Transcription Factor FoxO1 Is Essential for Enamel Biomineralization

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta

Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification

Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone. © 2010 American Society for Bone and Mineral Research

Hitomi (ASTRO-H) X-ray Astronomy Satellite

The Hitomi (ASTRO-H) mission is the sixth Japanese x-ray astronomy satellite developed by a large international collaboration, including Japan, USA, Canada, and Europe. The mission aimed to provide the highest energy resolution ever achieved at E  >  2  keV, using a microcalorimeter instrument, and to cover a wide energy range spanning four decades in energy from soft x-rays to gamma rays. After a successful launch on February 17, 2016, the spacecraft lost its function on March 26, 2016, but the commissioning phase for about a month provided valuable information on the onboard instruments and the spacecraft system, including astrophysical results obtained from first light observations. The paper describes the Hitomi (ASTRO-H) mission, its capabilities, the initial operation, and the instruments/spacecraft performances confirmed during the commissioning operations for about a month

Identification of a novel intracellular interaction domain essential for Bves function.

While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathione-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function

Expression of ZO-1 and E-cadherin in HCE cells.

<p>A, E, Parental HCE cells B, F, HCE cell lines transfected with WT Bves, C, G, KK-Mut Bves, and D, H, KK-Del Bves were stained with ZO-1 in panels A through D and E-cadherin in panels E through H. Note the loss of peripheral ZO-1 and E-cadherin staining in KK-Mut Bves and KK-Del Bves cells. (Scale Bar 100 µm)</p

Immunostaining with anti-cytokeratin and anti-vimentin.

<p>Immunostaining with anti-cytokeratin (panels A through D), anti-vimentin (panels E through H) and merge (panels I through L) are given. A, E, I, Parental cells show high levels of cytokeratin and little vimentin staining. Transfection with WT Bves (B, F, J) shows similar staining patterns as parental cells. In contrast, cell lines expressing KK-Mut Bves (C, G, K) have a subpopulation of vimentin-positive cells and KK-Del Bves expressing cells (D, H, L) show nearly complete conversion to a vimentin-positive phenotype. (Scale Bar 100 µm)</p

Bves constructs and GST pull-down assay.

<p>A, Diagram of GST-fusion proteins and deletion constructs. GST C- and N-terminal Bves proteins and serially deleted Flag-tagged Bves proteins. B, GST pull-down assay with C- or N-terminal Bves. Bacterial lysates of GST-Bves fusion proteins were reacted with COS-7 cell lysates transfected with Flag-tagged WT Bves and analyzed with Western blots using an anti-Flag antibody. The potential effects of glycosylation were also tested. Control band (WT Bves protein with or without glycosylation) are shown on lane a and b. Only GST C-terminal Bves reacted in this assay regardless of glycosylation state (lanes d and g) C, Serial deletion analysis of C-terminal interaction. GST C-terminal Bves was reacted with Del-5 Bves, Del-4 Bves and Del-3 Bves and processed for Western blotting to detect interactions. Del-5 Bves and Del-4 Bves (lanes a and b) interact with the GST C-terminal Bves while Del-3 Bves does not.</p