SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Yanagi, S. Articles by Yamamura, H. Search for Related Content PubMed PubMed Citation Articles by Yanagi, S. Articles by Yamamura, H. Related Collections Hemostasis, Thrombosis, and Vascular Biology Signal Transduction Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 November 2001, Vol. 98, No. 9, pp. 2869-2871 BRIEF REPORT Syk expression in endothelial cells and their morphologic defects in embryonic Syk-deficient mice Shigeru Yanagi, Ryoko Inatome, Junyi Ding, Hironori Kitaguchi, Victor L. J. Tybulewicz, and Hirohei Yamamura From the Departments of Biochemistry and Physiology, Kobe University School of Medicine, Chuo-ku, Japan; and National Institute for Medical Research, London, United Kingdom.     Abstract Top Abstract Introduction Study design Results and discussion References Mice deficient in the Syk tyrosine kinase showed severe petechiae in utero and died shortly after birth. The mechanism of this bleeding, however, remains unknown. Here it is shown that this bleeding is caused by morphologic defects of Syk-deficient endothelial cells during embryogenesis. Immunoblot and reverse transcriptase-polymerase chain reaction Northern blot analysis indicated that Syk is expressed in several endothelial cell lines. Immunocytochemical analysis also confirmed that Syk is expressed in the normal embryonic endothelial cells and is absent in Syk-deficient mice. Furthermore, electron microscopic analysis of Syk-deficient mice revealed an abnormal morphogenesis and a decreased number of endothelial cells. The results indicate a critical role for Syk in endothelial cell function and in maintaining vascular integrity in vivo. (Blood. 2001;98:2869-2871) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Syk protein-tyrosine kinase contains a C-terminal kinase domain and tandem N-terminal SH2 domains that bind phosphorylated immunoreceptor tyrosine-based activation motif and play critical roles in signaling through immune receptors.1,2 Syk is expressed by all hematopoietic cells; it is essential for lymphocyte development and signal transduction via immune receptors in nonlymphoid cells.3,4 On the other hand, Syk-deficient mice show a lethal phenotype associated with severe petechiae, diffuse hemorrhage, and chylous ascites.5,6 This latter phenotype is suggestive of loss of vascular integrity although Syk expression has not been examined in vascular endothelial cells (ECs). Remarkably, the bleeding phenotype is transferable when Syk-deficient bone marrow is transplanted into wild-type mice.7,8 Classical tests of platelet function such as bleeding time are normal in radiation chimeras reconstituted with Syk-deficient platelets.7,9 Thus, the mechanism of this bleeding is currently unclear. Here we report that Syk is expressed in ECs and the bleeding observed in Syk-deficient mice might be caused by EC dysfunction during embryogenesis.     Study design Top Abstract Introduction Study design Results and discussion References Western blot and RT-PCR Northern blot analysis Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) Northern blot analysis were performed as described previously.10 Immunohistology Wild-type and Syk-deficient mice at embryonic day 18.5 were used for immunohistochemical analysis. Both embryos were fixed in 10% neutral buffered formalin followed by Bouins solution. Sections were immunostained using Serial sections 4 µm thick were cut and stained on glass slides. For double staining, the cells were incubated with the polyclonal anti-Syk antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and the monoclonal anti-CD31 antibody (Caltag, Burlingame, CA) for 2 hours at room temperature, washed 3 times with 0.2% Triton X-100 in phosphate-buffered saline, and then incubated with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (Sigma, St Louis, MO) and Alexa Fluor 594-labeled goat antirat immunoglobulin G (Molecular Probes, Eugene, OR) for 1 hour. The samples were washed as before, mounted using Slowfade Antifade kit (Molecular Probes), and analyzed using an Olympus (Tokyo, Japan) fluorescence microscope. Electron microscopic analysis Subcutaneous tissues from wild-type and Syk-deficient mice at embryonic day 18.5 were fixed for 3 days with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). They were postfixed with 2% OsO4 in the same buffer. After dehydration with a graded series of ethanol, they were substituted by propylene oxide and embedded in epoxy resin. Silver to gold thin sections were doubly stained with uranyl acetate and lead citrate, examined with an H-7000 transmission electron microscope (Hitachi, Tokyo, Japan), and photographed at various magnifications as indicated.     Results and discussion Top Abstract Introduction Study design Results and discussion References To examine whether Syk was expressed in vascular ECs, we performed immunoblot analyses using anti-Syk antibody. As shown in Figure 1A, compared with the human Ramos B cells, a lower level of Syk expression was observed both in human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs). The specificity of the antibody was evidenced by the abolition of a 72-kd band in lysates from Syk-deficient fetal liver and Syk-deficient DT40 avian B cells (Figure 1A, lanes 2 and 5). To confirm Syk expression in HUVECs, we performed RT-PCR analysis using messenger RNA (mRNA) prepared from these cells. This analysis demonstrated that Syk mRNA was expressed in HUVECs (Figure 1B). Furthermore, immunofluorescence microscopic analysis of microvasculature of wild-type and Syk-deficient mice showed that Syk is expressed in mouse embryonic ECs and is lacking from the endothelium of mutant mice (Figure 1C). These results provide the first evidence of Syk expression in ECs. It has recently been reported that Syk is also expressed, albeit at a low level, in various nonhematopoietic cells, including hepatocytes, neuronal cells, and fibroblasts.11-14 Thus, Syk may be a multifunctional protein tyrosine kinase involved in numerous signal transduction pathways. View larger version (28K): [in this window] [in a new window]   Figure 1. Expression of Syk in vascular ECs. (A) Immunoblot analysis of Syk in vascular ECs. Cell and tissue extracts (10 µg protein) were analyzed by immunoblotting with anti-Syk antibody: wild-type (lane 1) and Syk-deficient mice liver (lane 2), HUVECs (lane 3), BAECs (lane 4), Syk-deficient DT40 chicken B cells (lane 5), wild-type DT40 cells (lane 6), and human Ramos B cells (lane 7). (B) RT-PCR Northern blot analysis of Syk mRNA in Ramos B cell line (lane 1) and HUVECs (lane 2). The positions of Syk and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control are indicated. (C) Expression of Syk in mouse embryonic ECs. Immunofluorescence microscopy of microvasculature of wild-type and Syk-deficient mice at embryonic day 18.5. ECs were double labeled with anti-Syk antibody and with anti-CD31 antibody as a marker for ECs. Bars = 1.0 µm To investigate whether the hemorrhage in Syk-deficient mice was caused by functional defect in microvascular ECs, we performed morphologic analyses of the microvasculature by electron microscopy using the subcutaneous tissues from the wild-type and Syk-deficient mice. Most surprisingly, electron microscopic analysis revealed the remarkable morphologic abnormality in microvasculature structure of Syk-deficient mice (Figure 2). ECs in particular showed unusually scanty cytoplasm (Figure 2B). The cytoplasm of ECs from the Syk-deficient mice barely traversed the inside of the vessel wall. It was very thin and thus appeared to be very fragile. As a consequence of the fragility of ECs, bleeding seemed to be caused by the collapse of blood vessel architecture (Figure 2D,E). View larger version (138K): [in this window] [in a new window]   Figure 2. Electron microscopic analysis of the microvasculature of Syk-deficient mice. Compared with panel A (wild-type [wt]), panel B shows remarkable morphologic abnormality and decreased numbers of ECs in Syk-deficient mice (Syk). Arrowheads highlight the scanty cytoplasm. Compared with panel C (wt), panel D shows EC shrinkage (arrowheads) and bleeding in Syk-deficient mice. The sections in panels A and C and in B and D are different sites. (F) A higher magnification image of the hemorrhagic area in panel E, which shows an erythrocyte leaking out from the vessel wall. (G,H) Tight junction structure between vascular ECs in wild-type and Syk-deficient mice, respectively (arrowheads). Bars = 2.5 µm (A-F) and 0.5 µm (G,H). Notably, in several thin sections from Syk-deficient mice there was a significant decrease in the number of ECs when compared with the wild-type controls (Figure 2B and data not shown). Indeed, Figure 2D shows EC shrinkage, a typical characteristic of apoptotic necrosis and bleeding in Syk-deficient mice. Thus, the abnormal morphogenesis of vascular ECs in the Syk-deficient mice may be the result of excessive stretching of a smaller number of ECs that could not retain the normal mechanical stability in the face of physical force such as blood flow. Alternatively, it is possible that the cytoplasmic content of ECs was decreased by the loss of Syk function. In this case, Syk may play an important regulatory role in the organization of the cytoskeletal system, which in turn affects the cytoplasmic structure of vascular ECs. Indeed, Syk has been reported to be translocated from cytosol to cytoskeleton and phosphorylate cortactin, an F-actin-binding protein that is involved in signaling events for the reorganization of the microfilaments.15 Similar hemorrhaging was also observed in mice deficient in platelet-derived growth factor-B, where the defect was postulated to be a loss of pericytes and formation of microaneurysm without a significant change in EC morphology and number.16 Pericytes form a part of the capillary wall and contribute to the mechanical stability of the capillary wall. In Syk-deficient mice, pericytes appeared well developed, without a decrease in their number (not shown), contrasting with phenotypes observed in platelet-derived growth factor-B-deficient mice. More importantly, however, there was no significant defect in tight junctions between vascular ECs in Syk-deficient mice (Figure 2H). Supporting this observation is that the point where the erythrocyte had leaked out from the vessel wall was not between ECs but the weak point between cell nucleus and cytoplasm, which had become structurally thin and obviously weak (Figure 2F). Taken together, our results suggest that the abnormal microvascular structure seen in Syk-deficient mice was mainly caused by the dysfunction of ECs and that Syk plays a critical role in physiologic regulation of ECs and contributes to maintaining vascular integrity in vivo.     Acknowledgments We thank Dr T. Terashima and Dr T. Hirase for discussion. We also thank Dr S. Jahangeer for critically reading the manuscript.     Footnotes Submitted April 11, 2001; accepted June 28, 2001. Supported in part by the Uehara Foundation of Japan, Fellowships of the Japan Society for the Promotion of Science for Young Scientists (R.I.), Grant-in-Aid for the Research for the Future Program from the Japan Society for the Promotion of Science, Grant-in-Aids for Scientific Research, and Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Hirohei Yamamura, Dept of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan; e-mail: yamamura{at}kobe-u.ac.jp.     References Top Abstract Introduction Study design Results and discussion References 1. Taniguchi T, Kobayashi T, Kondo J, et al. Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J Biol Chem. 1991;266:15790-15796[Abstract/Free Full Text]. 2. Yanagi S, Kurosaki T, Yamamura H. The structure and function of nonreceptor tyrosine kinase p72syk expressed in hematopoietic cells. Cell Signal. 1995;7:185-193[CrossRef][Medline] [Order article via Infotrieve]. 3. Kurosaki T. Functional dissection of BCR signaling pathways. Curr Opin Immunol. 2000;12:276-281[CrossRef][Medline] [Order article via Infotrieve]. 4. Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol Today. 2000;21:148-154[CrossRef][Medline] [Order article via Infotrieve]. 5. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature. 1995;378:303-306[CrossRef][Medline] [Order article via Infotrieve]. 6. Turner M, Mee PJ, Costello PS, et al. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature. 1995;378:298-302[CrossRef][Medline] [Order article via Infotrieve]. 7. Poole A, Gibbins JM, Turner M, et al. The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 1997;16:2333-2341[CrossRef][Medline] [Order article via Infotrieve]. 8. Kiefer F, Brumell J, Al Alawi N, et al. The Syk protein tyrosine kinase is essential for Fc receptor signaling in macrophages and neutrophils. Mol Cell Biol. 1998;18:4209-4220[Abstract/Free Full Text]. 9. Law DA, Nannizzi-Alaimo L, Ministri K, et al. Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets. Blood. 1999;93:2645-2652[Abstract/Free Full Text]. 10. Yanagi S, Sugawara H, Kurosaki M, Sabe H, Yamamura H, Kurosaki T. CD45 modulates phosphorylation of both autophosphorylation and negative regulatory tyrosines of Lyn in B cells. J Biol Chem. 1996;271:30487-30492[Abstract/Free Full Text]. 11. Tsuchida S, Yanagi S, Inatome R, et al. Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk: involvement of Syk in signaling events of hepatocytes. J Biochem. 2000;127:321-327. 12. Tsujimura T, Yanagi S, Inatome R, et al. Syk protein-tyrosine kinase is involved in neuron-like differentiation of embryonal carcinoma P19 cells. FEBS Lett. 2001;489:129-133[CrossRef][Medline] [Order article via Infotrieve]. 13. Wang H, Malbon CC. G(s) repression of adipogenesis via Syk. J Biol Chem. 1999;274:32159-32166[Abstract/Free Full Text]. 14. Yamada T, Fujieda S, Yanagi S, et al. Protein-tyrosine kinase p72Syk expressed in human nasal fibroblasts and its effect on RANTES production. J Immunol. 2001;166:538-543[Abstract/Free Full Text]. 15. Maruyama S, Kurosaki T, Sada K, Yamanashi Y, Yamamoto T, Yamamura H. Physical and functional association of cortactin with Syk in human leukemic cell line K562. J Biol Chem. 1996;271:6631-6635[Abstract/Free Full Text]. 16. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277:242-245[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Tohyama and H. Yamamura Protein Tyrosine Kinase, Syk: A Key Player in Phagocytic Cells J. Biochem., March 1, 2009; 145(3): 267 - 273. [Abstract] [Full Text] [PDF] K. M. Bijli, F. Fazal, M. Minhajuddin, and A. Rahman Activation of Syk by Protein Kinase C-{delta} Regulates Thrombin-induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Tyrosine Phosphorylation of RelA/p65 J. Biol. Chem., May 23, 2008; 283(21): 14674 - 14684. [Abstract] [Full Text] [PDF] S. Inubushi, M. Nagano-Fujii, K. Kitayama, M. Tanaka, C. An, H. Yokozaki, H. Yamamura, H. Nuriya, M. Kohara, K. Sada, et al. Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk J. Gen. Virol., May 1, 2008; 89(5): 1231 - 1242. [Abstract] [Full Text] [PDF] S. Luangdilok, C. Box, L. Patterson, W. Court, K. Harrington, L. Pitkin, P. Rhys-Evans, P. O-charoenrat, and S. Eccles Syk Tyrosine Kinase Is Linked to Cell Motility and Progression in Squamous Cell Carcinomas of the Head and Neck Cancer Res., August 15, 2007; 67(16): 7907 - 7916. [Abstract] [Full Text] [PDF] K. Taniguchi, R.-i. Kohno, T. Ayada, R. Kato, K. Ichiyama, T. Morisada, Y. Oike, Y. Yonemitsu, Y. Maehara, and A. Yoshimura Spreds Are Essential for Embryonic Lymphangiogenesis by Regulating Vascular Endothelial Growth Factor Receptor 3 Signaling Mol. Cell. Biol., June 15, 2007; 27(12): 4541 - 4550. [Abstract] [Full Text] [PDF] J.-W. Xu, I. Morita, K. Ikeda, T. Miki, and Y. Yamori C-Reactive Protein Suppresses Insulin Signaling in Endothelial Cells: Role of Spleen Tyrosine Kinase Mol. Endocrinol., February 1, 2007; 21(2): 564 - 573. [Abstract] [Full Text] [PDF] H.-S. Cha, D. L. Boyle, T. Inoue, R. Schoot, P. P. Tak, P. Pine, and G. S. Firestein A Novel Spleen Tyrosine Kinase Inhibitor Blocks c-Jun N-Terminal Kinase-Mediated Gene Expression in Synoviocytes J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 571 - 578. [Abstract] [Full Text] [PDF] J. Lu, W.-H. Lin, S.-Y. Chen, R. Longnecker, S.-C. Tsai, C.-L. Chen, and C.-H. Tsai Syk Tyrosine Kinase Mediates Epstein-Barr Virus Latent Membrane Protein 2A-induced Cell Migration in Epithelial Cells J. Biol. Chem., March 31, 2006; 281(13): 8806 - 8814. [Abstract] [Full Text] [PDF] E. Ryschich, P. Lizdenis, C. Ittrich, A. Benner, S. Stahl, A. Hamann, J. Schmidt, P. Knolle, B. Arnold, G. J. Hammerling, et al. Molecular Fingerprinting and Autocrine Growth Regulation of Endothelial Cells in a Murine Model of Hepatocellular Carcinoma Cancer Res., January 1, 2006; 66(1): 198 - 211. [Abstract] [Full Text] [PDF] M. Ulanova, L. Puttagunta, M. Marcet-Palacios, M. Duszyk, U. Steinhoff, F. Duta, M.-K. Kim, Z. K. Indik, A. D. Schreiber, and A. D. Befus Syk tyrosine kinase participates in {beta}1-integrin signaling and inflammatory responses in airway epithelial cells Am J Physiol Lung Cell Mol Physiol, March 1, 2005; 288(3): L497 - L507. [Abstract] [Full Text] [PDF] J. He, Y. Tohyama, K.-i. Yamamoto, M. Kobayashi, Y. Shi, T. Takano, C. Noda, K. Tohyama, and H. Yamamura Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function Genes Cells, January 1, 2005; 10(1): 23 - 35. [Abstract] [Full Text] [PDF] M. Steinberg, O. Adjali, L. Swainson, P. Merida, V. D. Bartolo, L. Pelletier, N. Taylor, and N. Noraz T-cell receptor-induced phosphorylation of the {zeta} chain is efficiently promoted by ZAP-70 but not Syk Blood, August 1, 2004; 104(3): 760 - 767. [Abstract] [Full Text] [PDF] S. M. S. Miah, K. Sada, P. T. Tuazon, J. Ling, K. Maeno, S. Kyo, X. Qu, Y. Tohyama, J. A. Traugh, and H. Yamamura Activation of Syk Protein Tyrosine Kinase in Response to Osmotic Stress Requires Interaction with p21-Activated Protein Kinase Pak2/{gamma}-PAK Mol. Cell. Biol., January 1, 2004; 24(1): 71 - 83. [Abstract] [Full Text] [PDF] E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow Tyrosine Residues Direct the Ubiquitination and Degradation of the NY-1 Hantavirus G1 Cytoplasmic Tail J. Virol., October 15, 2003; 77(20): 10760 - 10868. [Abstract] [Full Text] [PDF] K. Maeno, K. Sada, S. Kyo, S. M. S. Miah, K. Kawauchi-Kamata, X. Qu, Y. Shi, and H. Yamamura Adaptor Protein 3BP2 Is a Potential Ligand of Src Homology 2 and 3 Domains of Lyn Protein-tyrosine Kinase J. Biol. Chem., June 27, 2003; 278(27): 24912 - 24920. [Abstract] [Full Text] [PDF] A. Mocsai, H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C. A. Lowell G-protein-coupled receptor signaling in Syk-deficient neutrophils and mast cells Blood, May 15, 2003; 101(10): 4155 - 4163. [Abstract] [Full Text] [PDF] E. Geimonen, R. LaMonica, K. Springer, Y. Farooqui, I. N. Gavrilovskaya, and E. R. Mackow Hantavirus Pulmonary Syndrome-Associated Hantaviruses Contain Conserved and Functional ITAM Signaling Elements J. Virol., December 20, 2002; 77(2): 1638 - 1643. [Abstract] [Full Text] [PDF] K. Sada, S. M. S. Miah, K. Maeno, S. Kyo, X. Qu, and H. Yamamura Regulation of Fcepsilon RI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells Blood, August 28, 2002; 100(6): 2138 - 2144. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Yanagi, S. Articles by Yamamura, H. Search for Related Content PubMed PubMed Citation Articles by Yanagi, S. Articles by Yamamura, H. Related Collections Hemostasis, Thrombosis, and Vascular Biology Signal Transduction Brief Reports Social Bookmarking                   What's this?

	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020