Engulfment of Apoptotic Cells Is Negatively Regulated by Rho-mediated Signaling
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m306079200
ISSN1083-351X
AutoresAnnie‐Carole Tosello‐Trampont, Kumiko Nakada‐Tsukui, Kodi S. Ravichandran,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase. The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase. Apoptosis or programmed cell death occurs during development and throughout life as part of normal tissue homeostasis. The prompt removal of apoptotic cells by phagocytes prevents their secondary necrosis and inflammation and also plays a critical role in tissue remodeling and immune response regulation (1.Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2148) Google Scholar). The general features of apoptosis and engulfment of cell corpses are highly conserved through evolution, which further underscores their importance. It is noteworthy that, compared with other types of phagocytosis such as that mediated via the Fc receptor (FcR), 1The abbreviations used are: FcRFc receptorCRcomplement receptorGEFguanine nucleotide exchange factorGFPgreen fluorescent proteinCRIBRac interactive bindingONC4Aoncogenic form of the Rho-specific GEF Lbc composed of only the DH-PH regions of LbcMLCKmyosin light chain kinase. the phagocytosis of apoptotic cells does not normally lead to production of pro-inflammatory mediators. The failure to remove apoptotic bodies has been implicated as a cause for different types of chronic inflammation and a predisposition to cancer, as well as autoimmune diseases (2.Mevorach D. Mascarenhas J.O. Gershov D. Elkon K.B. J. Exp. Med. 1998; 188: 2313-2320Crossref PubMed Scopus (586) Google Scholar, 3.Hughes J. Liu Y. Van Damme J. Savill J. J. Immunol. 1997; 158: 4389-4397PubMed Google Scholar, 4.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1291) Google Scholar). There is now increasing evidence that a distinct set of evolutionarily conserved cellular receptors, ligands, and signaling molecules facilitates the uptake of apoptotic cells. Fc receptor complement receptor guanine nucleotide exchange factor green fluorescent protein Rac interactive binding oncogenic form of the Rho-specific GEF Lbc composed of only the DH-PH regions of Lbc myosin light chain kinase. In the past few years, a number of studies have shown a central role for the Rho-family GTPases and their downstream effectors during different types of phagocytosis (5.Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (371) Google Scholar, 6.Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar, 7.Chimini G. Chavrier P. Nat. Cell. Bio. 2000; 2: E191-E196Crossref PubMed Scopus (266) Google Scholar, 8.Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (206) Google Scholar, 9.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10.Albert M.L. Kim J.I. Birge R.B. Nat. Cell. Bio. 2000; 2: 899-905Crossref PubMed Scopus (333) Google Scholar, 11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13.Greenberg S. J. Cell Sci. 2001; 114: 1039-1040PubMed Google Scholar). An important role for Rac and its upstream activators has been demonstrated from worm to mammals in the engulfment of apoptotic cells (10.Albert M.L. Kim J.I. Birge R.B. Nat. Cell. Bio. 2000; 2: 899-905Crossref PubMed Scopus (333) Google Scholar, 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14.Leverrier Y. Lorenzi R. Blundell M.P. Brickell P. Kinnon C. Ridley A.J. Thrasher A.J. J. Immunol. 2001; 166: 4831-4834Crossref PubMed Scopus (106) Google Scholar, 15.Reddien P.W. Horwitz H.R. Nat. Cell. Bio. 2000; 2: 131-136Crossref PubMed Scopus (341) Google Scholar, 16.Wu Y.C. Tsai M.C. Cheng L.C. Chou C.J. Weng N.Y. Dev. Cell. 2001; 1: 491-502Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17.Zhou Z. Caron E. Hartwieg E. Hall A. Horvitz H.R. Dev. Cell. 2001; 1: 477-489Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Previously, we have observed that the CrkII/Dock180/ELMO proteins lead to activation of Rac and promote Rac-dependent phagocytosis (11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Studies using overexpression of a dominant-negative form of Cdc42 and mice lacking the Cdc42 effector WASP (Wiskott-Aldrich syndrome protein) have suggested a positive role for Cdc42 in the uptake of apoptotic cells (6.Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar, 9.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14.Leverrier Y. Lorenzi R. Blundell M.P. Brickell P. Kinnon C. Ridley A.J. Thrasher A.J. J. Immunol. 2001; 166: 4831-4834Crossref PubMed Scopus (106) Google Scholar). However, the role of RhoA in phagocytosis of apoptotic cells is less clear. RhoA and the activation of Rho-kinase have been shown to be critical for complement receptor (CR)-mediated phagocytosis but not FcR-mediated uptake (6.Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar, 18.Olazabal I.M. Caron E. May R.C. Schilling K. Knecht D.A. Machesky L.M. Curr. Biol. 2002; 12: 1413-1418Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Because FcR- and CR-mediated phagocytosis utilize different Rho-family members (6.Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar, 18.Olazabal I.M. Caron E. May R.C. Schilling K. Knecht D.A. Machesky L.M. Curr. Biol. 2002; 12: 1413-1418Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and because the outcome of engulfment of apoptotic cells is distinct from the above two types of phagocytosis (19.Fadok V.A. Henson P.M. Curr. Biol. 1998; 8: R693-R695Abstract Full Text Full Text PDF PubMed Google Scholar), we examined the contribution of RhoA during engulfment. Here we present evidence that RhoA activity is down-regulated during engulfment. Moreover, RhoA seems to negatively affect basal engulfment, such that inhibition of RhoA-mediated signaling enhances the uptake of apoptotic cells. Among the Rho effectors, the Rho-kinase seems to be primarily responsible for this inhibitory effect on engulfment. Cell Culture—The phagocytic LR73 Chinese hamster ovary cell line was cultured as described previously (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The J774 macrophage cell line was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mm HEPES, pH 7.4, 0.05 μm β-mercaptoethanol, 4.5 g/liter glucose, and antibiotics. Plasmids and Antibodies—The plasmids encoding the following proteins were kindly provided by the indicated investigators: Myc-tagged wt CrkII (M. Matsuda, Japan); C3 exoenzyme (A. Reynolds, Vanderbilt University); FLAG-tagged Rac1Q61L, Rac1T17N, RhoQ63L, RhoT19N, Myc-tagged Cdc42Q61L, and Cdc42T17N (T. Parsons, University of Virginia); ONC4A, the DH-PH region of Rho-specific guanine exchange factor (GEF) Lbc and PS 1, the DH/PH deleted mutant of Lbc (D. Toksoz, Tufts University School of Medicine, Boston); oncogenic Vav2-GFP encoding the PH/DH regions of Vav2, and C-term Vav2-GFP encoding the DH/PH-deleted mutant of Vav2 (B. P. Liu and K. Burridge, University of North Carolina); DH-PH region of Cdc42-specific GEF intersectin (T. Karnoub and C. Der); Scar-WA plasmid (Laura Machesky); GFP-tagged G14VRhoA and its mutants, and GFP-DAD (A. Palazzo and G. Gunderson); and various mDia plasmids. The sources of the antibodies used were as follows: anti-GFP and anti-Cdc42 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG M2 (Sigma), anti-Crk (Transduction Laboratories, Lexington, KY), anti-Rac (Upstate Biotechnology, Lake Placid, NY) and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). All immunoblots were developed by using enhanced chemiluminescence (Pierce). Rac-GTP and Rho-GTP Pull-down Assay—Bacterially produced glutathione S-transferase-Cdc42/CRIB domain of p21-activated kinase or glutathione S-transferase-Rho-binding domain of rhotekin proteins bound to glutathione-Sepharose beads was incubated with lysates of LR73 for 1 h at 4 °C. The beads were washed, and the binding of Rac-GTP and Rho-GTP in the lysates to the beads was analyzed by immunoblotting for Rac and Rho, respectively. Transfection and Immunoblotting—For transient transfections, LipofectAMINE 2000 reagent (Invitrogen) was used as per the manufacturer's recommendations. Briefly, 100,000 LR73 cells were plated in 24-well plates. The cells were transfected the next day by incubation with plain medium containing 1.5 μl of LipofectAMINE 2000 and the indicated plasmids for 7 h before washing and were incubated with fresh medium for 16–20 h before performing the phagocytosis assays. When cotransfecting multiple plasmids, carrier DNA was added to match plasmid concentration in the different samples. In some experiments, the transfection was done in triplicates (using the same master mix of transfection reagent plus plasmids), with two wells used for the engulfment assays and the third well being used to analyze the expression of proteins by immunoblots. The lysis and immunoblotting were performed as described previously (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Phagocytosis Assays—16–20 h after transfection, the cells were incubated with 2 μm carboxylate-modified red fluorescent beads (Sigma and Molecular Probes, Eugene, OR) or with apoptotic thymocytes labeled with CM-Orange (Molecular Probes), as described previously (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 40–50% of thymocytes were Annexin V positive (i.e. apoptotic), and less than 5% of Annexin V positive cells were propidium iodide positive (i.e. necrotic). The thymocytes were washed and plated (at 0.7–1 × 106 cells/well in 300 μl of growth medium) on top of LR73 cells or J774 cells. Routinely, the assays were carried out in duplicates or triplicates for each condition with a 50-min incubation for thymocytes. The wells were then aspirated and washed twice with cold phosphate-buffered saline. The cells on the plate were trypsinized, resuspended in cold medium (with 0.1% sodium azide), and analyzed by flow cytometry. Unengulfed thymocytes were gated out by their forward and side scatter. Routinely, 10,000–20,000 events were collected, and the data were analyzed using CellQuest software. The controls included the use of live thymocytes (not treated with dexamethasone) and apoptotic thymocytes incubated at 4 °C. Although we trypsinized the cells prior to analysis, the fluorescence-activated cell sorter assay could not distinguish between fluorescence derived from bound and that from engulfed thymocytes. However, as determined by confocal microscopy, the majority of fluorescent phagocytes scored in the fluorescence-activated cell sorter assay represent cells that have engulfed the thymocytes or are in the process of engulfment (data not shown). For engulfment assays with 2 μm carboxylate-modified beads, the cells were incubated with beads in serumfree medium for 2 h. After washing, the cells on the plate were trypsinized, resuspended in cold medium with 0.5% sodium azide, and 10,000–20,000 cells were analyzed for each point by two-color flow cytometry (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Forward and side-scatter parameters were used to distinguish the unbound beads from cells. The transfected cells were recognized by their GFP fluorescence (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The ADP-ribosyltransferase C3 (intact toxin, Sigma) was pre-incubated with the phagocytes at 5 μg/ml for 16 h, and the beads were added with fresh serum-free medium for 2 h. The intact Clostridium difficile toxin B (provided by Dr. Chang Hahn, University of Virginia; Dr. Charalabos Pothoulakis, Harvard Medical School; and Dr. Popoff, Institut Pasteur) was pre-incubated at 10 ng/ml for 15 min and was present throughout the engulfment assay. Y-27632 (Calbiochem, San Diego, CA) was used at 10 μm or 30 μm (as indicated) and added along with the beads onto the cells for 2 h during uptake. ML-7 (Calbiochem) was used at 5, 10, and 30 μm (as indicated) and added along with the beads onto the cells for 2 h during uptake. Microscopy—The indicated plasmids were transfected into LR73 cells using LipofectAMINE 2000 reagent. 20 h after transfection, the cells were incubated for 2 h in the presence or absence of the Rho inhibitor Y-27632 in serum-free medium. LR73 cells were fixed in 3.7% paraformaldehyde and permeabilized with phosphate-buffered saline/0.1% bovine serum albumin/0.1% Triton X-100. The permeabilized cells were blocked with phosphate-buffered saline/1% bovine serum albumin prior to staining with Alexa Fluor 568-phalloidin (Molecular Probes) and analyzed by using an Olympus confocal microscope. The images shown are representative of multiple cells with similar phenotype on the same slide and are representative of two independent experiments. Down-regulation of Rho Activity during Engulfment—To assess the role of Rho in the phagocytosis of apoptotic cells, we examined the activity of endogenous Rho during engulfment by performing a Rho-GTP pull-down assay. Phagocytic LR73 cell lines were incubated for different times with 2 μm carboxylate-modified latex beads, which we have shown previously can function as a simplified target that mimics the negative charge on apoptotic cells (11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This simplified target was chosen to avoid contamination by Rho proteins derived from the target cells. The level of Rho-GTP in the phagocyte began to decrease after 30 min and was dramatically decreased after 2 h of incubation with the targets (Fig. 1A). Interestingly, we had observed previously that maximal uptake of these surrogate apoptotic targets occurs at the 2-h time point in our assays (11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In contrast, the level of Rac-GTP was increased in the phagocytes at the 30-min and 2-h time points under similar conditions (Fig. 1B). This result suggested a reciprocal regulation of Rho-GTP and Rac-GTP levels and raised the possibility that inhibition of Rho activity might be beneficial during phagocytosis. Inhibition of Endogenous Rho Promotes Phagocytosis of Apoptotic Cells—To further examine the benefit of inhibition of Rho activity during phagocytosis, a phagocytosis assay was performed to test the effect of C3 exoenzyme (from Clostridium botulinum) (20.Aktories K. Hall A. Trends Pharmacol. Sci. 1989; 10: 415-418Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 21.Richard J.F. Petit L. Gibert M. Marvaud J.C. Bouchaud C. Popoff M.R. Int. Microbiol. 1999; 2: 185-194PubMed Google Scholar, 22.Just I. Hofmann F. Genth H. Gerhard R. Int. J. Med. Microbiol. 2001; 291: 243-250Crossref PubMed Scopus (37) Google Scholar), which specifically inactivates Rho proteins through ADP-ribosylation but does not affect Rac or Cdc42. Pre-treatment of J774 murine macrophage cells with C3 exoenzyme for 16 h increased their ability to engulf apoptotic thymocytes (Fig. 2A). Pre-treatment of LR73 cells with the C3 exoenzyme likewise significantly enhanced the engulfment (fold increase of 1.23 + 0.09, p < 0.01, n = 3). In comparison, C. difficile toxin B, which inhibits all Rho family GTPases-inhibited engulfment, similar to what we have seen previously (Fig. 2B; Ref. 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Transient transfection of LR73 cells with a plasmid encoding the C3 exoenzyme also led to a similar enhancement of uptake (Fig. 2B). In the same experiment, transient transfection of an activated form of Rho (RhoQ63L) partially inhibited the uptake of apoptotic cells (Fig. 2B and see below). In addition to the effect on uptake of apoptotic cells, C3 pre-treatment of LR73 phagocytes also enhanced the uptake of 2 μm carboxylate-modified beads (Fig. 2C). Because the uptake of beads and apoptotic cells were both regulated similarly by Rho-mediated signaling, we have used the carboxylate-modified 2-μm beads in many of the subsequent experiments. Activation of Endogenous Rho Inhibits the Engulfment—The above data implied that inactivation of endogenous Rho promotes engulfment, possibly because of the removal of a basal inhibitory effect due to Rho. We then asked whether forced activation of endogenous Rho, for example through Rho-specific GEFs, would lead to inhibition of phagocytosis. To achieve this, we expressed an oncogenic form of the Rho-specific GEF Lbc, denoted ONC4A (composed of only the DH-PH regions of Lbc; see supplemental Fig. 1), which has been shown to lead to Rho-GTP loading within cells and subsequent oncogenic transformation (23.Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (69) Google Scholar). Overexpression of ONC4A is expected to allow the cycling of RhoA between GTP and GDP bound states and also to permit regulation by Rho-GAPs during phagocytosis (and thereby overcome the limitations of constitutively active mutants of RhoA). Expression of the ONC4A form of Lbc in LR73 phagocytes, but not a DH-PH-deleted mutant of Lbc (PS1; see supplemental Fig. 1), resulted in significant inhibition of engulfment (Fig. 3A). In contrast, expression of an oncogenic version of Vav2 (that promotes Rac activation as well as other Rho-family GTPases; see supplemental Fig. 1; Ref. 24.Liu B.P. Burridge K. Mol. Cell. Biol. 2000; 20: 7160-7169Crossref PubMed Scopus (171) Google Scholar) enhanced phagocytosis (Fig. 3B), whereas a mutant of Vav2 lacking the catalytic activity (denoted C-ter; see supplemental Fig. 1) had no effect. We then examined whether the inhibition of phagocytosis observed above was caused by a defect in the binding of targets to the phagocytes or their internalization. Overexpression of the ONC4A or PS1 did not affect particle binding to the phagocytes compared with control vector-transfected cells (assessed by performing the phagocytosis assay in the presence of 1% azide or at 4 °C to prevent internalization but not binding). This finding suggested that forced activation of endogenous Rho in the phagocytes causes inhibition of uptake rather than inhibition of the initial binding of the targets (Fig. 3C). Overexpression of Constitutively Active RhoA but Not Rac and Cdc42 Inhibits Engulfment—Transient transfection of LR73 cells with an activated form of Rho (RhoQ63L) partially inhibited the uptake of apoptotic cells (Fig. 2B) as well as 2-μm carboxylate-modified beads (Fig. 4A). In contrast, a weak dominant-negative form of Rho (RhoT19N) did not inhibit engulfment. Although in some experiments RhoT19N increased the uptake, this was not consistently observed. The better and more consistent enhancement with C3 likely reflects a higher degree of endogenous RhoA inhibition, as the RhoT19N was not very potent in our hands. We have previously shown that expression of the adapter protein CrkII (homologue of the worm CED-2) significantly enhances engulfment by a Rac-dependent mechanism (12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Coexpression of RhoQ63L with CrkII potently inhibited the CrkII-mediated enhanced uptake (Fig. 4A). In contrast, the RhoT19N did not inhibit the CrkII-mediated uptake. As a control, the dominant-negative RacT17N inhibited the CrkII-mediated uptake, as has been shown previously (Fig. 4A; Refs. 11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 12.Tosello-Trampont A.-C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Comparable inhibition of engulfment was also seen when the RhoQ63L plasmid was transfected into LR73 cells stably expressing CrkII (data not shown). As another approach, coexpression of RhoQ63L also inhibited uptake mediated by ELMO1/Dock180/CrkII proteins (which have been shown to promote maximal Rac activation and maximal phagocytosis) (Ref. 11.Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar and data not shown). These data suggested that the inhibition of phagocytosis caused by overexpression of activated Rho are dominant under these conditions. It is noteworthy that overexpression of activated Rho did not affect particle binding to the phagocytes (see Fig. 3C). When we compared the effect due to constitutively active Rho with other small GTPases, the inhibitory effect due to active Rho appeared specific, because expression of a constitutively active Rac1 (RacQ61L) or constitutively active Cdc42 (Cdc42Q61L) did not cause an inhibition (Fig. 4B). Moreover, overexpression of Intersectin, a Cdc42-specific GEF (25.Karnoub A.E. Worthylake D.K. Rossman K.L. Pruitt W.M. Campbell S.L. Sondek J. Der C.J. Nat. Struct. Biol. 2001; 8: 1037-1041Crossref PubMed Scopus (84) Google Scholar, 26.Hussain N.K. Jenna S. Glogauer M. Quinn C.C. Wasiak S. Guipponi M. Antonarakis S.E. Kay B.K. Stossel T.P. Lamarche-Vane N. McPherson P.S. Nat. Cell. Bio. 2001; 3: 927-932Crossref PubMed Scopus (304) Google Scholar), promoted engulfment (Fig. 4B). This is in distinct contrast to the effect seen with activation of endogenous Rho through ONC4A. The difference in the ability to engulf between activation of endogenous Cdc42 through Intersectin and activated Cdc42Q61L likely reflects the lower potency of the "constitutively active" Cdc42 mutant (as has been observed by others with this mutant). Thus, forced activation of endogenous Rho or overexpression of constitutively active forms of Rho, but not Rac1 or Cdc42, inhibited engulfment. Taken together with the enhanced uptake seen when Rho was inactivated through the C3 toxin, these data suggested that Rho proteins negatively regulate basal uptake, which may need to be overcome for optimal engulfment. Rho-kinase as a Key Rho Effector in Negative Regulation of Engulfment—GTP-bound Rho has been shown to bind a number of different downstream effectors such as p140mDia, Rho-kinases, citron, rhotekin, rhophilin, and protein kinase N to mediate various functional outcomes (Fig. 5A; Refs. 27.Van Aelst L. Symons M. Genes Dev. 2002; 16: 1032-1054Crossref PubMed Scopus (191) Google Scholar, 28.Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 29.Frame M.C. Brunton V.G. Curr. Opin. Genet. Dev. 2002; 12: 36-43Crossref PubMed Scopus (76) Google Scholar). Expression of a different constitutively active form of RhoA (RhoAG14V) in J774 macrophage cells again led to a potent inhibition of uptake (similar to that seen with RhoAQ63L). However, a RhoAG14V mutant carrying a concurrent T37Y mutation, which fails to interact with all of its known effectors, abolished the inhibitory effect due to RhoAG14V (Fig. 5B). This result suggested that inhibition by the active forms of RhoA involves interaction of RhoA with its effectors. Other investigators have successfully used effector domain mutants of RhoA, which can interact with some effectors but not others, to test the involvement of particular downstream effectors (Fig. 5A; Refs. 30.Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (231) Google Scholar, 31.Palazzo A.F. Cook T.A. Alberts A.S. Gundersen G.G. Nat. Cell. Bio. 2001; 3: 723-729Crossref PubMed Scopus (489) Google Scholar). We observed that the RhoAG14V/F39V mutant, which can interact with mDia and Rho-kinase but not the other known effectors (32.Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (949) Google Scholar, 33.Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (798) Google Scholar, 34.Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar), was still able to potently inhibit engulfment of beads when overexpressed in J774 macrophages (Fig. 5B). However, the RhoAG14V/F39A mutant that retains the ability to activate mDia, but not Rho-kinase, completely lost its inhibitory effect on engulfment. This pointed to Rho-kinase (also referred to as ROK and ROCK) as a potential mediator of the inhibitory effect due to Rho. Essentially similar results were obtained with the same set of Rho mutants when LR73 cells were used as phagocytes (Fig. 5C). Although the above data suggested that Rho-kinase was likely the primary mediator of the Rho-dependent inhibition of engulfment, the F39A mutant of Rho can also bind mDia (31.Palazzo A.F. Cook T.A. Alberts A.S. Gundersen G.G. Nat. Cell. Bio. 2001; 3: 723-729Crossref PubMed Scopus (489) Google Scholar, 35.Fujisawa K. Madaule P. Ishizaki T. Watanabe G. Bito H. Saito Y. Hall A. Narumiya S. J. Biol. Chem. 1998; 273: 18943-18949Abstract Full Text Full Text P
Referência(s)