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      Determination of the CD148-Interacting Region in Thrombospondin-1

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          Abstract

          CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1 st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1 st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1 st, but not the 2 nd and 3 rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1 st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1 st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1 st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis in vivo. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1 st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.

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          Most cited references43

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          Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.

          Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.
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            A novel role for p120 catenin in E-cadherin function

            Îndirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120–E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.
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              Thrombospondins function as regulators of angiogenesis

              Thrombospondins (TSPs) -1 and -2 were among the first protein inhibitors of angiogenesis to be identified, a property that was subsequently attributed to the interactions of sequences in their type I repeats with endothelial cell-surface receptors. The interactions of TSPs-1 and -2 with cell-surface receptors, proteases, growth factors, and other bioactive molecules, coupled with the absence of direct structural functions that can be attributed to these matrix proteins, qualify them for inclusion in the category of ‘matricellular proteins’. The phenotypes of TSP-1, TSP-2, and double TSP-1/2-null mice confirm the roles that these proteins play in the regulation of angiogenesis, and provide clues to some of the other important functions of these multi-domain proteins. One of these functions is the ability of TSP-1 to activate the latent TGFβ1 complex, a property that is not shared by TSP-2. A major pathway by which TSP1 or TSP2 inhibits angiogenesis involves an interaction with CD 36 on endothelial cells, which leads to apoptosis of both the liganded and adjacent cells. However a homeostatic mechanism, which inhibits endothelial cell proliferation, and may be physiologically preferable under some circumstances, has also been elucidated, and involves interaction with the very low density lipoprotein receptor (VLDLR). The interaction of TSP1with its receptor, CD47, further inhibits angiogenesis by antagonizing nitric oxide signaling in endothelial and vascular smooth muscle cells. Paradoxically, there is also evidence that TSP-1 can function to promote angiogenesis. This apparent contradiction can be explained by the presence of sequences in different domains of the protein that interact with different receptors on endothelial cells. The anti-angiogenic function of TSPs has spurred interest in their use as anti-tumor agents. Currently, peptide mimetics, based on sequences in the type I repeats of TSPs that have been shown to have anti-angiogenic properties, are undergoing clinical testing.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 May 2016
                2016
                : 11
                : 5
                : e0154916
                Affiliations
                [1 ]Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [2 ]Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [3 ]Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                Center for Cancer Research, National Cancer Institute, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: TT RLM. Performed the experiments: KT KS RK RJ. Analyzed the data: KT KS RK TT. Contributed reagents/materials/analysis tools: DMBS JC. Wrote the paper: KT KS RK TT.

                Article
                PONE-D-15-55087
                10.1371/journal.pone.0154916
                4858292
                27149518
                9c3fff1b-7f96-4e7a-a354-102d982500ad
                © 2016 Takahashi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 24 December 2015
                : 21 April 2016
                Page count
                Figures: 5, Tables: 0, Pages: 22
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute;
                Award ID: HL109715
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases;
                Award ID: DK097332
                Award Recipient :
                This work was supported by National Institutes of Health (NIH) grants DK97332 (TT) and HL109715 (TT); National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [ http://www.niddk.nih.gov/]; National Heart, Lung, and Blood Institute (NHLBI) [ http://www.nhlbi.nih.gov/]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Proliferation
                Biology and Life Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Medicine and Health Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Biology and Life Sciences
                Developmental Biology
                Angiogenesis
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Epithelial Cells
                Endothelial Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Endothelial Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Endothelial Cells
                Research and Analysis Methods
                Precipitation Techniques
                Immunoprecipitation
                Biology and Life Sciences
                Molecular Biology
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                Molecular Probe Techniques
                Immunoblotting
                Research and Analysis Methods
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                Biology and Life Sciences
                Biochemistry
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                Post-Translational Modification
                Phosphorylation
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
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                Tyrosine
                Physical Sciences
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                Biology and Life Sciences
                Biochemistry
                Proteins
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                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
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                Hydroxyl Amino Acids
                Tyrosine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Hydroxyl Amino Acids
                Tyrosine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Hydroxyl Amino Acids
                Tyrosine
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblot Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblot Analysis
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