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Distinct Signaling Pathways for MCP-1-dependent Integrin Activation and Chemotaxis

2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês

10.1074/jbc.m009068200

1083-351X

Noboru Ashida, Hidenori Arai, Masahide Yamasaki, Toru Kita,

Glycosylation and Glycoproteins Research

Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated β1 integrin-dependent, but not β2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells. Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated β1 integrin-dependent, but not β2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells. monocyte chemoattractant protein-1 mitogen-activated protein kinase cysteine-cysteine chemokine receptor bovine serum albumin extracellular regulated kinase mitogen-activated protein kinase kinase c-Jun N-terminal kinase intercellular adhesion molecule-1 vascular cell adhesion molecule-1 4-morpholineethanesulfonic acid arginine-glycine-aspartate-serine Several lines of evidence indicate that monocyte chemoattractant protein-1 (MCP-1)1 is involved in the pathogenesis of atherosclerosis by promoting directed migration of inflammatory cells, such as monocytes and T lymphocytes (1Libby P. J. Intern. Med. 2000; 247: 349-358Crossref PubMed Scopus (518) Google Scholar, 2Reape T.J. Groot P.H. Atherosclerosis. 1999; 147: 213-225Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). During the progression of atherosclerosis, there is an accumulation of low-density lipoprotein within macrophages present in the intimal layer. Deposition of lipids within these cells leads to the formation and eventual enlargement of atherosclerotic lesions. Boringet al. (3Boring L. Gosling J. Cleary M. Charo I.F. Nature. 1998; 394: 894-897Crossref PubMed Scopus (1668) Google Scholar) noted an overall decrease in atherosclerotic lesion size in mice deficient for the MCP-1 receptor, CCR2, when they are crossed with ApoE knockout mice. Gu et al. (4Gu L. Okada Y. Clinton S.K. Gerard C. Sukhova G.K. Libby P. Rollins B.J. Mol. Cell. 1998; 2: 275-281Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar) also found decreased atherosclerotic lesions in MCP-1-deficient mice when they are crossed with the low-density lipoprotein receptor knockout mice. These studies have demonstrated that MCP-1 and CCR2 play a crucial role in the initiation of atherosclerosis by recruiting monocytes to the vessel wall.According to the multistep theory, monocytes roll on the endothelial cells, interact with E-selectin, adhere to the endothelial cells by firm adhesion to ICAM-1 and VCAM-1, and then migrate into the subendothelium (5Butcher E.C. Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2503) Google Scholar). Rolling of monocytes on endothelial cells is dependent on the binding of E-selectin and sialyl Lewis X, and adhesion to the endothelium is dependent on the interaction of integrins on monocytes and adhesion molecules on the endothelial cells, such as VCAM-1 and ICAM-1. Integrins consist of several subtypes, and each subtype is specific for its ligand. For example, α4β1 integrin, very late antigen-4, binds to VCAM-1, and β2 integrins bind to ICAM-1. Fibronectin, one of the extracellular matrix proteins, is also known to bind to β1 integrins, mainly to α5β1 integrin. In these serial events of the multistep theory, MCP-1 can play a key role in monocyte recruitment by both integrin activation and by promoting migration to the vessel wall. However, the signal transduction pathways leading to integrin activation and chemotaxis have not been fully elucidated.We recently demonstrated that the βγ subunit of heterotrimeric G protein, Gi, plays a key role in MCP-1-induced chemotaxis (6Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar). In that study, we reported that activation of ERK was not involved in chemotaxis by MCP-1. MAPK family members, ERK, JNK, and p38-MAPK, have been implicated in events necessary for proliferation, differentiation, apoptosis, and certain kinds of stress responses (7Lopez-Ilasaca M. Biochem. Pharmacol. 1998; 56: 269-277Crossref PubMed Scopus (237) Google Scholar). These MAPKs are activated by specific cascades responsible for certain stimuli and eventually induce a variety of cell responses. Recently, several groups have reported on the involvement of MAPK and Rho in chemotaxis (8Ayala J.M. Goyal S. Liverton N.J. Claremon D.A. O'Keefe S.J. Hanlon W.A. J. Leukocyte Biol. 2000; 67: 869-875Crossref PubMed Scopus (51) Google Scholar,9Jing Q. Xin S.M. Zhang W.B. Wang P. Qin Y.W. Pei G. Circ. Res. 2000; 87: 52-59Crossref PubMed Scopus (73) Google Scholar). Most of the studies on signal transduction of chemotaxis and cell adhesion have been conducted in adherent cells. However, in adherent cells it would be difficult to separate chemotaxis and integrin activation, because these two functions are closely connected. Therefore, the aim of our study was to examine whether integrin activation and chemotaxis can be separated by studying the signaling cascades leading to integrin activation and chemotaxis mediated by MCP-1 in human monocytic THP-1 cells and to elucidate the role of MAPK and Rho in these biological functions.DISCUSSIONIn this study we have elucidated the role of MAPK and Rho GTPase in MCP-1-mediated cell adhesion and chemotaxis. We show that MCP-1 induced activation of the integrins α4β1 and α5β1 in THP-1 cells and that the integrin activation is dependent on ERK activation. In contrast, MCP-1-dependent chemotaxis was dependent on activation of Rho and p38-MAPK. Thus, as depicted in Fig.10 two important biological functions mediated by MCP-1 utilize two distinct MAPK-dependent signaling pathways.In this study we used sensitive cell adhesion assays to demonstrate important functions of MCP-1. Although Weber et al. (16Weber C. Alon R. Moser B. Springer T.A. J. Cell Biol. 1996; 134: 1063-1073Crossref PubMed Scopus (203) Google Scholar) demonstrated that binding of monocytes to VCAM-1 was reduced at 15 min under stimulation with MCP-1 in a similar assay, we found greater than a 2-fold increase in cell adhesion to this molecule at 10 to 20 min. In preliminary experiments, we have found a basal increase in cell adhesion after labeling the cells with fluorescent dye and washing the cells. We speculate that this is because of some stress on the cells. Further, a basal increase in cell adhesion after spinning down and washing the cells might be because of increased MAPK activation during these procedures. In support of this hypothesis, MacKenna et al. (17MacKenna D.A. Dolfi F. Vuori K. Ruoslahti E. J. Clin. Invest. 1998; 101: 301-310Crossref PubMed Scopus (273) Google Scholar) reported that in cardiac fibroblasts ERK and JNK are activated by mechanical stretch. Therefore, it would be important to avoid stress on the cells as much as possible in this experiment. Work is now in progress to determine the effect of mechanical stress on MAPK activation and cell adhesion.We found that in the monocytic cell line β1 integrins but not β2 integrins are activated by MCP-1. However, Weber et al. (18Weber K.S. Klickstein L.B. Weber C. Mol. Biol. Cell. 1999; 10: 861-873Crossref PubMed Scopus (80) Google Scholar) have reported that MCP-1 induces a prolonged increase in the binding of monocytes to ICAM-1 in a static adhesion assay. This difference may be because of a difference in the way to remove nonadherent cells. We removed nonadherent cells by centrifugation, but they did it by plate washer. So we speculate that binding of β2 integrin and ICAM-1 is not strong enough to overcome the centrifugation force. Further, Chanet al. (19Chan J.R. Hyduk S.J. Cybulsky M.I. J. Immunol. 2000; 164: 746-753Crossref PubMed Scopus (101) Google Scholar) have reported that activation of β1 integrin by chemokines might be much stronger than that of β2 integrin and that β1 integrin/VCAM-1 interaction activates β2 integrin-mediated cell adhesion in human T cells. Therefore, in in vivosituations, activation of β1 integrins might be stronger and more important in the early phase of cell migration.In the cell adhesion assay, we could not abrogate MCP-1-dependent adhesion to fibronectin by the RGDS peptide, even though we have used sufficient concentrations of the peptide. We speculate that the inhibitory effect of this peptide on the interaction between fibronectin and integrins is not so strong (20Ruppert M. Aigner S. Hubbe M. Yagita H. Altevogt P. J. Cell Biol. 1995; 131: 1881-1891Crossref PubMed Scopus (130) Google Scholar). Because RGDS-independent adhesion of fibronectin has been reported (21Silletti S. Mei F. Sheppard D. Montogomery A.M.P. J. Cell Biol. 2000; 149: 1485-1501Crossref PubMed Scopus (131) Google Scholar), it is also possible that RGDS-independent cell adhesion is induced by MCP-1.In this study we showed that in THP-1 cells ERK is responsible for integrin activation by MCP-1 but not p38-MAPK or Rho. Laudanna et al. (22Laudanna C. Campbell J.J. Butcher E.C. Science. 1996; 271 (.-): 981Crossref PubMed Scopus (431) Google Scholar), however, reported that Rho is also involved in integrin activation by interleukin-8 in neutrophils and lymphocytes. The reasons for these differences are not clear, but signaling through integrin activation in response to chemokines might be cell type-specific. It is also possible that different types of integrins in leukocytes might be activated in response to each chemokine.Recently, several reports have shown that p38-MAPK is involved in chemotaxis induced by serum, lysophosphatidylcholine, and chemokines in leukocytes and smooth muscle cells (8Ayala J.M. Goyal S. Liverton N.J. Claremon D.A. O'Keefe S.J. Hanlon W.A. J. Leukocyte Biol. 2000; 67: 869-875Crossref PubMed Scopus (51) Google Scholar, 9Jing Q. Xin S.M. Zhang W.B. Wang P. Qin Y.W. Pei G. Circ. Res. 2000; 87: 52-59Crossref PubMed Scopus (73) Google Scholar). Our study has also shown that p38-MAPK is involved in chemotaxis induced by MCP-1 in THP-1 cells. In contrast, Yen et al. (23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) showed that ERK is responsible for MCP-1-mediated chemotaxis. Knall et al.(24Knall, C., Worthen, G. S., and Johnson, G. L. (1997)Proc. Natl. Acad. Sci. U. S. A.Google Scholar), on the other hand, have shown that ERK or p38-MAPK is not involved in interleukin-8-mediated chemotaxis. The reason for these differences is not clear, but in the system of Yen et al.(23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) the activation of integrin might have been required for monocyte chemotaxis. Rho family GTPases have also been shown to be involved in cell migration (25Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. Cell Biol. 1998; 141: 1147-1157Crossref PubMed Scopus (445) Google Scholar). Thus our data are consistent with the others that p38-MAPK and Rho are involved in chemotaxis. However, the relationship between Rho and MAPK is quite complicated. For example, Zhang et al. (26Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar) have shown that p38-MAPK is downstream of Rho in interleukin-1-mediated signaling. In contrast, Hippenstiel et al. (27Hippenstiel S. Soeth S. Kellas B. Fuhrmann O. Seybold J. Krull M. Eichel-Streiber C. Goebeler M. Ludwig S. Suttorp N. Blood. 2000; 95: 3044-3051Crossref PubMed Google Scholar) have reported that LPS-induced activation of p38-MAPK is not affected by Clostridium difficile toxin B-10463, a specific inhibitor of Rho. In terms of ERK and Rho, some reports have claimed that Rho is upstream of ERK (28Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (183) Google Scholar, 29Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (215) Google Scholar, 30Lassus P. Roux P. Zugasti O. Philips A. Fort P. Hibner U. Oncogene. 2000; 19: 2377-2385Crossref PubMed Scopus (37) Google Scholar), whereas others have demonstrated that Rho and ERK are activated independently (31Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, few studies have been conducted to examine whether ERK and p38-MAPK are differentially affected by Rho. In this paper, we clearly show that MCP-1 phosphorylates ERK and p38-MAPK in THP-1 cells and that Rho is upstream of p38-MAPK but not of ERK in MCP-1-mediated signal transduction. We have also shown that the Rho kinase (32Matsui 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 (934) Google Scholar) is between Rho and p38-MAPK and is a key molecule for chemotaxis. However, downstream targets of p38-MAPK leading to chemotaxis still remain to be determined. Although we found that MCP-1 also activated JNK (data not shown), the role of JNK activation in MCP-1-mediated signaling was not determined in this study.In summary, we have provided clear evidence that two distinct signaling cascades are present to mediate MCP-1-induced activation of β1 integrins and chemotaxis in THP-1 cells. These two distinct signaling cascades would be important for transmigration of monocytes through endothelial cells. The most intriguing aspect of this study is that we could separate two important biological functions of leukocytes, integrin activation and chemotaxis, by different assays and found that two distinct signaling cascades mediate these two functions. In adherent cells, however, segregation of integrin activation and chemotaxis would be very difficult to assess. As depicted in Fig. 10, identification of signaling molecules located at the bifurcation to ERK and p38-MAPK would be important to delineate the signaling cascades through CCR2. Further, it would be intriguing to determine the signaling cascades in a condition closer to in vivosituations. Several lines of evidence indicate that monocyte chemoattractant protein-1 (MCP-1)1 is involved in the pathogenesis of atherosclerosis by promoting directed migration of inflammatory cells, such as monocytes and T lymphocytes (1Libby P. J. Intern. Med. 2000; 247: 349-358Crossref PubMed Scopus (518) Google Scholar, 2Reape T.J. Groot P.H. Atherosclerosis. 1999; 147: 213-225Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). During the progression of atherosclerosis, there is an accumulation of low-density lipoprotein within macrophages present in the intimal layer. Deposition of lipids within these cells leads to the formation and eventual enlargement of atherosclerotic lesions. Boringet al. (3Boring L. Gosling J. Cleary M. Charo I.F. Nature. 1998; 394: 894-897Crossref PubMed Scopus (1668) Google Scholar) noted an overall decrease in atherosclerotic lesion size in mice deficient for the MCP-1 receptor, CCR2, when they are crossed with ApoE knockout mice. Gu et al. (4Gu L. Okada Y. Clinton S.K. Gerard C. Sukhova G.K. Libby P. Rollins B.J. Mol. Cell. 1998; 2: 275-281Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar) also found decreased atherosclerotic lesions in MCP-1-deficient mice when they are crossed with the low-density lipoprotein receptor knockout mice. These studies have demonstrated that MCP-1 and CCR2 play a crucial role in the initiation of atherosclerosis by recruiting monocytes to the vessel wall. According to the multistep theory, monocytes roll on the endothelial cells, interact with E-selectin, adhere to the endothelial cells by firm adhesion to ICAM-1 and VCAM-1, and then migrate into the subendothelium (5Butcher E.C. Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2503) Google Scholar). Rolling of monocytes on endothelial cells is dependent on the binding of E-selectin and sialyl Lewis X, and adhesion to the endothelium is dependent on the interaction of integrins on monocytes and adhesion molecules on the endothelial cells, such as VCAM-1 and ICAM-1. Integrins consist of several subtypes, and each subtype is specific for its ligand. For example, α4β1 integrin, very late antigen-4, binds to VCAM-1, and β2 integrins bind to ICAM-1. Fibronectin, one of the extracellular matrix proteins, is also known to bind to β1 integrins, mainly to α5β1 integrin. In these serial events of the multistep theory, MCP-1 can play a key role in monocyte recruitment by both integrin activation and by promoting migration to the vessel wall. However, the signal transduction pathways leading to integrin activation and chemotaxis have not been fully elucidated. We recently demonstrated that the βγ subunit of heterotrimeric G protein, Gi, plays a key role in MCP-1-induced chemotaxis (6Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar). In that study, we reported that activation of ERK was not involved in chemotaxis by MCP-1. MAPK family members, ERK, JNK, and p38-MAPK, have been implicated in events necessary for proliferation, differentiation, apoptosis, and certain kinds of stress responses (7Lopez-Ilasaca M. Biochem. Pharmacol. 1998; 56: 269-277Crossref PubMed Scopus (237) Google Scholar). These MAPKs are activated by specific cascades responsible for certain stimuli and eventually induce a variety of cell responses. Recently, several groups have reported on the involvement of MAPK and Rho in chemotaxis (8Ayala J.M. Goyal S. Liverton N.J. Claremon D.A. O'Keefe S.J. Hanlon W.A. J. Leukocyte Biol. 2000; 67: 869-875Crossref PubMed Scopus (51) Google Scholar,9Jing Q. Xin S.M. Zhang W.B. Wang P. Qin Y.W. Pei G. Circ. Res. 2000; 87: 52-59Crossref PubMed Scopus (73) Google Scholar). Most of the studies on signal transduction of chemotaxis and cell adhesion have been conducted in adherent cells. However, in adherent cells it would be difficult to separate chemotaxis and integrin activation, because these two functions are closely connected. Therefore, the aim of our study was to examine whether integrin activation and chemotaxis can be separated by studying the signaling cascades leading to integrin activation and chemotaxis mediated by MCP-1 in human monocytic THP-1 cells and to elucidate the role of MAPK and Rho in these biological functions. DISCUSSIONIn this study we have elucidated the role of MAPK and Rho GTPase in MCP-1-mediated cell adhesion and chemotaxis. We show that MCP-1 induced activation of the integrins α4β1 and α5β1 in THP-1 cells and that the integrin activation is dependent on ERK activation. In contrast, MCP-1-dependent chemotaxis was dependent on activation of Rho and p38-MAPK. Thus, as depicted in Fig.10 two important biological functions mediated by MCP-1 utilize two distinct MAPK-dependent signaling pathways.In this study we used sensitive cell adhesion assays to demonstrate important functions of MCP-1. Although Weber et al. (16Weber C. Alon R. Moser B. Springer T.A. J. Cell Biol. 1996; 134: 1063-1073Crossref PubMed Scopus (203) Google Scholar) demonstrated that binding of monocytes to VCAM-1 was reduced at 15 min under stimulation with MCP-1 in a similar assay, we found greater than a 2-fold increase in cell adhesion to this molecule at 10 to 20 min. In preliminary experiments, we have found a basal increase in cell adhesion after labeling the cells with fluorescent dye and washing the cells. We speculate that this is because of some stress on the cells. Further, a basal increase in cell adhesion after spinning down and washing the cells might be because of increased MAPK activation during these procedures. In support of this hypothesis, MacKenna et al. (17MacKenna D.A. Dolfi F. Vuori K. Ruoslahti E. J. Clin. Invest. 1998; 101: 301-310Crossref PubMed Scopus (273) Google Scholar) reported that in cardiac fibroblasts ERK and JNK are activated by mechanical stretch. Therefore, it would be important to avoid stress on the cells as much as possible in this experiment. Work is now in progress to determine the effect of mechanical stress on MAPK activation and cell adhesion.We found that in the monocytic cell line β1 integrins but not β2 integrins are activated by MCP-1. However, Weber et al. (18Weber K.S. Klickstein L.B. Weber C. Mol. Biol. Cell. 1999; 10: 861-873Crossref PubMed Scopus (80) Google Scholar) have reported that MCP-1 induces a prolonged increase in the binding of monocytes to ICAM-1 in a static adhesion assay. This difference may be because of a difference in the way to remove nonadherent cells. We removed nonadherent cells by centrifugation, but they did it by plate washer. So we speculate that binding of β2 integrin and ICAM-1 is not strong enough to overcome the centrifugation force. Further, Chanet al. (19Chan J.R. Hyduk S.J. Cybulsky M.I. J. Immunol. 2000; 164: 746-753Crossref PubMed Scopus (101) Google Scholar) have reported that activation of β1 integrin by chemokines might be much stronger than that of β2 integrin and that β1 integrin/VCAM-1 interaction activates β2 integrin-mediated cell adhesion in human T cells. Therefore, in in vivosituations, activation of β1 integrins might be stronger and more important in the early phase of cell migration.In the cell adhesion assay, we could not abrogate MCP-1-dependent adhesion to fibronectin by the RGDS peptide, even though we have used sufficient concentrations of the peptide. We speculate that the inhibitory effect of this peptide on the interaction between fibronectin and integrins is not so strong (20Ruppert M. Aigner S. Hubbe M. Yagita H. Altevogt P. J. Cell Biol. 1995; 131: 1881-1891Crossref PubMed Scopus (130) Google Scholar). Because RGDS-independent adhesion of fibronectin has been reported (21Silletti S. Mei F. Sheppard D. Montogomery A.M.P. J. Cell Biol. 2000; 149: 1485-1501Crossref PubMed Scopus (131) Google Scholar), it is also possible that RGDS-independent cell adhesion is induced by MCP-1.In this study we showed that in THP-1 cells ERK is responsible for integrin activation by MCP-1 but not p38-MAPK or Rho. Laudanna et al. (22Laudanna C. Campbell J.J. Butcher E.C. Science. 1996; 271 (.-): 981Crossref PubMed Scopus (431) Google Scholar), however, reported that Rho is also involved in integrin activation by interleukin-8 in neutrophils and lymphocytes. The reasons for these differences are not clear, but signaling through integrin activation in response to chemokines might be cell type-specific. It is also possible that different types of integrins in leukocytes might be activated in response to each chemokine.Recently, several reports have shown that p38-MAPK is involved in chemotaxis induced by serum, lysophosphatidylcholine, and chemokines in leukocytes and smooth muscle cells (8Ayala J.M. Goyal S. Liverton N.J. Claremon D.A. O'Keefe S.J. Hanlon W.A. J. Leukocyte Biol. 2000; 67: 869-875Crossref PubMed Scopus (51) Google Scholar, 9Jing Q. Xin S.M. Zhang W.B. Wang P. Qin Y.W. Pei G. Circ. Res. 2000; 87: 52-59Crossref PubMed Scopus (73) Google Scholar). Our study has also shown that p38-MAPK is involved in chemotaxis induced by MCP-1 in THP-1 cells. In contrast, Yen et al. (23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) showed that ERK is responsible for MCP-1-mediated chemotaxis. Knall et al.(24Knall, C., Worthen, G. S., and Johnson, G. L. (1997)Proc. Natl. Acad. Sci. U. S. A.Google Scholar), on the other hand, have shown that ERK or p38-MAPK is not involved in interleukin-8-mediated chemotaxis. The reason for these differences is not clear, but in the system of Yen et al.(23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) the activation of integrin might have been required for monocyte chemotaxis. Rho family GTPases have also been shown to be involved in cell migration (25Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. Cell Biol. 1998; 141: 1147-1157Crossref PubMed Scopus (445) Google Scholar). Thus our data are consistent with the others that p38-MAPK and Rho are involved in chemotaxis. However, the relationship between Rho and MAPK is quite complicated. For example, Zhang et al. (26Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar) have shown that p38-MAPK is downstream of Rho in interleukin-1-mediated signaling. In contrast, Hippenstiel et al. (27Hippenstiel S. Soeth S. Kellas B. Fuhrmann O. Seybold J. Krull M. Eichel-Streiber C. Goebeler M. Ludwig S. Suttorp N. Blood. 2000; 95: 3044-3051Crossref PubMed Google Scholar) have reported that LPS-induced activation of p38-MAPK is not affected by Clostridium difficile toxin B-10463, a specific inhibitor of Rho. In terms of ERK and Rho, some reports have claimed that Rho is upstream of ERK (28Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (183) Google Scholar, 29Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (215) Google Scholar, 30Lassus P. Roux P. Zugasti O. Philips A. Fort P. Hibner U. Oncogene. 2000; 19: 2377-2385Crossref PubMed Scopus (37) Google Scholar), whereas others have demonstrated that Rho and ERK are activated independently (31Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, few studies have been conducted to examine whether ERK and p38-MAPK are differentially affected by Rho. In this paper, we clearly show that MCP-1 phosphorylates ERK and p38-MAPK in THP-1 cells and that Rho is upstream of p38-MAPK but not of ERK in MCP-1-mediated signal transduction. We have also shown that the Rho kinase (32Matsui 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 (934) Google Scholar) is between Rho and p38-MAPK and is a key molecule for chemotaxis. However, downstream targets of p38-MAPK leading to chemotaxis still remain to be determined. Although we found that MCP-1 also activated JNK (data not shown), the role of JNK activation in MCP-1-mediated signaling was not determined in this study.In summary, we have provided clear evidence that two distinct signaling cascades are present to mediate MCP-1-induced activation of β1 integrins and chemotaxis in THP-1 cells. These two distinct signaling cascades would be important for transmigration of monocytes through endothelial cells. The most intriguing aspect of this study is that we could separate two important biological functions of leukocytes, integrin activation and chemotaxis, by different assays and found that two distinct signaling cascades mediate these two functions. In adherent cells, however, segregation of integrin activation and chemotaxis would be very difficult to assess. As depicted in Fig. 10, identification of signaling molecules located at the bifurcation to ERK and p38-MAPK would be important to delineate the signaling cascades through CCR2. Further, it would be intriguing to determine the signaling cascades in a condition closer to in vivosituations. In this study we have elucidated the role of MAPK and Rho GTPase in MCP-1-mediated cell adhesion and chemotaxis. We show that MCP-1 induced activation of the integrins α4β1 and α5β1 in THP-1 cells and that the integrin activation is dependent on ERK activation. In contrast, MCP-1-dependent chemotaxis was dependent on activation of Rho and p38-MAPK. Thus, as depicted in Fig.10 two important biological functions mediated by MCP-1 utilize two distinct MAPK-dependent signaling pathways. In this study we used sensitive cell adhesion assays to demonstrate important functions of MCP-1. Although Weber et al. (16Weber C. Alon R. Moser B. Springer T.A. J. Cell Biol. 1996; 134: 1063-1073Crossref PubMed Scopus (203) Google Scholar) demonstrated that binding of monocytes to VCAM-1 was reduced at 15 min under stimulation with MCP-1 in a similar assay, we found greater than a 2-fold increase in cell adhesion to this molecule at 10 to 20 min. In preliminary experiments, we have found a basal increase in cell adhesion after labeling the cells with fluorescent dye and washing the cells. We speculate that this is because of some stress on the cells. Further, a basal increase in cell adhesion after spinning down and washing the cells might be because of increased MAPK activation during these procedures. In support of this hypothesis, MacKenna et al. (17MacKenna D.A. Dolfi F. Vuori K. Ruoslahti E. J. Clin. Invest. 1998; 101: 301-310Crossref PubMed Scopus (273) Google Scholar) reported that in cardiac fibroblasts ERK and JNK are activated by mechanical stretch. Therefore, it would be important to avoid stress on the cells as much as possible in this experiment. Work is now in progress to determine the effect of mechanical stress on MAPK activation and cell adhesion. We found that in the monocytic cell line β1 integrins but not β2 integrins are activated by MCP-1. However, Weber et al. (18Weber K.S. Klickstein L.B. Weber C. Mol. Biol. Cell. 1999; 10: 861-873Crossref PubMed Scopus (80) Google Scholar) have reported that MCP-1 induces a prolonged increase in the binding of monocytes to ICAM-1 in a static adhesion assay. This difference may be because of a difference in the way to remove nonadherent cells. We removed nonadherent cells by centrifugation, but they did it by plate washer. So we speculate that binding of β2 integrin and ICAM-1 is not strong enough to overcome the centrifugation force. Further, Chanet al. (19Chan J.R. Hyduk S.J. Cybulsky M.I. J. Immunol. 2000; 164: 746-753Crossref PubMed Scopus (101) Google Scholar) have reported that activation of β1 integrin by chemokines might be much stronger than that of β2 integrin and that β1 integrin/VCAM-1 interaction activates β2 integrin-mediated cell adhesion in human T cells. Therefore, in in vivosituations, activation of β1 integrins might be stronger and more important in the early phase of cell migration. In the cell adhesion assay, we could not abrogate MCP-1-dependent adhesion to fibronectin by the RGDS peptide, even though we have used sufficient concentrations of the peptide. We speculate that the inhibitory effect of this peptide on the interaction between fibronectin and integrins is not so strong (20Ruppert M. Aigner S. Hubbe M. Yagita H. Altevogt P. J. Cell Biol. 1995; 131: 1881-1891Crossref PubMed Scopus (130) Google Scholar). Because RGDS-independent adhesion of fibronectin has been reported (21Silletti S. Mei F. Sheppard D. Montogomery A.M.P. J. Cell Biol. 2000; 149: 1485-1501Crossref PubMed Scopus (131) Google Scholar), it is also possible that RGDS-independent cell adhesion is induced by MCP-1. In this study we showed that in THP-1 cells ERK is responsible for integrin activation by MCP-1 but not p38-MAPK or Rho. Laudanna et al. (22Laudanna C. Campbell J.J. Butcher E.C. Science. 1996; 271 (.-): 981Crossref PubMed Scopus (431) Google Scholar), however, reported that Rho is also involved in integrin activation by interleukin-8 in neutrophils and lymphocytes. The reasons for these differences are not clear, but signaling through integrin activation in response to chemokines might be cell type-specific. It is also possible that different types of integrins in leukocytes might be activated in response to each chemokine. Recently, several reports have shown that p38-MAPK is involved in chemotaxis induced by serum, lysophosphatidylcholine, and chemokines in leukocytes and smooth muscle cells (8Ayala J.M. Goyal S. Liverton N.J. Claremon D.A. O'Keefe S.J. Hanlon W.A. J. Leukocyte Biol. 2000; 67: 869-875Crossref PubMed Scopus (51) Google Scholar, 9Jing Q. Xin S.M. Zhang W.B. Wang P. Qin Y.W. Pei G. Circ. Res. 2000; 87: 52-59Crossref PubMed Scopus (73) Google Scholar). Our study has also shown that p38-MAPK is involved in chemotaxis induced by MCP-1 in THP-1 cells. In contrast, Yen et al. (23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) showed that ERK is responsible for MCP-1-mediated chemotaxis. Knall et al.(24Knall, C., Worthen, G. S., and Johnson, G. L. (1997)Proc. Natl. Acad. Sci. U. S. A.Google Scholar), on the other hand, have shown that ERK or p38-MAPK is not involved in interleukin-8-mediated chemotaxis. The reason for these differences is not clear, but in the system of Yen et al.(23Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukoc. Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) the activation of integrin might have been required for monocyte chemotaxis. Rho family GTPases have also been shown to be involved in cell migration (25Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. Cell Biol. 1998; 141: 1147-1157Crossref PubMed Scopus (445) Google Scholar). Thus our data are consistent with the others that p38-MAPK and Rho are involved in chemotaxis. However, the relationship between Rho and MAPK is quite complicated. For example, Zhang et al. (26Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar) have shown that p38-MAPK is downstream of Rho in interleukin-1-mediated signaling. In contrast, Hippenstiel et al. (27Hippenstiel S. Soeth S. Kellas B. Fuhrmann O. Seybold J. Krull M. Eichel-Streiber C. Goebeler M. Ludwig S. Suttorp N. Blood. 2000; 95: 3044-3051Crossref PubMed Google Scholar) have reported that LPS-induced activation of p38-MAPK is not affected by Clostridium difficile toxin B-10463, a specific inhibitor of Rho. In terms of ERK and Rho, some reports have claimed that Rho is upstream of ERK (28Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (183) Google Scholar, 29Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (215) Google Scholar, 30Lassus P. Roux P. Zugasti O. Philips A. Fort P. Hibner U. Oncogene. 2000; 19: 2377-2385Crossref PubMed Scopus (37) Google Scholar), whereas others have demonstrated that Rho and ERK are activated independently (31Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, few studies have been conducted to examine whether ERK and p38-MAPK are differentially affected by Rho. In this paper, we clearly show that MCP-1 phosphorylates ERK and p38-MAPK in THP-1 cells and that Rho is upstream of p38-MAPK but not of ERK in MCP-1-mediated signal transduction. We have also shown that the Rho kinase (32Matsui 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 (934) Google Scholar) is between Rho and p38-MAPK and is a key molecule for chemotaxis. However, downstream targets of p38-MAPK leading to chemotaxis still remain to be determined. Although we found that MCP-1 also activated JNK (data not shown), the role of JNK activation in MCP-1-mediated signaling was not determined in this study. In summary, we have provided clear evidence that two distinct signaling cascades are present to mediate MCP-1-induced activation of β1 integrins and chemotaxis in THP-1 cells. These two distinct signaling cascades would be important for transmigration of monocytes through endothelial cells. The most intriguing aspect of this study is that we could separate two important biological functions of leukocytes, integrin activation and chemotaxis, by different assays and found that two distinct signaling cascades mediate these two functions. In adherent cells, however, segregation of integrin activation and chemotaxis would be very difficult to assess. As depicted in Fig. 10, identification of signaling molecules located at the bifurcation to ERK and p38-MAPK would be important to delineate the signaling cascades through CCR2. Further, it would be intriguing to determine the signaling cascades in a condition closer to in vivosituations. We thank Dr. Israel F. Charo for critical reading of the manuscript.