• The Role of Polycomb Repressive Complex 2 in Epidermal Homeostasis and Hair Growth

      Botchkareva, Natalia V.; Mardaryev, Andrei N.; Asamaowei, Inemo E. (University of BradfordFaculty of Life Sciences, 2017)
      Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.
    • 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.