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.
ABSTRACT
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 α5β1 and α2β1. 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. FASEB J. 32, 4889–4898 (2018). www.fasebj.org
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. FASEB J. 28, 1386–1397 (2014). www.fasebj.org
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.
What's new?
Matricellular protein fibulin‐7 has recently been suggested to play a role in glioblastoma, but its tissue localization and biological function remain to be clarified. Here, the authors show that fibulin‐7 is mainly overexpressed by endothelial cells and pericytes composing the aberrant microvasculature in glioblastoma. Fibulin‐7 selectively binds to angiopoietin‐1, leading to suppression of Tie2 activation in endothelial cells, and contributes to the formation of aberrant endothelial cell tubelike structures following stimulation with VEGF. The findings support a role of fibulin‐7 in aberrant blood vessel formation in glioblastoma through modulation of the angiopoietin1‐Tie2 axis.
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.
•HYBID plays a pro-tumorigenic role in glioblastoma cells.•Host-derived HYBID increases glioblastoma progression.•HYBID promotes infiltration of macrophages into glioblastoma and contributes to ...glioblastoma progression.
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.
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.
•Teneurin-4 forms molecular complexes with all teneurin family members.•Teneurin-4 promotes homophilic and heterophilic cell binding with all teneurins.•Oligodendrocyte lineage cells adhere to the extracellular domain of teneurins.•The homophilic interaction of teneurin-4 promotes oligodendrocyte differentiation.
Destruction of articular cartilage in osteoarthritis (OA) is initiated by depletion of the hyaluronan (HA)-aggrecan network, followed by degradation of the collagen fibrils. Previously, we reported ...the implications of HA-binding protein involved in HA depolymerization (HYBID), alias cell migration-inducing protein (CEMIP) and KIAA1199, for HA degradation. However, transmembrane protein 2 (TMEM2), which is ~ 50% homologous to HYBID, was discovered as another hyaluronidase, but their expression and regulation by OA chondrocytes remain elusive. Here we report that the absolute mRNA copy numbers of HYBID are significantly (7.1-fold) higher in OA cartilage than normal cartilage, whereas TMEM2 levels are not different between the groups. HA-degrading activity of cultured OA chondrocytes disappeared by siRNA-mediated knockdown of HYBID, but not TMEM2. HYBID expression was significantly up-regulated by treatment with interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α) and additively increased by the combined treatment. No significant changes in the TMEM2 expression were seen by the factors examined. IL-1α remarkably enhanced IL-6 production and increased HYBID expression when soluble IL-6 receptor was supplemented. These results demonstrate that in stark contrast to the constitutive expression of TMEM2 and its negligible HA-degrading activity, HYBID is overexpressed in OA cartilage and up-regulated by IL-6 and TNF-α in OA chondrocytes.
Perlecan (HSPG2; Perl) is expressed in all basement membranes and cartilage. Perl has been reported to have anti‐atherogenic activity; however, its role in the aortic wall is unknown. In this report, ...we analyzed the structure of the aortic wall in Perlecan deficient mice (HSPG2‐/‐‐Tg).
Previously, we created the lethality rescued HSPG2‐/‐‐Tg mice model. Aorta samples were prepared from the thoracic aorta of HSPG2‐/‐‐Tg (Perl KO) and control HSPG+/+‐Tg mice. We analyzed the histology of these aortic tissues by Elastica Van Gieson (EVG) staining, immunohistochemistry (IHC), and electron microscope (EM). In IHC, we observed that perlecan is localized at the intersection between elastin and fibrillin‐1. EVG staining showed no difference in aorta histology between Perl KO and control mice, however, EM analysis showed that the elastic fibers in Perl KO mice were partially torn and thinner. In addition, we found that Perl KO mice had an aortic dissection with a frequency of about 15% in 10‐weeks mice. Real‐time PCR analysis showed no significant differences in the expression of genes for the major components of elastic fibers and matrix metalloproteinases (MMPs).
These results suggest that Perl is likely to contribute to the maintainance of elastic fibers strength against mechanical stress, such as blood flow, vasodilation, and contraction.
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