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

      Botchkareva, Natalia V.; Ahmed, Mohammed I. (University of BradfordCentre for Skin Sciences, Division of Biomedical Sciences, School of life Sciences, 2011-11-14)
      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.
    • Identification of human hair follicle antigens targeted in the presumptive autoimmune hair follicle disorder Alopecia Areata and their potential functional relevance In Vitro. Methods development for isolation and identification of Alopecia Areata-relevant human hair follicle antigens using a proteomics approach and their functional assessment using an Ex Vivo hair follicle organ culture model.

      Tobin, Desmond J.; Leung, Man Ching (University of BradfordDepartment of Biomedical Sciences, 2010-06-09)
      Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle¿s, less in Huxley¿s/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.
    • An integrative bioinformatics approach for analyses of multi-level transcriptional regulation and three-dimensional organization in the epidermis and skin appendages. Exploring genomic transcriptional profiles of the distinct stages of hair follicle and sweat gland development and analyses of mechanism integrating the transcriptional regulation, linear and high-order genome organization within epidermal differentiation complex in keratinocytes.

      Botchkarev, Vladimir A.; Peng, Yonghong; Poterlowicz, Krzysztof (University of BradfordSchool of Life Sciences, 2013-11-04)
      The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.
    • The role of the SWI/SNF ATP dependent chromatin remodelling complex in the regulation of the human hair follicle cell proliferation and control of the human cutaneous wound healing

      Fessing, Michael Y.; Botchkareva, Natalia V.; Kellett, Carl W. (University of BradfordFaculty of Life sciences, 2018)
      Epigenetic regulation of gene expression occurs at a number of levels including covalent DNA and histone modifications, nucleosome positioning and ATP-dependent chromatin remodelling as well as higher order chromatin folding and 3D genome organisation. ATP-dependent chromatin remodelling complexes modulate nucleosome structure, positioning and chromatin de-compaction and are involved in gene activation and repression. SWI/SNF ATP-dependent chromatin remodelling complexes contain either BRG1 or BRM as the core ATPase together with other common and variable subunits. BRG1 is required for terminal epidermal differentiation in mice and humans and for hair follicle stem cell activation during mouse hair follicle regeneration and cutaneous wound healing. However, the role of SWI/SNF complexes in human hair growth and wound healing remain unknown. Here it is demonstrated that genes encoding SWI/SNF complex subunits are expressed in human hair follicles. It also highlights that siRNA mediated suppression of SWI/SNF complexes in hair follicle culture has no effect on hair growth, or anagen-catagen transition in the short term, but a significant increase in proliferation of the outer root sheath keratinocytes was seen. The data also documents the expression of several SWI/SNF subunits in human epidermis and that siRNA mediated SMARCA4 gene suppression in primary human keratinocyte monolayers defined the requirements of BRG1 for wound closure through control of cell migration, but not proliferation. In summary, this data revealed a diverse SWI/SNF complex subunit composition in human epidermis and hair follicle, and an essential role of the core complex ATPase BRG1 in keratinocyte migration during wound closure and re-epithelisation.
    • Unravelling novel molecular targets for photobiomodulation in human hair follicle towards the development of more effective light-based therapies for hair growth

      Botchkareva, Natalia V.; Mardaryev, Andrei N.; Buscone, Serena (University of BradfordFaculty of Life Sciences, 2017)
      Light and optical techniques have made a profound impact on modern medicine both in diagnostics and in therapy. Therapeutic action of light is based on photomechanical, photothermal, photochemical and photobiological interactions, depending on the wavelength, power density, exposure time and optical properties of tissue and cells. Last decade experienced a growing rise of commercial devices for management of hair growth, where all of them are based on low levels of light resulting into photobiological, non-thermal interaction of photons with cells, a process that recently has received an official term ‘photobiomodulation’. However, the design and analysis of the reported clinical studies are highly debated in a wider scientific community. The picture is further complicated by a virtual lack of proof about the exact molecular targets that mediate the physiological response of skin and hair follicles (HF) to low levels of light. The goal of this project was to investigate the expression of light-sensitive receptors in the human HF and to study the impact of UV-free blue light on hair growth ex vivo. The expression of Cryptochromes 1 and 2 (CRY1, 2), Opsin 2 and 3 (OPN2 and OPN3), but not other Opsins 1, 4 and 5 was detected in the distinct compartments of skin and anagen HF. Evaluation of the physiological role of detected light-sensitive receptors on hair growth was performed by the modulation of photoreceptors activity in HF ex vivo model. HFs treated with KL001, a stabilizer of CRY1 protein that lengthens the circadian period, delayed HF anagen-catagen transition; while silencing of CRY1 induced premature catagen development accompanied by reduced cell proliferation. Silencing of CRY1 in the HF outer root sheath (ORS) cells in vitro caused downregulation of ii genes involved in the control of proliferation; including the cyclin dependent kinase 6 (CDK6). OPN3 also had a positive effect on metabolic activity and proliferation of the ORS cells in vitro. OPN3 silencing resulted in the altered expression of genes involved in the control of proliferation and apoptosis. Investigated CRY1, OPN2 and 3 greatly absorb in the blue to green-region of the visible spectrum. This led us to investigate the effect of blue light on HF growth. Daily treatment with blue light (453 nm, 3.2 J/cm2, 16 nm full width half maximum) prolonged anagen phase in HF ex vivo that was associated with sustained proliferation. In addition, blue light (3.2 J/cm2) significantly stimulated proliferation of ORS cells in vitro. This effect was abrogated by silencing of OPN3. To summarize, CRY 1, OPN 2 and OPN 3 are expressed in the distinct compartments of the HF, including HF stem cells. Blue light (453 nm) at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. The further research should be conducted to decipher interactions between blue light and the investigated receptors in the HFs. In addition, the beneficial effect of blue light at low radiant exposure on hair growth raises a possibility of increasing therapeutic efficacy when combined with topical chemistry used for management of hair growth.