Recognizing scientific excellence in the biology of cell adhesion

The prestigious 2005 Japan Prize for Cell Biology has been awarded to Dr. Masatoshi Takeichi, Director of RIKEN Developmental Biology, Kobe, Japan, and Dr. Erkki Ruoslahti, Distinguished Professor, The Burnham Institute, La Jolla, USA for their "Fundamental contribution in elucidating the molecular mechanisms of cell adhesion". The award is scheduled to be presented to the scientists in ceremonies in Tokyo on April 20, 2005 as part of a week-long celebration of "Japan Prize Week"

Signaling through Raf-1 in the Neovasculature and Target Validation by Nanoparticles

A recent study demonstrated that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) activate Raf-1 kinase in an experimental neovasculature system. The study showed that bFGF and VEGF activate p21-activated protein kinase-1 (PAK-1) and Src kinase, respectively. PAK-1 and Src kinases phosphorylate specific serine and tyrosine residues within the activation loop of Raf-1 kinase. Their findings further suggest that phosphorylation at these sites protects endothelial cells from apoptosis induced by both intrinsic and extrinsic factors. The tumor neovasculature provides specific molecular markers or "zip codes". This group of investigators has previously shown that nanosphere-aided targeting of the neovasculature with mutant Raf-1 causes regression of the tumor vasculature. Thus, nanoparticles coated with "zip code"-specific homing biomolecules may be useful for delivering anti-angiogenic molecules that can induce tumor regression

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

Rationale The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. Methods and results Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2\u27-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network- like structures in the presence of VEGF. Conclusion Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tubeforming cells in a 2D matrix in VEGF-dependent manner

Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF- and VEGF-induced capillary morphogenesis of endothelial cells

BACKGROUND: Angiogenesis, or the remodeling of existing vasculature serves as a lifeline to nourish developing embryos and starved tissues, and to accelerate wound healing, diabetic retinopathy, and tumor progression. Recent studies indicate that angiogenesis requires growth factor activity as well as cell adhesion events mediated by α(5)β(1 )and α(v)β(3 )integrins. We previously demonstrated that human lipid phosphate phosphohydrolase-3 (LPP3) acts as a cell-associated ligand for α(5)β(1 )and α(v)β(3 )integrins. Here, we test the hypothesis that an anti-LPP3 antibody can inhibit basic fibroblast growth factor (bFGF)-and vascular endothelial growth factor (VEGF)-induced capillary morphogenesis of endothelial cells (ECs). RESULTS: We report that bFGF and VEGF up-regulate LPP3 protein expression in ECs. Immunoprecipitation analyses show that LPP3 is a cell surface protein and undergoes N-glycosylation. Fluorescent activated cell sorting (FACS) data suggest that anti-LPP3-RGD detects native neoepitope on the surface of activated ECs. Moreover, we demonstrate LPP3 protein expression in tumor endothelium alongside VEGF. The embedding of ECs into three-dimensional type I collagen in the presence of bFGF and VEGF induce capillary formation. Importantly, we show that the addition of an anti-LPP3 antibody specifically and significantly blocks bFGF- and VEGF-induced capillary morphogenesis of ECs. CONCLUSION: These data suggest that activated ECs as well as tumor endothelium express LPP3 protein. In an in vitro assay, the anti-LPP3-RGD specifically blocks bFGF and VEGF induced capillary morphogenesis of ECs. Our results, therefore, suggest a role for LPP3 in angiogenesis

Analysis of VEGF-responsive Genes Involved in the activation of endothelial cells

BACKGROUND: Identification of the genes and pathways associated with the activation of endothelial cells (ECs) could help uncover the role of ECs in wound healing, vascular permeability, blood brain barrier function, angiogenesis, diabetic retinopathy, atherosclerosis, psoriasis, and growth of solid tumors. DESIGN: Herein, we embedded ECs in 3D type I collagen gel, left unstimulated or stimulated with VEGF(165), and subjected to suppression subtractive hybridization followed by differential display (SSHDD). Gene fragments obtained from SSHDD were subjected to DNA sequence analysis. Database search with nucleotide sequence were performed using the BLAST algorithm and expression of candidate genes determined by northern blot analysis. RESULTS: A total of ~32 cDNA fragments, including known regulators of angiogenesis, and a set of genes that were not reported to be associated with activation of ECs and angiogenesis previously were identified. We confirmed the mRNA expression of KDR, α(2 )integrin, Stanniocalcin, including a set of 11 candidate genes. Western immunoblotting results indicated that KDR, α(2 )integrin, MMP-1, MMP-2, and VE-cadherin genes were indeed active genes. CONCLUSION: We have identified a set of 11 VEGF-responsive endothelial cell candidate genes. Their expression in endothelial cell is confirmed by northern blot analyses. This preliminary report forms as a foundation for functional studies to be performed to reveal their roles in EC activation and pathophysiological events associated with the vasculature including tumor growth

Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

<p>Abstract</p> <p>Background</p> <p>The acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis <it>via </it>the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells.</p> <p>Results</p> <p>We observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that <it>LPP3</it>-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that <it>LPP3</it>-knockdown reduced β-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated β-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth <it>via </it>increased β-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells.</p> <p>Conclusions</p> <p>These results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying β-catenin and CYCLIN-D1 activities.</p

Histone Demethylases KDM4A and KDM4C Regulate Differentiation of Embryonic Stem Cells to Endothelial Cells

SummaryUnderstanding epigenetic mechanisms regulating embryonic stem cell (ESC) differentiation to endothelial cells may lead to increased efficiency of generation of vessel wall endothelial cells needed for vascular engineering. Here we demonstrated that the histone demethylases KDM4A and KDM4C played an indispensable but independent role in mediating the expression of fetal liver kinase (Flk)1 and VE-cadherin, respectively, and thereby the transition of mouse ESCs (mESCs) to endothelial cells. KDM4A was shown to bind to histones associated with the Flk1 promoter and KDM4C to bind to histones associated with the VE-cadherin promoter. KDM4A and KDM4C were also both required for capillary tube formation and vasculogenesis in mice. We observed in zebrafish that KDM4A depletion induced more severe vasculogenesis defects than KDM4C depletion, reflecting the early involvement of KDM4A in specifying endothelial cell fate. These findings together demonstrate the essential role of KDM4A and KDM4C in orchestrating mESC differentiation to endothelial cells through the activation of Flk1 and VE-cadherin promoters, respectively

Distinct Roles of the Adaptor Protein Shc and Focal Adhesion Kinase in Integrin Signaling to ERK

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf