Exploring Molecular Mechanisms Controlling Skin Homeostasis and Hair Growth. MicroRNAs in Hair-cycle-Dependent Gene Regulation, Hair Growth and Associated Tissue Remodelling.

Abstract

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.