/fisiologia , Melanócitos/metabolismo , Melanócitos/patologia , Senilidade Prematura/metabolismo , Animais , Proteína 7 Relacionada à Autofagia , Proliferação de Células/fisiologia , Células Cultivadas , Homeostase/fisiologia , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/fisiologia , Melaninas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Autophagy ; 11(2): 298-313, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25484081

RESUMO

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.


Assuntos
Autofagia/fisiologia , Glândula de Harder/metabolismo , Lisossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glândula de Harder/efeitos dos fármacos , Lisossomos/patologia , Camundongos , Inibidores de Proteassoma/metabolismo , Vacúolos/metabolismo
10.
J Dermatol Sci ; 71(1): 67-75, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23669018

RESUMO

BACKGROUND: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects. OBJECTIVE: This study tested whether autophagy is essential for epidermal barrier formation and function. METHODS: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7Δepi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays. RESULTS: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7Δepi epidermis. Atg7Δepi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7Δepi mice. CONCLUSION: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin.


Assuntos
Autofagia , Epiderme/metabolismo , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Absorção Cutânea , Animais , Proteína 7 Relacionada à Autofagia , Diferenciação Celular , Células Cultivadas , Difusão , Epiderme/ultraestrutura , Proteínas de Fluorescência Verde , Queratina-14/genética , Queratinócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Permeabilidade , Regiões Promotoras Genéticas , Perda Insensível de Água
11.
J Invest Dermatol ; 133(6): 1629-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23340736

RESUMO

The skin is exposed to environmental insults such as UV light that cause oxidative damage to macromolecules. A centerpiece in the defense against oxidative stress is the Nrf2 (nuclear factor (erythroid-derived-2)-like 2)-mediated transcriptional upregulation of antioxidant and detoxifying enzymes and the removal of oxidatively damaged material. Autophagy has an important role in the intracellular degradation of damaged proteins and entire organelles, but its role in the epidermis has remained elusive. Here, we show that both UVA and UVA-oxidized phospholipids induced autophagy in epidermal keratinocytes. Oxidative stressors induced massive accumulation of high-molecular-weight protein aggregates containing the autophagy adaptor protein p62/SQSTM1 in autophagy-deficient (autophagy-related 7 (ATG7) negative) keratinocytes. Strikingly, even in the absence of exogenous stress, the expression of Nrf2-dependent genes was elevated in autophagy-deficient keratinocytes. Furthermore, we show that autophagy-deficient cells contained significantly elevated levels of reactive oxidized phospholipids. Thus, our data demonstrate that autophagy is crucial for both the degradation of proteins and lipids modified by environmental UV stress and for limiting Nrf2 activity in keratinocytes. Lipids that promote inflammation and tissue damage because of their reactivity and signaling functions are commonly observed in aged and diseased skin, and thus targeting autophagy may be a promising strategy to counteract the damage promoted by excessive lipid oxidation.


Assuntos
Autofagia/efeitos da radiação , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Fosfolipídeos/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia , Células Cultivadas , Epiderme/patologia , Epiderme/efeitos da radiação , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Fosfolipídeos/genética , Regulação para Cima/fisiologia , Regulação para Cima/efeitos da radiação
12.
Biochem Biophys Res Commun ; 430(2): 689-94, 2013 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-23211599

RESUMO

Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.


Assuntos
Autofagia/imunologia , Queratina-5/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Timo/imunologia , Animais , Autoimunidade , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Peso Corporal/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Deleção de Genes , Marcação de Genes , Queratina-15 , Queratina-5/genética , Camundongos , Camundongos Transgênicos , Timo/citologia , Ganho de Peso/genética
13.
PLoS One ; 7(6): e38933, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719991

RESUMO

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.


Assuntos
Autofagia , Modelos Animais , Tolerância a Antígenos Próprios , Timo/imunologia , Animais , Western Blotting , Feminino , Proteínas de Fluorescência Verde/genética , Camundongos
14.
J Invest Dermatol ; 131(6): 1208-15, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21307874

RESUMO

Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.


Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Desoxirribonuclease I/fisiologia , Cabelo/metabolismo , Casco e Garras/metabolismo , Queratinócitos/enzimologia , Animais , Apoptose , DNA Mitocondrial/metabolismo , Camundongos , Estresse Mecânico
15.
Am J Pathol ; 173(3): 689-99, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18688025

RESUMO

Primary abnormalities in permeability barrier function appear to underlie atopic dermatitis and epidermal trauma; a concomitant barrier dysfunction could also drive other inflammatory dermatoses, including psoriasis. Central to this outside-inside view of disease pathogenesis is the epidermal generation of cytokines/growth factors, which in turn signal downstream epidermal repair mechanisms. Yet, this cascade, if sustained, signals downstream epidermal hyperplasia and inflammation. We found here that acute barrier disruption rapidly stimulates mRNA and protein expression of epidermal vascular endothelial growth factor-A (VEGF-A) in normal hairless mice, a specific response to permeability barrier requirements because up-regulation is blocked by application of a vapor-impermeable membrane. Moreover, epidermal vegf(-/-) mice display abnormal permeability barrier homeostasis, attributable to decreased VEGF signaling of epidermal lamellar body production; a paucity of dermal capillaries with reduced vascular permeability; and neither angiogenesis nor epidermal hyperplasia in response to repeated tape stripping (a model of psoriasiform hyperplasia). These results support a central role for epidermal VEGF in the maintenance of epidermal permeability barrier homeostasis and a link between epidermal VEGF production and both dermal angiogenesis and the development of epidermal hyperplasia. Because psoriasis is commonly induced by external trauma [isomorphic (Koebner) phenomenon] and is associated with a prominent permeability barrier abnormality, excess VEGF production, prominent angiogenesis, and epidermal hyperplasia, these results could provide a potential outside-inside mechanistic basis for the development of psoriasis.


Assuntos
Epiderme/metabolismo , Epiderme/patologia , Homeostase/fisiologia , Neovascularização Patológica/metabolismo , Psoríase/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Northern Blotting , Derme/irrigação sanguínea , Derme/metabolismo , Hiperplasia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Pelados , Camundongos Knockout , Permeabilidade
16.
FASEB J ; 21(14): 3994-4004, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17625068

RESUMO

To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo-alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up-regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC-labeled albumin injected intravenously (i.v.) into lactating mice extravasated rapidly and accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombinant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium-derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.


Assuntos
Células Epiteliais/metabolismo , Inativação Gênica/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Diferenciação Celular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lactação/fisiologia , Masculino , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Transgênicos , Leite/metabolismo , Gravidez , Fator A de Crescimento do Endotélio Vascular/genética
17.
Differentiation ; 73(8): 406-13, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16316411

RESUMO

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.


Assuntos
Caspases/metabolismo , Epiderme/embriologia , Epiderme/enzimologia , Queratinócitos/metabolismo , Animais , Caspase 14 , Caspase 3 , Diferenciação Celular , Ativação Enzimática , Queratinócitos/enzimologia , Camundongos
18.
Cancer Res ; 64(10): 3508-16, 2004 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15150105

RESUMO

The angiogenic cytokine vascular endothelial growth factor (VEGF)-A plays a central role in both wound healing and tumor growth. In the skin, epidermal keratinocytes are a major source of this growth factor. To study the contribution of keratinocyte-derived VEGF-A to these angiogenesis-dependent processes, we generated mice in which this cytokine was inactivated specifically in keratin 5-expressing tissues. The mutant mice were macroscopically normal, and the skin capillary system was well established, demonstrating that keratinocyte-derived VEGF-A is not essential for angiogenesis in the skin during embryonic development. However, healing of full-thickness wounds in adult animals was appreciably delayed compared with controls, with retarded crust shedding and the appearance of a blood vessel-free zone underneath the newly formed epidermis. When 9,12-dimethyl 1,2-benzanthracene was applied as both tumor initiator and promoter, a total of 143 papillomas developed in 20 of 23 (87%) of control mice. In contrast, only three papillomas arose in 2 of 17 (12%) of the mutant mice, whereas the rest merely displayed epidermal thickening and parakeratosis. Mutant mice also developed only 2 squamous cell carcinomas, whereas 11 carcinomas were found in seven of the control animals. These data demonstrate that whereas keratinocyte-derived VEGF-A is dispensable for skin vascularization under physiological conditions, it plays an important albeit nonessential role during epidermal wound healing and is crucial for the development of 9,12-dimethyl 1,2-benzanthracene-induced epithelial skin tumors.


Assuntos
Queratinócitos/fisiologia , Papiloma/irrigação sanguínea , Papiloma/prevenção & controle , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/fisiologia , Cicatrização/fisiologia , Animais , Éxons , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Papiloma/induzido quimicamente , Papiloma/metabolismo , Pele/irrigação sanguínea , Pele/citologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genética
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