The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and β-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of β-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that β-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of β-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of β-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and ¿-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of ¿-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that ¿-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of ¿-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of ¿-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T22:05:12Z (GMT). No. of bitstreams: 1 Talero_LuisAlbertoPedroza_D.pdf: 1538530 bytes, checksum: 6dc94ec71cdd6f03d096be015a2b1757 (MD5) Previous issue date: 2009 / Resumo: A Poliendocrinopatia autoimune associada a candidíase e distrofia ectodérmica (APECED) é um síndrome caracterizado pela presença de pelo menos dois sintomas clínicos, endocrinopatia autoimune, sendo que as mais comuns são hipoparatiroidismo, doença de Addison, além de candidíase mucocutânea crônica. É também comum nos pacientes o desenvolvimento de distrofia ectodérmica, como distrofia nas unhas ou alopécia. O APECED é produzido por mutações no gene AIRE, que codifica uma proteína com propriedades reguladoras na transcrição de proteínas ectópicas no timo, o que estaria envolvido na seleção negativa de células T auto-reativas, e conseqüentemente no desenvolvimento da doença autoimune. No entanto a associação da deficiência da proteína AIRE com a suscetibilidade a candidíase ou a distrofia ectodérmica permanecem obscuras. No presente trabalho, investigamos a possibilidade que esta associação esteja envolvida com a expressão e função da proteína AIRE no ambiente extra-tímico. Usando células de sangue periférico de pacientes com mutações no AIRE, e a técnica de SiRNA para silenciar este gene em células de linhagem mielomonocítica THP-1, demonstramos que a proteína AIRE é importante para a resposta via KF-kB dos TLRs e Dectina-1, sendo que AIRE está presente num complexo com Dectina-1, Syk e Card-9, formado após o estímulo com Curdlan. Além disso demonstramos que a formação deste complexo pode acontecer no citoplasma ou membrana citoplasmática, uma vez que após este estímulo, a proteína AIRE é exportada do núcleo permanecendo temporariamente na membrana. Finalmente usando a técnica de espectroscopia de massa e microscopia confocal, mostramos que AIRE interage com a proteína Queratina 17, tanto em células THP-1 como em células HaCaT (linhagem de queratinócitos), quando as células entram num estágio de espraiamento e migração. Assim, a presença da proteína AIRE na via de sinalização da Dectina-1, pode estar relacionada com a susceptibilidade a infecções crônicas por C. albicans observada nestes pacientes. A resposta imune via Dectina-1 é importante na resposta a este fungo e defeitos na molécula CARD9 e Dectina-1 podem estar associados a Candidíase mucocutânea crônica. Por outro lado, a descrição da associação de AIRE com K17 pode ser relevante, já que pacientes com mutações no gene que codifica para a proteína K17 desenvolvem uma doença chamada paquioníquia congênita, caracterizada por distrofia das unhas e alopécia, características clínicas observadas também nos pacientes com APECED. Deste modo, neste trabalho apresentamos evidências que apontam para um novo papel funcional da proteína AIRE no ambiente extratímico, que poderia explicar em parte algumas características clínicas dos pacientes com APECED, como a elevada suscetibilidade a infecções por C. albicans, e a distrofia ectodérmica / Abstract: The autoimmune polyendocrinopathy candidiasis and ectodermal dystrophy (APECED) is characterized by the presence of two from three major clinical symptoms: Addison's disease, and/or hypoparathyroidism, and/or chronic mucocutaneous candidiasis. These patients develop also ectodermal dystrophies like nail dystrophy and alopecia. APECED is caused by mutations in the autoimmune regulator gene (AIRE). This gene encodes a protein with DNA binding capacity that can transcriptionally modulate ectopic peripheral tissue antigen (PTA) expression in the thymus, facilitating T cell negative selection. Defects in AIRE may be related with the development of multipleendocrine failure of autoimmune origin in patients with APECED. In spite of this, the role of AIRE deficiency in the C. albicans susceptibility or ectodermal dystrophy, common features in APECED patients, remains to be elucidated. In the present work we explored the hypothesis that candidiasis and ectodermal dystrophy are associated with the extra-thymic role of AIRE. For this we used peripheral blood mononuclear cells from APECED patients, and also THP-1 cells treated with SiRNA for AIRE gene to obtain AIRE deficient cells. We demonstrated that AIRE is required for Dectin-1- and TLR-ligand-induced inflammatory response and complexes with Dectin-1, Syk, and CARD9 after Curdlan stimulation. In addition, we showed that this complex formation takes place outside the nucleus, once that after Curdlan stimulation AIRE seems to be exported to the cytoplasm and transiently locate at the cytoplasmic membrane. Finally using mass spectra and confocal microscopy, we showed an interaction between AIRE and the intermediate filament protein Keratin-17, in both THP-1 cells and the keratinocyte cell line HaCaT. Therefore, the presence of AIRE protein in Dectin-1 pathway seems to be important on the C. albicans response, and the absence of this protein could be a risk factor important for developing candidiasis, commonly observed in APECED patients. This observation is supported by the fact that Dectin-1 is important for C albicans response, and also the recently description of mutations in Dectin-1 and CARD9 and its association with chronic mucocutaneous candidiasis. On the other hand, the description of AIRE and K17 association is important, since patients with defects on K17 gene develop congenital pachyonychia, a disease characterized by nail dystrophy and alopecia, also observed in APECED patients. Thus we provided evidence for a new role of AIRE protein in the extrathymic environment, which in may explain, at least in part, some of the common clinical features other than autoimmunity, observed in APECED patients / Doutorado / Saude da Criança e do Adolescente / Doutor em Saude da Criança e do Adolescente