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The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-β markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-β, or IL-1α, TNF-α and TGF-β. The signaling molecules’ inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-β-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-β-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-β was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-β in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA. Aggrecan degradation by ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is essential to cartilage destruction in osteoarthritis, but its expression profile and regulation are unclear. The authors show overexpression of ADAMTS4 in osteoarthritis synovium and synergistic upregulation by interleukin-1α, tumor necrosis factor-α, and transforming growth factor-β in osteoarthritis synovial fibroblasts. These findings suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID) is involved in cartilage destruction via HA depolymerization in human knee osteoarthritis. However, roles of HYBID in the progression of osteoarthritis remain elusive. This study sought to examine whether genetic depletion of Hybid could suppress surgically induced osteoarthritis of mouse knee joints. In osteoarthritis induced by medial collateral ligament transection with meniscus removal, articular cartilage destruction and osteophyte formation at the medial femoral-tibial joint were significantly inhibited in Hybid-deficient (Hybid-/-) mouse group compared to wild-type group. Hybid was highly produced by synovial cells and articular chondrocytes in wild-type mouse osteoarthritis joint. Interleukin-1β, interleukin-6 and tumor necrosis factor-α were up-regulated in the osteoarthritis joint tissues of both wild-type and Hybid-/- mice. Vascular density at the synovial and periosteal junction was significantly reduced in Hybid-/- mice compared to wild-type mice. High-molecular-weight HA was accumulated in osteoarthritis joint tissues of Hybid-/- mice. Injections of high-molecular-weight HA to knee joints attenuated the cartilage destruction and osteophyte formation in wild-type mouse osteoarthritis group. Inhibition of cartilage destruction and osteophyte formation in Hybid-/- mice was also observed in destabilization of the medial meniscus model. These data are the first to demonstrate that cartilage destruction and osteophyte formation are suppressed in Hybid-/- mice and suggest that Hybid-mediated HA depolymerization is implicated for the progression of mechanically induced knee osteoarthritis.

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein–coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop–derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), plays a role in HA degradation. CEMIP2, also known as transmembrane protein 2 (TMEM2), possessing a sequence similarity with HYBID, is reported as a hyaluronidase in mice. However, the expression of these molecules in osteoarthritic synovium and their involvement in HA degradation in synovial fluid (SF) from patients with knee osteoarthritis remain elusive. This study examined their expression in synovial tissue and the relationship with molecular weight of HA in SF in knee osteoarthritis patients. Quantification of mRNA demonstrated that HYBID expression is significantly (5.5-fold) higher in osteoarthritic synovium than in normal control synovium, whereas TMEM2 expression level is similar between the two groups. By immunohistochemistry, HYBID was localized mainly to CD68-negative and fibroblast-specific protein 1-positive synovial lining cells and sublining fibroblasts in osteoarthritic synovium. The mRNA expression levels of HYBID, but not TMEM2, in osteoarthritic synovium positively correlated with distribution of lower-molecular-weight HA with below 1000 kDa in SF. HA-degrading activity in osteoarthritic synovial fibroblasts was abrogated by siRNA-mediated knockdown of HYBID. Among the 12 factors examined, IL-6 significantly up-regulated the HYBID expression and HA-degrading activity in osteoarthritic synovial fibroblasts. These data suggest that HYBID overexpressed by IL-6-stimulated synovial fibroblasts is implicated in HA degradation in osteoarthritic synovium.

Cell adhesion between oligodendrocytes and neuronal axons is a critical step for myelination that enables the rapid propagation of action potential in the central nervous system. Here, we show that the transmembrane protein teneurin-4 plays a role in the cell adhesion required for the differentiation of oligodendrocytes. We found that teneurin-4 formed molecular complexes with all of the four teneurin family members and promoted cell-cell adhesion. Oligodendrocyte lineage cells attached to the recombinant extracellular domain of all the teneurins and formed well-branched cell processes. In an axon-mimicking nanofibers assay, nanofibers coated with the recombinant teneurin-4 extracellular domain increased the differentiation of oligodendrocytes. Our results show that teneurin-4 binds to all teneurins through their extracellular domain, which facilitates the oligodendrocyte-axon adhesion, and promotes oligodendrocyte differentiation via its homophilic interaction.

Glioblastoma (GBM) is pathologically characterized by highly malignant neoplastic cells, focal necrosis and aberrant blood vessels composed of disorganized endothelial cells and pericytes. The recent cancer microarray database revealed upregulation of fibulin-7 (Fbln7), a member of the fibulin family, but provided no information on the tissue localization or biological function. In the present study, we demonstrated that Fbln7 is markedly overexpressed by the GBM tissue among astrocytic tumors, and immunolocalized mainly to endothelial cells and pericytes of the glomeruloid and hypertrophied microvessels. The production of Fbln7 by endothelial cells and pericytes was confirmed in cultured human umbilical vein endothelial cells (HUVEC) and human brain vascular pericytes (HBVP) and vascular endothelial growth factor (VEGF) stimulated the Fbln7 expression in HUVEC. Fbln7 bound to angiopoietin-1, but not angiopoietin-2 or Tie2 receptor, through interaction between the N-terminal portions of Fbln7 and angiopoietin-1, and it blocked phosphorylation of Tie2 receptor in HUVEC. In a coculture assay using HUVEC and HBVP, multilayered and irregular-shaped tube-like structures of HUVEC were induced by treatment with a high concentration of VEGF. This was accompanied by Fbln7 overproduction by HUVEC and angiopoietin-1 expression by HBVP. The production of aberrant VEGF-induced tube-like structures was attenuated by treatment with antibody or synthetic peptides specific to the Fbln7 N-terminal domain or knockdown of Fbln7. These data demonstrate that Fbln7 is overexpressed by endothelial cells and pericytes of the abnormal microvessels in GBM, and suggest that Fbln7 may contribute to the aberrant vessel formation by modulation of the angiopoietin-1/angiopoietin-2-Tie2 axis.

The development of ectodermal organs, such as teeth, requires epithelial–mesenchymal interactions. Basic helix–loop–helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial–mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), vascular endothelial growth factor

Glioblastoma is the most malignant tumor of the brain associated with poor prognosis and outcome, and hence there is an urgent need to develop novel treatments for glioblastoma. In this study, we focused on hyaluronan binding protein (HYBID, as known as CEMIP/KIAA1199), a protein involved in hyaluronan depolymerization in chondrocytes and synoviocytes. We previously reported that Hybid-deficient (KO) mice show accumulation of hyaluronan in the brain, and memory impairment. To elucidate the role of HYBID in glioblastoma pathogenesis, we knocked down HYBID in human glioblastoma cells using siRNAs and developed a murine orthotopic xenograft model in the Hybid KO mice. Downregulation of HYBID in glioblastoma cells resulted in inhibition of cell proliferation and migration, and increased cell death. The growth of glioblastoma cells implanted in the mouse brain was suppressed in Hybid KO mice compared to that in the wild-type mice. Interestingly, infiltration of macrophages in the glioblastoma tissue was decreased in Hybid KO mice. Using intraperitoneal macrophages derived from Hybid KO mice and glioma cell supernatants, we examined the role of HYBID in macrophages in the tumor environment. We showed that HYBID contributes to macrophage migration and the release of pro-tumor factors. Moreover, we revealed that HYBID can be a poor prognostic factor in glioma patients by bioinformatics approaches. Our study provides data to support that HYBID expressed by both glioblastoma cells and tumor-associated macrophages may contribute to glioblastoma progression and suggests that HYBID may be a potential target for therapy that focuses on the tumor microenvironment of glioblastoma.

Hyaluronan (HA), a major component of the extracellular matrix in vertebrate tissues, provides structural and functional integrity to cells and organs. Biological functions of HA are dependent on the molecular size of HA and the interaction with a wide range of HA-binding proteins, i.e., hyaladherins. In this book chapter, we introduce hyaladherins and focus on HYBID (Hyaluronan-binding protein involved in hyaluronan depolymerization, alias KIAA1199 and CEMIP), which is one of the hyaladherins and plays a central role in HA degradation in human dermal and arthritic synovial fibroblasts. The protocols describe the preparation of the stable transfectants expressing HYBID, the assays of HYBID-mediated HA depolymerization, and the binding assay of HYBID to HA. These methods will be helpful to further study the HYBID-mediated biological activities and its relevance on HA degradation and turnover under various physiological and pathological conditions such as wound healing, ageing, arthritis, and cancer.

Hyaluronan (HA), a major component of the extracellular matrix in vertebrate tissues, provides structural and functional integrity to cells and organs. Biological functions of HA are dependent on the molecular size of HA and the interaction with a wide range of HA-binding proteins, i.e., hyaladherins. In this book chapter, we introduce hyaladherins and focus on HYBID (Hyaluronan-binding protein involved in hyaluronan depolymerization, alias KIAA1199 and CEMIP), which is one of the hyaladherins and plays a central role in HA degradation in human dermal and arthritic synovial fibroblasts. The protocols describe the preparation of the stable transfectants expressing HYBID, the assays of HYBID-mediated HA depolymerization, and the binding assay of HYBID to HA. These methods will be helpful to further study the HYBID-mediated biological activities and its relevance on HA degradation and turnover under various physiological and pathological conditions such as wound healing, ageing, arthritis, and cancer.

The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin β1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.

Angiogenesis is crucial for tissue development and homeostasis; however, excessive angiogenesis can lead to diseases, including arthritis and cancer metastasis. Some antiangiogenic drugs are available, but side effects remain problematic. Thus, alternative angiogenesis inhibition strategies are needed. Fibulin-7 (Fbln7) is a newly discovered member of the fibulin protein family, a group of cell-secreted glycoproteins, that functions as a cell adhesion molecule and interacts with other extracellular matrix (ECM) proteins as well as cell receptors. We previously showed that a recombinant C-terminal Fbln7 fragment (Fbln7-C) inhibits tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In the present study, we examined the in vivo antiangiogenic activity of recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins using a rat corneal angiogenesis model. We found that both Fbln7-FL and Fbln7-C inhibited neovascularization. Fbln7-C bound to vascular endothelial growth factor receptor 2 (VEGFR2), inhibiting VEGFR2 and ERK phosphorylation and resulting in reduced HUVEC motility. HUVEC attachment to Fbln7-C occurred through an interaction with integrin α5β1 and regulated changes in cellular morphology. These results suggest that Fbln7-C action may target neovascularization by altering cell/ECM associations. Therefore, Fbln7-C could have potential as a therapeutic agent for diseases associated with angiogenesis.

Fibulin-7 (Fbln7) has been identified as the latest member of the fibulin family of secreted glycoproteins in developing teeth, functioning as a cell adhesion molecule and interacting with other matrix proteins, receptors, and growth factors. More recently, we have shown that the C-terminal Fbln7 fragment (Fbln7-C) has antiangiogenic activity in vitro. Fbln7 is also expressed in immune-privileged tissues, such as eye and placenta, but its functional significance is unknown. In the current study, we show that human monocytes adhere to both full-length Fbln7 (Fbln7-FL) and Fbln7-C, in part, via integrins α(5)β(1) and α(2)β(1). Morphologic studies and surface expression analyses of CD14, mannose receptor (CD206), major histocompatibility complex II, and CD11b receptors revealed that both Fbln7-FL and Fbln7-C inhibit M-CSF–induced monocyte differentiation. Fbln7-C had significantly greater negative effects on cell spreading and stress fiber formation, including the production of IL-6 and metalloproteinase-1/-9 compared with Fbln7-FL. Furthermore, in an LPS-induced systemic inflammation model, Fbln7-C and Fbln7-FL reduced the infiltration of immune cells, such as neutrophils and macrophages, to the inflamed peritoneum. Thus, these results suggest that Fbln7 and Fbln7-C could modulate the activity of immune cells and have therapeutic potential for inflammatory diseases.—Sarangi, P. P., Chakraborty, P., Dash, S. P., Ikeuchi, T., de Vega, S., Ambatipudi, K., Wahl, L., Yamada, Y. Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo.

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.

The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research.

Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.

El pdf del artículo es el manuscrito de autor (PMCID: PMC3538143).-- et al., The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. In the present work, we have investigated the role of Epiprofin (Epfn/Sp6), a member of the SP/KLF transcription factor family that is expressed in the limb ectoderm and the AER, during limb development. Epfn mutant mice have a defective autopod that shows mesoaxial syndactyly in the forelimb and synostosis (bony fusion) in the hindlimb and partial bidorsal digital tips. Epfn mutants also show a defect in the maturation of the AER that appears flat and broad, with a double ridge phenotype. By genetic analysis, we also show that Epfn is controlled by WNT/b-CATENIN signaling in the limb ectoderm. Since the less severe phenotypes of the conditional removal of b-catenin in the limb ectoderm strongly resemble the limb phenotype of Epfn mutants, we propose that EPFN very likely functions as a modulator of WNT signaling in the limb ectoderm., This work was supported by grant BFU2008-00397 from the Spanish Ministry of Education and Science and grant API 05/27 from the IFIMAV to M.R., the Intramural Research Program of the NIDCR, NIH to Y.Y. and grant GIU05/27 from the University of the Basque Country to F.U. A.T. had a postdoctoral fellowship from the Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV).

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin β1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Kruppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5′ Cap capture method. Except for its 5′-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Kruppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank™ by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.

Perlecan is a major heparan sulfate proteoglycan found in the subendothelial extracellular matrix of the vascular wall. The aim of this study was to investigate the role of perlecan in the regulation of vascular tone. A previously developed conditional perlecan‐deficient mouse model was used to measure changes in the isometric force of isolated aortic rings. The vessels were first precontracted with phenylephrine, and then treated with increasing concentrations of vasorelaxants. Endothelium‐dependent relaxation, elicited by acetylcholine, was significantly reduced in the perlecan‐deficient aortas, whereas endothelium‐independent relaxation caused by the exogenous nitric oxide donor sodium nitroprusside remained well preserved. The expression of the endothelial nitric oxide synthase (eNOS) gene, detected by real‐time polymerase chain reaction, was significantly decreased in the perlecan‐deficient aortas. The expression of eNOS protein detected using Western blotting was also significantly decreased in the perlecan‐deficient aortas. We examined the role of perlecan in eNOS gene expression by creating perlecan knockdown human aortic endothelial cells using small interfering RNA (siRNA) for perlecan. Perlecan gene expression was significantly reduced in the perlecan siRNA‐treated cells, resulting in a significant decrease in eNOS gene expression. Perlecan deficiency induced endothelial dysfunction, as indicated by a reduction in endothelium‐dependent relaxation due, at least partly, to a reduction in eNOS expression. These findings suggest that perlecan plays a role in the activation of eNOS gene expression during normal growth processes., Perlecan deficiency induced endothelial dysfunction at least partly, to a reduction in eNOS expression. These findings suggest that perlecan plays a role in the activation of the eNOS expression during normal growth processes.

Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.—Suzuki, N., Numakawa, T., Chou, J., de Vega, S., Mizuniwa, C., Sekimoto, K., Adachi, N., Kunugi, H., Arikawa-Hirasawa, E., Yamada, Y., Akazawa, C. Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling.

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the "core matrisome" in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins.

Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designatedfurue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of thefuruemice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that thefuruemutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in thefuruemice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from thefuruemice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.

Odontogenesis is governed by a complex network of intercellular signaling events between the dental epithelium and mesenchyme. This network leads to the progressive determination of tooth shape, and to the differentiation of these tissues into enamel-producing ameloblasts and dentin-producing odontoblasts respectively. Among the main signaling pathways involved in the regulation of tooth development, Bone Morphogenetic Protein (BMP), Sonic hedgehog (Shh) and Wingless-type MMTV integration site (Wnt) pathways have been reported to play significant roles. Recently, the phenotype of mice deficient in Epiprofin/Sp6 (Epfn) has been found to present striking dental abnormalities, including a complete lack of differentiated ameloblasts and consequently no enamel, highly altered molar cusp patterns and the formation of multiple supernumerary teeth. In this article, we review the interaction of Epfn with the BMP, Shh and Wnt pathways in the regulation of tooth development, based on the data obtained from the study of several genetically modified mice.

TGF-beta has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-beta in skeletal development is unclear. In this study, we investigated the role of TGF-beta signaling in growth plate development by creating mice with a conditional knockout of the TGF-beta type I receptor ALK5 (ALK5(CKO)) in skeletal progenitor cells using Dermo1-Cre mice. ALK5(CKO) mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5(CKO) growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5(CKO) growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-beta signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-beta signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.