Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23814号 / 医科博第135号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 松村 由美, 教授 森本 尚樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase-PCR (qRT-PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)-mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

No / Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H2O2 in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 μM H2O2 increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH4Cl and elevated l-tyrosine, H2O2-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H2O2-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca2+-chelator BAPTA. Thus, BAPTA reduced the level of H2O2-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca2+ and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca2+ binding capacity and, in addition, correlated inversely with H2O2-induced increases in intracellular Ca2+. Our results show that melanin may have an important role in regulating intracellular Ca2+ homeostasis and it is suggested that melanin protects against H2O2-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca2+.

No / The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21WAF1/Cip1 gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-J¿ protein to the p21 promoter. We show here that Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

Vitiligo is a skin disease with 2 % prevalence in a worldwide population. It is characterised by loss or decrease in activity of epidermal melanocytes, which lead to skin and hair depigmentation. It has negative impact on psyche, social relationships of patients and reduces the protection of the organism against UV radiation. One of the treatment methods is autologous transplantation of melanocytes or suspension of melanocytes with keratinocytes. Use of the biocompatible membrane, which allows the cultivation of these cells with resulting transplantation on the depigmented lesion, could improve treatment and make it more efficient. The main goal of this work was to create the biocompatible membrane from nanofiber layers of polyvinylalcohol (PVA) which can stand as a carrier for cell transplants in vitiligo therapy. PVA scaffolds were prepared by electrostatic spinning and later on modified by cold methane plasma (CH4) for lowering their hydrofility. Samples of modified nanofiber carriers were analysed according to their physical and chemical characteristics (visualization fiber morphology by SEM, XPS and surface Zeta potential analysis and contact angle). Consequently, adhesion, proliferation and metabolic activity of cultivating mice cell lines of melanocytes (Melan-a) and keratinocytes (XB2) were examined...