No / Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined. A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively. BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing. BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair. Level of evidence: Not ratable.

No description available.

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.

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.

The Spemann organizer in amphibians gives rise to the anterior mesendoderm (AME) and is capable of inducing neural tissues. This inductive activity is thought to occur largely via the antagonism of Bone Morphogenetic Protein (BMP) signaling in the organizer. In the mouse, BMP antagonists Chordin and Noggin function redundantly in the AME and are required during forebrain maintenance. However, the timing of forebrain initiation and the function of BMP antagonism in forebrain initiation remained unclear prior to this study. In addition, the Transforming Growth Factor β (TGFβ) ligand Nodal patterns the forebrain via its function in the anterior primitive streak (APS), the precursor tissue of the AME. Whether BMP and Nodal signaling pathways interact has not been previously investigated. The goal of this dissertation was to investigate the cellular and molecular mechanisms involved in early mammalian forebrain establishment by embryonic and genetic manipulations. This study determined that forebrain initiation occurs during early gastrulation and requires signals from the AVE and AME. The AVE was identified as a source of active BMP antagonism in vivo, and the BMP antagonism supplied by exogenous tissues was capable to promote forebrain initiation and maintenance in the murine ectoderm. It is likely that BMP antagonism enhances forebrain gene expression via inhibiting posteriorization. This study further identified a possible crosstalk between BMP and Nodal signaling. Loss of Chordin or Noggin in combination with heterozygosity for Nodal or Smad3 results in holoprosencephaly. Molecular analyses suggest that the BMP-Nodal interaction occurs in the APS and/or the AME. Failure of this interaction results in an imbalance of BMP and Nodal signal levels that devastate APS and AME patterning during early forebrain establishment, ultimately leading to holoprosencephaly in mid-gestation. This interaction is likely to occur extracellularly, possibly by formation of a BMP-Nodal heteromeric complex. Furthermore, the spatiotemporal expression of phospho-Smad1/5/8, an effector of BMP signaling pathway, was characterized during early mouse embryogenesis. Distribution of phospho-Smad1/5/8 serves as a faithful readout of BMP signaling activity and helps to better understand how BMPs are involved in patterning early embryos. The implication of phospho-Smad1/5/8 expression in both wildtype and mutant embryos is also discussed. / Dissertation

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv(-/-) mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2(-/-) mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13031号 / 論医博第2113号 / 新制||医||1016(附属図書館) / 32989 / (主査)教授 妻木 範行, 教授 安達 泰治, 教授 開 祐司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (β-catenin/Lef1) markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways.

O presente estudo utilizou o modelo fatia-dental/matriz-polimérica para avaliar a influência do tratamento dentinário e das BMPs dentinárias na diferenciação das células-tronco da polpa de dentes decíduos (SHED). Secções transversais (1mm) foram preparadas a partir de terceiros molares humanos extraídos. Matrizes poliméricas a base de ácido poli-L-lático (PLLA) foram criadas no interior da cavidade pulpar das secções dentinárias, tratadas com solução de EDTA a 10%; NaOCl a 5.25%; ou permanecendo sem tratamento. Matrizes poliméricas confeccionadas sem as fatias dentais foram utilizadas como controle. As células (5x104) foram semeadas nas matrizes e, após 7, 14, 21 e 28 dias de cultura in vitro, a expressão de marcadores de diferenciação odontoblástica (DSPP, DMP1 e MEPE) e a proliferação celular (WST-1) foram avaliadas. Células (5x105) semeadas nas matrizes foram transplantadas em camundongos imunodeficientes e cultivadas in vivo por um período de 14 e 28 dias. Para avaliar a atividade das BMPs dentinárias, 5x104 células foram semeadas em matrizes poliméricas com fatia dental e cultivadas na presença de anticorpos anti-BMP- 2, -4, ou -7 (2 μg/ml) durante 14 dias. Adicionalmente, 5x105 células foram tratadas com rhBMP-2, -4, ou -7 (100ng/mL) por 24hs. As células cultivadas in vitro e in vivo alteraram sua expressão genética durante o curso do tempo. DSPP, DMP-1 e MEPE foram expressos por células cultivadas in vitro após 14 dias (tratamento com EDTA e dentina sem tratamento) e in vivo após 28 dias (EDTA), não sendo detectados nos grupos NaOCl e nas células cultivadas nas matrizes sem fatia dental. A proliferação foi reduzida com a diferenciação celular (p<0.05). A utilização de BMP-2/4Ab no meio de cultura exerceu um efeito inibitório na expressão dos marcadores de diferenciação celular, não ocorrendo quando do cultivo das SHED na presença de BMP-7Ab. DSPP, DMP-1 e MEPE foram expressos por células tratadas com rhBMP-2, e DSPP e DMP-1 por células tratadas com rhBMP-4 e -7. Células sem tratamento não expressaram os marcadores. O modelo fatia-dental/matriz-polimérica demonstrou ser adequado para o estudo da diferenciação de células-tronco pulpares, sugerindo que a dentina possa fornecer um microambiente favorável para a diferenciação de celular. As proteínas ósseas morfogenéticas dentinárias BMP-2 e BMP-4 parecem exercer um papel relevante nesse processo. / The effect of dentin pre-treatments and dentin-derived BMPs on SHED differentiation was tested using the Tooth-Slice Scaffold model (TSS). Dentin slices (1mm thickness) were prepared from extracted human third molars. Biodegradable PLLA scaffolds were prepared inside the pulp chamber of the tooth-slices, treated alternatively with a 5.25% NaOCl or 10% EDTA solution, or remaining untreated (WO-T). PLLA sponge scaffolds with no tooth-slice (PSS) were used as control. SHED (5x104) were seeded in TSS and PSS and after 7, 14, 21 and 28 days in culture, RT-PCR (DSPP, DMP1 and MEPE) and WST-1 proliferation assay were performed. Additionally, cells (5x105) were seeded in TSS and PSS and transplanted into SCID mice (14 and 28 days). To verify the dentinderived BMPs bioactivity, SHED (5x104) were cultured in TSS in the presence of antihuman BMP-2, -4, and -7 antibodies for 14 days. Besides, cells in culture were treated with rhBMP-2; -4; or -7 for 24 hours. After in vitro and in vivo time course, SHED altered their genetic expression. The cells cultured in vitro in the TSS (EDTA or WO-T) expressed the differentiation markers after 14 days and maintained expression thereafter. Cell proliferation rate was reduced following the differentiation (p<0.05). Cells transplanted in vivo expressed DSPP, DMP-1 and MEPE after 28 days (EDTA). No transcripts were found in tooth-slices treated with NaOCl or in PSS groups. BMP-2/4Ab prevented the differentiation process and no inhibitory effect was detected for BMP-7Ab. After 24 hours, expression of DSPP, DMP-1 and MEPE was found for rhBMP-2, and DSPP and DMP-1 for rhBMP-4 and rhBMP-7 treated SHED, but not for untreated cells. The tooth slice scaffold model suggests that dentin can provide the environment for SHED differentiation and dentin-derived morphogenic signals BMP-2 and BMP-4 play an important role in this process.

O presente estudo utilizou o modelo fatia-dental/matriz-polimérica para avaliar a influência do tratamento dentinário e das BMPs dentinárias na diferenciação das células-tronco da polpa de dentes decíduos (SHED). Secções transversais (1mm) foram preparadas a partir de terceiros molares humanos extraídos. Matrizes poliméricas a base de ácido poli-L-lático (PLLA) foram criadas no interior da cavidade pulpar das secções dentinárias, tratadas com solução de EDTA a 10%; NaOCl a 5.25%; ou permanecendo sem tratamento. Matrizes poliméricas confeccionadas sem as fatias dentais foram utilizadas como controle. As células (5x104) foram semeadas nas matrizes e, após 7, 14, 21 e 28 dias de cultura in vitro, a expressão de marcadores de diferenciação odontoblástica (DSPP, DMP1 e MEPE) e a proliferação celular (WST-1) foram avaliadas. Células (5x105) semeadas nas matrizes foram transplantadas em camundongos imunodeficientes e cultivadas in vivo por um período de 14 e 28 dias. Para avaliar a atividade das BMPs dentinárias, 5x104 células foram semeadas em matrizes poliméricas com fatia dental e cultivadas na presença de anticorpos anti-BMP- 2, -4, ou -7 (2 μg/ml) durante 14 dias. Adicionalmente, 5x105 células foram tratadas com rhBMP-2, -4, ou -7 (100ng/mL) por 24hs. As células cultivadas in vitro e in vivo alteraram sua expressão genética durante o curso do tempo. DSPP, DMP-1 e MEPE foram expressos por células cultivadas in vitro após 14 dias (tratamento com EDTA e dentina sem tratamento) e in vivo após 28 dias (EDTA), não sendo detectados nos grupos NaOCl e nas células cultivadas nas matrizes sem fatia dental. A proliferação foi reduzida com a diferenciação celular (p<0.05). A utilização de BMP-2/4Ab no meio de cultura exerceu um efeito inibitório na expressão dos marcadores de diferenciação celular, não ocorrendo quando do cultivo das SHED na presença de BMP-7Ab. DSPP, DMP-1 e MEPE foram expressos por células tratadas com rhBMP-2, e DSPP e DMP-1 por células tratadas com rhBMP-4 e -7. Células sem tratamento não expressaram os marcadores. O modelo fatia-dental/matriz-polimérica demonstrou ser adequado para o estudo da diferenciação de células-tronco pulpares, sugerindo que a dentina possa fornecer um microambiente favorável para a diferenciação de celular. As proteínas ósseas morfogenéticas dentinárias BMP-2 e BMP-4 parecem exercer um papel relevante nesse processo. / The effect of dentin pre-treatments and dentin-derived BMPs on SHED differentiation was tested using the Tooth-Slice Scaffold model (TSS). Dentin slices (1mm thickness) were prepared from extracted human third molars. Biodegradable PLLA scaffolds were prepared inside the pulp chamber of the tooth-slices, treated alternatively with a 5.25% NaOCl or 10% EDTA solution, or remaining untreated (WO-T). PLLA sponge scaffolds with no tooth-slice (PSS) were used as control. SHED (5x104) were seeded in TSS and PSS and after 7, 14, 21 and 28 days in culture, RT-PCR (DSPP, DMP1 and MEPE) and WST-1 proliferation assay were performed. Additionally, cells (5x105) were seeded in TSS and PSS and transplanted into SCID mice (14 and 28 days). To verify the dentinderived BMPs bioactivity, SHED (5x104) were cultured in TSS in the presence of antihuman BMP-2, -4, and -7 antibodies for 14 days. Besides, cells in culture were treated with rhBMP-2; -4; or -7 for 24 hours. After in vitro and in vivo time course, SHED altered their genetic expression. The cells cultured in vitro in the TSS (EDTA or WO-T) expressed the differentiation markers after 14 days and maintained expression thereafter. Cell proliferation rate was reduced following the differentiation (p<0.05). Cells transplanted in vivo expressed DSPP, DMP-1 and MEPE after 28 days (EDTA). No transcripts were found in tooth-slices treated with NaOCl or in PSS groups. BMP-2/4Ab prevented the differentiation process and no inhibitory effect was detected for BMP-7Ab. After 24 hours, expression of DSPP, DMP-1 and MEPE was found for rhBMP-2, and DSPP and DMP-1 for rhBMP-4 and rhBMP-7 treated SHED, but not for untreated cells. The tooth slice scaffold model suggests that dentin can provide the environment for SHED differentiation and dentin-derived morphogenic signals BMP-2 and BMP-4 play an important role in this process.