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TNF Cdc42-Mediated Filopodium Multiple Signal Transduction Pathways Regulate TNF-Induced Actin Reorganization in Macrophages: Inhibition of Cdc42-Mediated Filopodium Formation by This information is current as TNF of September 28, 2021. Maikel Peppelenbosch, Elke Boone, Gareth E. Jones, S.J.H. van Deventer, Guy Haegeman, Walter Fiers, Johan Grooten and Anne J. Ridley J Immunol 1999; 162:837-845; ; Downloaded from http://www.jimmunol.org/content/162/2/837 References This article cites 48 articles, 17 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/162/2/837.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Multiple Signal Transduction Pathways Regulate TNF-Induced Actin Reorganization in Macrophages: Inhibition of Cdc42-Mediated Filopodium Formation by TNF Maikel Peppelenbosch,1*†‡§ Elke Boone,‡ Gareth E. Jones,¶ S.J.H. van Deventer,† Guy Haegeman,‡ Walter Fiers,‡ Johan Grooten,‡ and Anne J. Ridley*| TNF is known to regulate macrophage (Mf) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased Downloaded from pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mf cytoskeleton, and that the ability of TNF to inhibit Mf chemotaxis may be due to decreased filopodium formation downstream of Cdc42. The Journal of Immunology, 1999, 162: 837–845. http://www.jimmunol.org/ umor necrosis factor (TNF), originally identified as an which is also observed in TNF-stimulated neutrophils, seems to be antitumoral activity, is now recognized as a major regu- mediated by an TNF-induced loss of chemotactic responses (7– T lator of inflammatory and immune responses (reviewed in 10). Indeed, we recently showed that TNF abrogated Mf chemo- Ref. 1). The proinflammatory action of TNF can be attributed to a taxis toward CSF-1 but had no effect on CSF-1-stimulated chemo- large extent to its effects on phagocytic cells of the monocytic and kinesis (11). Therefore, TNF action in Mf includes specific effects myeloid lineages. Macrophages (Mf)2 in particular function as on Mf locomotion. important mediators of TNF in pathophysiology. In these cells, TNF acts by stimulating two different types of plasma mem- TNF triggers the synthesis and secretion of many proinflammatory brane-localized receptors: TNF receptor 1 (TNFR-1) and TNF re- cytokines, including TNF itself, as well as lipid mediators such as ceptor 2 (TNFR-2), both of which are expressed on Mf. Although by guest on September 28, 2021 platelet-activating-factor and prostaglandins (2, 3). Mf activation these TNFRs are independently active in TNF signaling (12), the is itself dependent upon stimulation by the membrane-bound form large majority of TNF-induced signaling events can be mediated of TNF expressed by CD41 Th1 cells (4, 5), which promotes im- solely by TNFR-1 (reviewed in Ref. 13). The addition of TNF to portant Mf effector functions such as the production of oxygen TNFR-1 results in receptor aggregation. Subsequently, a variety of radicals and nitric oxide, the synthesis of antibiotic enzymes, and adapter molecules is recruited to the intracellular part of the re- the fusion of phagosomes with lysosomes. Thus, TNF-induced ac- ceptor, different domains apparently interacting with specific tivation is essential for the Mf-mediated destruction of pathogens adapter molecules, which in turn activate distinct signal transduc- and tumor cells (6). In addition, TNF has profound effects on tion pathways. Among the receptor associating-proteins identified phagocyte locomotion, inhibiting the migratory response of Mf is a protein called FAN, which binds the TNFR at amino acids toward chemotactic stimuli, an effect that probably serves to retain 307–321 and activates neutral sphingomyelinase activity (14). Di- Mf at the actual site of infection. This migration-inhibitory effect, rectly adjacent to the FAN-binding part of the receptor is the death domain (amino acids 326–413), which can bind the TNFR-1- associated death domain protein (15). The latter protein can sub- *Ludwig Institute for Cancer Research, London, United Kingdom; †Laboratory for sequently recruit other signaling proteins, including the TNFR- Experimental Internal Medicine, Academic Medical Centre, Amsterdam, The Neth- associated proteins 1 and 2 (TNFR-associated factor-1 and TNFR- ‡ erlands; Laboratory for Molecular Biology, Flemish Institute for Biotechnology, associated factor-2) (16, 17), the Fas-associated protein with death Gent, Belgium; ¶The Randall Institute, Kings College, London, United Kingdom; and \Department of Biochemistry and Molecular Biology, University College of London, domain (17), receptor-interacting protein (18), and others. The pro- London, United Kingdom tein complex thus formed mediates a number of responses, includ- Received for publication May 28, 1998. Accepted for publication October 5, 1998. ing TNF-induced activation of NF-kB, c-Jun N-terminal kinase The costs of publication of this article were defrayed in part by the payment of page (stress-activated protein kinase), caspases, and apoptosis (19). Fi- charges. This article must therefore be hereby marked advertisement in accordance nally, the membrane proximal region of the receptor (amino acids with 18 U.S.C. Section 1734 solely to indicate this fact. 205–258) binds and, upon receptor activation, stimulates a phos- 1 Address correspondence and reprint requests to Dr. Maikel Peppelenbosch, Labo- ratory for Experimental Internal Medicine, G2-130, Academic Medical Centre, phatidylinositol-4-phosphate 5-kinase (20). In addition, this part of Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail address: the receptor together with the death domain induces nitric oxide [email protected] synthase (21). 2 Abbreviations used in this paper: Mf, macrophage(s); TNFR, TNF receptor; TPA, The role of the different TNFR-1 domains and associated signal 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; MAPK, mitogen-ac- f tivated protein kinase; PAK, p21-activated kinase; TRITC, tetramethylrhodamine B transduction pathways in mediating the effects of TNF on M isothiocyanate. migration has not been investigated. However, a group of proteins Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 838 TNF SIGNAL TRANSDUCTION TO THE CYTOSKELETON IN Mf known as the Rho family of small GTPases has been implicated in England Biolabs, Beverly, MA), or 100 ng/ml human recombinant TNF regulating cell migration. Members of this family, including (Genzyme, Cambridge, MA) for 5 min, and subsequently stimulated with Cdc42, Rac, and Rho, play a major role in remodeling the actin 100 ng/ml human recombinant CSF-1 (Chiron Corporation, Emeryville, CA) for 10 min (J774.2 cells), 15 min (Bac1.2F5 cells and 4-4 cells), or 20 cytoskeleton in response to external stimuli (reviewed in Refs. 22 min (P388D1 cells), or stimulated for 30 min with 1000 infectious units/ml and 23). Cdc42 specifically induces the formation of filopodia in murine TNF (4-4 cells; murine TNF was produced in Escherichia coli, Swiss 3T3 cells (24, 25), whereas Rac and Rho regulate lamelli- purified to $99% homogeneity in our laboratory, and contained 4 ng of podium and stress fiber formation, respectively (26–27). Recently, endotoxin/mg protein). For testing the action of mutated human TNFRs, 4-4 cells (107 cells in 1 ml of RPMI 1640 containing 5% FCS) were trans- we demonstrated that Rho family members have similar activities fected with a total of 5 mg of cDNA by electroporation (300 V/950 mF/10 in Mf: Cdc42 mediates filopodium formation, whereas Rac reg- ms), and seeded on coverslips. After 24 h, cells were stimulated for 30 min ulates lamellipodium production (28). As changes in cell locomo- with the htr-1 mAb (100 ng/ml; a gift of Dr. M. Brockhaus, Hoffman-La tion are dependent upon controlled remodeling of the actin cy- Roche, Basel, Switzerland), which clusters and thus activates the trans- toskeleton, it is generally assumed that Rho family members have fected human TNFRs but not the endogenous murine receptors (31, 32). To investigate pinocytosis, fluorescently labeled BSA (prepared using Flu- an important function in directing cell motility (29). Therefore, the orlink; Amersham, Arlington Heights, IL) was added to the culture me- TNF-induced inhibition of Mf chemotaxis might well be brought dium in some experiments together with htr-1 Ab. about by interference with either the activation of Rho family pro- After stimulation, cells were fixed in 3.5% formaldehyde/PBS (v/v).
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