of May 28, 2018.This information is current as BκProtein Kinase and NF-

Mitogen-ActivatedMacrophages: Role of p38 Chemokine Production in HumanIsoform b with Collagen Up-Regulates Activation of Discoidin Domain Receptor 1

Faure and Teizo YoshimuraWataru Matsuyama, Lihua Wang, William L. Farrar, Michel

http://www.jimmunol.org/content/172/4/2332doi: 10.4049/jimmunol.172.4.2332

2004; 172:2332-2340; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/4/2332.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Activation of Discoidin Domain Receptor 1 Isoform b withCollagen Up-Regulates Chemokine Production in HumanMacrophages: Role of p38 Mitogen-Activated Protein Kinaseand NF-�B

Wataru Matsuyama,1* Lihua Wang,* William L. Farrar,* Michel Faure, †

and Teizo Yoshimura2*

Macrophages produce an array of proinflammatory mediators at sites of inflammation and contribute to the development ofinflammatory responses. Important roles for cytokines, such as IL-1 or TNF-�, and bacterial products, such as LPS, in this processhave been well documented; however, the role for the extracellular matrix proteins, such as collagen, remains unclear. Wepreviously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, is expressed during differentiationof human monocytes into macrophages, and the interaction of the DDR1b isoform with collagen facilitates their differentiation viathe p38 mitogen-activated protein kinase (MAPK) pathway. In this study, we report that the interaction of DDR1b with collagenup-regulates the production of IL-8, macrophage inflammatory protein-1�, and monocyte chemoattractant protein-1 in humanmacrophages in a p38 MAPK- and NF-�B-dependent manner. p38 MAPK was critical for DDR1b-mediated, increased NF-�Btrans-activity, but not for I �B degradation or NF-�B nuclear translocation, suggesting a role for p38 MAPK in the modificationof NF-�B. DDR1b-mediated I�B degradation was mediated through the recruitment of the adaptor protein Shc to the LXNPXYmotif of the receptor and the downstream TNFR-associated factor 6/NF-�B activator 1 signaling cascade. Taken together, ourstudy has identified NF-�B as a novel target of DDR1b signaling and provided a novel mechanism by which tissue-infiltratingmacrophages produce large amounts of chemokines during the development of inflammatory diseases. Intervention of DDR1bsignaling may be useful to control inflammatory diseases in which these proteins play an important role.The Journal of Immu-nology, 2004, 172: 2332–2340.

M acrophages play an important role in the developmentof inflammatory responses by secreting an array ofcytokines and chemokines in a tissue microenviron-

ment. Proinflammatory cytokines, such as IL-1 and TNF-�, arepotent activators of macrophages and up-regulate the expressionand production of cytokines/chemokines. At sites of inflammation,they also interact with the components of the extracellular matrix(ECM)3 through receptors, such as integrins, and are activated forincreased release or production of cytokines/chemokines. For in-stance, activation of monocytes with collagen, the most abundantprotein in the ECM, induced IL-1 release (1). Adherence of PBMCto type I collagen also enhanced the production of IL-8 in responseto LPS, IL-1�, or TNF-� (2). Collagen-induced IL-1 release was

only partially inhibited by an Ab against �2�1 integrin, a classiccell-surface collagen receptor (1), suggesting the presence of an asyet unidentified receptor(s) involved in collagen-induced cytokine/chemokine production by macrophages.

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagenreceptor (receptor tyrosine kinase) with a unique extracellular do-main homologous to discoidin 1 of Dictyostelium discoideum (3).DDR1 is constitutively expressed in epithelial cells of normal tis-sues, such as lung, kidney, colon, and brain, and also in tumor cellsof epithelial origins, such as mammary, ovarian, and lung carci-nomas (3). Five DDR1 isoforms (a, b, c, d, and e) can be generatedby alternative splicing of the DDR1 gene (3, 4). Disruption of theDDR1 gene in mice resulted in viable animals that were signifi-cantly smaller than their littermates, and female DDR1-null miceshowed defects in blastocyst implantation and mammary glanddevelopment (5). Primary vascular smooth muscle cells isolatedfrom the DDR1-null mice showed decreased proliferation, colla-gen attachment, and migration in vitro (6, 7). In contrast, primarymesangial cells isolated from the kidney of the DDR1-null miceshowed enhanced proliferation (8). These previous observationshave indicated that DDR1 plays a role in cell attachment, migra-tion and proliferation; however, the underlying molecular mecha-nisms of DDR1 activation remain unclear.

We previously reported that the expression of two DDR1 iso-forms, DDR1a and DDR1b, could be induced in human leuko-cytes, including neutrophils, monocytes, and lymphocytes in vitro.In vivo, tissue-infiltrating mononuclear cells, especially macro-phages, were positive for DDR1 mRNA (9). The DDR1a andDDR1b isoforms differ from each other by an in-frame insertion of

* Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick,MD 21702; and † SUGEN, South San Francisco, CA 94080.

Received for publication September 5, 2003. Accepted for publication December 1,2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Current address: Third Department of Internal Medicine, Faculty of Medicine, Ka-goshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.2 Address correspondence and reprint requests to Dr. Teizo Yoshimura, Laboratory ofMolecular Immunoregulation, National Cancer Institute, Building 559, Room 9, Fred-erick, MD 21702. E-mail address: yoshimur@mail.ncifcrf.gov3 Abbreviations used in this paper: ECM, extracellular matrix; DDR1, discoidin do-main receptor 1; MAPK, mitogen-activated protein kinase; TRAF6, TNFR-associatedfactor 6; TAK1, TGF-�-activated protein kinase 1; TAB1, TAK1 binding protein 1;MIP-1�, macrophage inflammatory protein-1�; MCP-1, monocyte chemoattractantprotein-1; CAPE, caffeic acid phenethyl ester; TLR, Toll-like receptor; DN, dominantnegative; IKK, I�B kinase; siRNA, small interfering RNA.

The Journal of Immunology

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111-bp coding for an additional 37-aa peptide in the proline-richjuxtamembrane region. The 37-aa insertion in DDR1b contains theLXNPXY motif corresponding to the consensus-binding motif ofthe Shc phosphotyrosine binding domain (3). To study the biolog-ical role for DDR1 expressed in leukocytes, we produced DDR1-overexpressing cell lines by transducing the human monocyticTHP-1 cells with retroviruses expressing either DDR1a or DDR1b.Interestingly, overexpression of DDR1a, but not DDR1b, pro-moted the migration of THP-1 cells through three-dimensional col-lagen lattices (9).

THP-1 cells are known to differentiate along the monocyticlineage following exposure to PMA (10), and to acquire charac-teristics of macrophages, including loss of proliferation andexpression of the MHC class II molecule, HLA-DR (11, 12). Thus,PMA-treated THP-1 cells provided a useful model to study themechanisms of macrophage differentiation. Using this model, wepreviously demonstrated that collagen activation of DDR1b, butnot DDR1a, facilitates PMA-induced differentiation of THP-1cells via activation of the p38 mitogen-activated protein kinase(MAPK) pathway. Activation of neither DDR1a nor DDR1b withcollagen affected THP-1 cell differentiation (13). We further iden-tified that activation of DDR1b induces the formation of a proteincomplex containing TNFR-associated factor 6 (TRAF6), TGF-�-activated protein kinase 1 (TAK1)-binding protein 1� (TAB1�),and p38� MAPK, and subsequent autophosphorylation of p38�MAPK in PMA-treated THP-1 cells (14), indicating that theDDR1b signaling targets the p38 MAPK pathway through an al-ternative mechanism for p38 MAPK activation in these cells (15,16). We confirmed our data with PMA-treated THP-1 cells usingGM-CSF-induced, human monocyte-derived primary macro-phages (GM macrophages) (13, 14). It is well known that TRAF6mediates the signaling of other receptors, including IL-1R, Toll-like receptors (TLRs) TRANCE-R, and CD40 (17, 18). Thus,DDR1b is a new member of the receptor family that uses TRAF6as a critical signaling molecule to transduce signals to its down-stream pathways.

The p38 MAPK pathway plays an important role in the expres-sion of several cytokine and chemokine genes, including IL-1�,IL-8, and MCP-1 (19, 20). Our previous finding that the interactionof DDR1b with collagen activates p38 MAPK in PMA-induceddifferentiated THP-1 cells, a model for macrophages, and in mono-cyte-derived human primary macrophages (13), led us to the hy-pothesis that activation of DDR1b also up-regulates the expressionand release of cytokines and chemokines in macrophages. In thepresent study, we tested our hypothesis using PMA-induced, dif-ferentiated DDR1b-overexpressing THP-1 cells and GM macro-phages. Collagen-activation of DDR1b markedly up-regulated theexpression and release of IL-1�, IL-8, macrophage inflammatoryprotein-1� (MIP-1�), and monocyte chemoattractant protein-1(MCP-1) in a p38 MAPK-dependent manner. Up-regulation ofIL-8, MIP-1�, and MCP-1 release was also dependent on NF-�B,indicating that NF-�B is a novel target of DDR1b signaling. In-terestingly, I�B degradation induced by DDR1b activation wasregulated through a unique signaling cascade involving the adaptorprotein Shc, TRAF6, and NF-�B activator 1 (Act1). Thus, DDR1b-collagen interaction up-regulates the production of chemokines bymacrophages and it is likely to contribute to the development of in-flammatory responses in a tissue microenvironment.

Materials and MethodsReagents

A mouse monoclonal anti-�1-integrin blocking Ab (DE9) was obtainedfrom Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal Absagainst human DDR1 (C-20) and Act1 (H-300), and mouse mAbs against

human TRAF6 (D-10 and C-20) and I�B� were from Santa Cruz Biotech-nology (Santa Cruz, CA). The production of the agonistic anti-DDR1mouse mAb 513 (IgM) was previously described (13). Rabbit polyclonalAbs against p38 MAPK, phosphorylated p38 MAPK, or p38� MAPK werefrom Cell Signaling Technology (Beverly, MA). Sheep anti-mouse or anti-rabbit IgGs coupled with HRP were from Amersham Pharmacia Biotech(Piscataway, NJ). An anti-human IL-1� neutralizing Ab was from R&DSystems (Minneapolis, MN). GM-CSF was from PeproTech (Rocky Hill,NJ). PBS, RPMI 1640, G418, recombinant protein G-agarose and TRIzolreagent were from Invitrogen (Gaithersburg, MD). Protease inhibitor mix-ture tablets, Complete mini, were from Roche (Indianapolis, IN). FCS wasfrom HyClone Laboratories (Logan, UT). Paraformaldehyde, formamide,PMA, anti-Flag Ab, and bovine collagen type I solution were from Sigma-Aldrich (St. Louis, MO). SB203580, PD98059, and caffeic acid phenethylester (CAPE) were from Biochem-Novabiochem (San Diego, CA). TheNF-�B-Luc plasmid containing five copies of the NF-�B site of the humanIg �-chain gene and the luciferase gene was from Stratagene (La Jolla,CA). Accu-prep was from Accurate Chemical & Scientific (Westbury,NY). [�-32P]dCTP and [�-32P]ATP were from Amersham Biosciences(Piscataway, NJ). Human �-actin cDNA was from Clontech Laboratories(Palo Alto, CA).

Mock-transduced, DDR1a-, or DDR1b-overexpressingTHP-1 cells

The production of DDR1a- or DDR1b-overexpressing THP-1 cells waspreviously described (9). Briefly, the human monocytic leukemic cell line,THP-1, was transduced with retrovirus expressing DDR1a or DDR1b, orcontrol retrovirus containing the vector only (mock-transduced). THP-1cells transduced by each virus were selected with G418, and cloned bylimiting dilution. The cloned THP-1 cells were maintained in RPMI 1640medium supplemented with 100 �g/ml gentamycin and 10% FCS (com-plete medium).

Preparation of monocyte-derived macrophages

Human PBMC were obtained from leukapheresis preparations obtained bythe Blood Bank, Clinical Center, National Institutes of Health (Bethesda,MD). The leukocyte-rich preparation was overlaid on Accu-prep in 50-mltubes and the tubes were centrifuged at 800 � g for 20 min at roomtemperature. PBMC fractions were collected, washed once with PBS atroom temperature and twice with complete medium at 4°C, and resus-pended in the complete medium. Monocytes were further purified by usingiso-osmotic Percoll gradient. At this stage, the purity of monocytes washigher than 90% (21). The cells (5 � 105/ml) were allowed to adhere to thesurface of six-well plates. Nonadherent cells were removed after a 5-hincubation at 37°C, and remaining adherent cells were cultured in the pres-ence of GM-CSF (50 ng/ml) for an additional 5 days to produce monocyte-derived macrophages (GM macrophages).

Northern blotting

THP-1 cells were cultured for various times at the cell density of 5 � 106

cells/ml in complete medium in the presence of 10 nM PMA in six-welltissue culture plates that were either collagen-coated or uncoated. TotalRNA was extracted from each culture by using TRIzol reagent, and North-ern blotting was performed as previously described (22). The cloning ofhuman MCP-1 cDNA (23) and the sources of IL-8 and MIP-1� cDNAs(24) were previously described. IL-1� cDNA was obtained by RT-PCRfrom a human monocyte cDNA library using a primer pair, 5�-CGTATGGCAGGACAAATGCTTCTTC-3� and 5�-TTCCCTCCAGGCTGCCATGAG-3�. Each cDNA was labeled with [�-32P]dCTP using the RediprimeII random prime labeling system (Amersham Biosciences).

ELISA

The concentrations of IL-1�, IL-8, MIP-1�, and MCP-1 were measured inthe Lymphokine Testing Laboratory, Clinical Services Program, ScienceApplications International Corporation-Frederick, National Cancer Insti-tute-Frederick (Frederick, MD), by using ELISA kits (R&D Systems) spe-cific for each human cytokine and chemokine. The sensitivity of the assaywas 10.0 (IL-8), 10.0 (MIP-1�), 1.0 (IL-1�), and 5.0 pg/ml (MCP-1),respectively.

Western blotting

Cell lysates were prepared from 1 � 107 cells using a lysis buffer contain-ing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, anda mixture of protease inhibitors. Each sample was mixed with double-strength sample buffer and boiled for 10 min. Electrophoresis was per-formed on 10% polyacrylamide gels by SDS-PAGE, and proteins were

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transferred electrophoretically to nitrocellulose membranes at 150 mA for1 h in a semidry system. Membranes were incubated with an Ab specificto each protein, followed by sheep anti-rabbit or anti-mouse IgG coupledwith HRP. Peroxidase activity was visualized by the Enhanced Chemilu-minescence Detection System (Amersham Biosciences). To study the deg-radation of I�B�, mock-transduced or DDR1b-overexpressing THP-1 cellswere incubated with PMA for 12 h, serum-starved for 12 h, and thenactivated with 50 �g/ml collagen for various times. To evaluate the asso-ciation of Act1 with TRAF6, cell lysates were subjected to immunopre-cipitation with anti-TRAF6 or anti-Act1Ab, and coimmunoprecipitation ofAct1 or TRAF6 was examined with anti-Act1 or anti-TRAF6 Ab.

Expression of Y513F DDR1b mutant and dominant negative(DN)-TRAF6

Production of the Y513F DDR1b mutant expression vector was previouslydescribed (13). The numbering for the tyrosine residue is based on theamino acid sequence available from GenBank (accession numberNP_054699). Parental THP-1 cells were transfected with either wild-typeDDR1b or Y513F mutant using Effectene Transfection Reagent (Qiagen,Valencia, CA) for 12 h, rinsed with PBS, and incubated for an additional12 h in complete medium. The transfected cells were treated with 10 nMPMA for 12 h, starved in RPMI 1640 for 10 h, rinsed three times with PBS,and subsequently activated with 50 �g/ml collagen for 3 h. One hundredmillion cells were lysed and the degradation of I�B� was examined byWestern blotting.

Construction of a mammalian expression vector for the Flag-DN-TRAF6 (289–522) fusion protein (25) was previously described (14).DDR1b-overexpressing THP-1 cells were treated with 10 nM PMA for12 h and then transfected with the vector with or without insert, usingEffectene Transfection Reagent (Qiagen) for 24 h, rinsed with PBS, andincubated for an additional 12 h in RPMI 1640 containing 1% FCS, andsubsequently activated with 50 �g/ml collagen for 60 min or 3 h. Onehundred million cells were lysed and the cell lysates were subjected toWestern blotting.

EMSA

Mock-transduced or DDR1b-overexpressing THP-1 cells were incubatedwith 10 nM PMA on collagen-coated plates for 6 h, nuclear extracts wereprepared as previously described (26), and aliquots were frozen at �80°C.For EMSA, end-labeled 32P-oligonucleotide probes corresponding to theNF-�B binding site of the Ig �-chain gene (5�-AGTTGAGGGGACTTTCCCAGGC-3�) was incubated with 5 �g of nuclear extracts in a 20-�lbinding mixture (50 mM Tris-HCl, pH 7.4, 25 mM MgCl2, 0.5 mM DTT,50% glycerol) at 4°C for 15 min. The DNA-protein complexes were re-solved on a 5% polyacrylamide gel. Gels were dried and then exposed tox-ray films.

Luciferase assay

Mock-transduced or DDR1b-overexpressing THP-1 cells were transfectedwith 10 �g of the NF-�B-Luc, pGLM-ENH, or pGLM-MA1MA2 (26) and5 �g of the pSV-�-galactosidase (Promega, Madison, WI) per 100-mmtissue culture plate, using Effectene Transfection Reagent (Qiagen). Aftera 12-h incubation, cells were rinsed with PBS, and then incubated with 10nM PMA on collagen-coated plates for an additional 12 h. Cells werecollected, lysed using the Reporter Lysis Buffer (Promega), and luciferaseand �-galactosidase activities were measured according to the protocolprovided by the manufacturer. The resulting luciferase activity was cor-rected, based on the �-galactosidase activity in the same cell extract.

RNA interference

A mixture of four small interfering RNA (SMARTPool siRNA) specific forAct1 was purchased from Dharmacon (Lafayette, CO). DDR1b-overex-pressing THP-1 cells were cultured at a density of 5 � 106 cells/ml incomplete medium in six-well plates in the presence of 10 nM PMA for12 h. Cells were washed three times with PBS and transfected with thesiRNA at the final concentration of 100 nM by using OligofectamineTMReagent (Invitrogen) according to the manufacturer’s protocol. After a48-h incubation, cells were rinsed with PBS, starved in RPMI 1640 for10 h, plated onto collagen-coated plates at 5 � 106 cells/ml, and incubatedfor 3 h. After three washes with PBS, 1 � 108 cells were lysed and usedfor Western blotting.

ResultsCollagen-activation of DDR1b up-regulates the expression andproduction of IL-1�, IL-8, MIP-1�, and MCP-1 in PMA-induceddifferentiated THP-1 cells

To examine whether collagen-activation of DDR1b affects theexpression of proinflammatory cytokines and chemokines bymacrophages, we first performed a cDNA array analysis usingPMA-treated, differentiated, mock-transduced, and DDR1b-over-expressing THP-1 cells. The expression of IL-1�, IL-8, MIP-1�,and MCP-1 mRNA was markedly up-regulated in DDR1b-over-expressing, but not mock-transduced, THP-1 cells that were incu-bated in the presence of PMA on collagen-coated plates for 12 h(data not shown).

To confirm our data obtained by the cDNA analysis, we per-formed Northern blotting (Fig. 1). Parental, mock-transduced, andDDR1a-overexpressing THP-1 cells expressed IL-1�, IL-8, MIP-1�, and MCP-1 mRNA after a 16-h incubation in the presence ofPMA on collagen-coated plates. The slow kinetics of MCP-1mRNA expression by these cell lines on collagen-coated plateswere identical to those previously detected by PMA-treated paren-tal THP-1 cells on uncoated plates (26). Therefore, collagen didnot appear to have any effect on the expression of the cytokine/chemokine mRNA by these three cell lines. The mechanisms reg-ulating the delayed up-regulation of the cytokine/chemokinemRNA expression in PMA-treated THP-1 cells remain uncharac-terized. In contrast, the expression of all four mRNA was rapidlyinduced in collagen-activated, PMA-treated DDR1b-overexpress-ing cells on collagen-coated plates, and the expression level ofeach mRNA in these cells was much higher than that in other celllines at 12 h, consistent with the cDNA array result. The expressionlevel of IL-1� rapidly decreased at 20 h, whereas the expression ofIL-8, MIP-1�, and MCP-1 mRNA was sustained up to 24 h.

The concentrations of IL-1�, IL-8, MIP-1�, and MCP-1 in theculture supernatants of each cell line were quantified by ELISA(Fig. 2). The concentration of IL-1� in the supernatants ofDDR1b-overexpressing cells was significantly higher than that inthe culture supernatants of other cell lines at 24 h, but there was nosignificant difference at 48 h. This agrees with the mRNA expres-sion data showing that activation of DDR1b caused only a shift inthe kinetics (Fig. 1). The concentrations of IL-8, MIP-1�, andMCP-1 were significantly higher in the culture supernatants ofdifferentiated DDR1b-overexpressing cells at 24 and 48 h in com-parison with other cell lines. Because IL-1� is known to up-reg-ulate the expression of IL-8, MIP-1�, and MCP-1 (27, 28), weadded anti-IL-1� neutralizing Ab to the cultures and determinedwhether early production of IL-1� was responsible for the in-creased production of these chemokines. The release of these che-mokines was not affected by the neutralizing Ab, indicating thatthe up-regulated production of these chemokines by differentiatedDDR1b-overexpressing cells was independent of IL-1� releasedby the same cells (data not shown).

Activation of DDR1 up-regulates the release of IL-8, MIP-1�,and MCP-1 from human primary macrophages in p38 MAPK-and NF-�B-dependent manners

Using in vitro-generated GM macrophages, we next evaluated theeffect of DDR1 activation on the production of these proinflam-matory mediators by primary macrophages. GM macrophages arereported to show characteristics that resemble those of alveolarmacrophages (29), and alveolar macrophages are a well-knownsource of cytokines and chemokines in vivo (28). GM macro-phages also express a high level of endogenous DDR1, predomi-nantly DDR1b (13). Thus, GM macrophages provided an excellent

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model to study the role for DDR1b-collagen interaction in theproduction of cytokines and chemokines. As shown in Fig. 3, GMmacrophages released only low levels of IL-1�, IL-8, MIP-1�, andMCP-1 in complete medium. Activation of DDR1 with agonisticanti-DDR1 Ab (513) slightly but significantly increased the releaseof these proteins. Activation with LPS increased the release of allfour proteins, and DDR1 activation markedly augmented LPS-in-duced release of these proteins. Control mouse IgM had no effect.Activation with collagen also augmented LPS-induced release ofthese proteins. The effects of collagen on the release of IL-1�,IL-8, and MIP-1� were higher than that of 513 Ab, and addition ofanti-�1 integrin blocking Ab reduced the levels of these three pro-teins to those obtained with 513 Ab, indicating that the effect ofcollagen on the release of IL-1�, IL-8, and MIP-1� was also reg-ulated through activation of �1 integrins. In contrast, collagen-mediated up-regulation of MCP-1 release was totally dependent onDDR1. Pretreatment of cells with the p38 MAPK inhibitorSB203580 or NF-�B inhibitor CAPE, but not DMSO, inhibitedcollagen-induced and 513 Ab-induced up-regulation of IL-8, MIP-1�, and MCP-1 release by GM macrophages, whereas collagen- or513 Ab-mediated up-regulation of IL-1� release was inhibited byonly SB203580. These results indicated that activation of DDR1,most likely DDR1b, up-regulates the production of these chemo-kines not only in differentiated THP-1 cells, a model for macro-phages, but also in primary macrophages in both p38 MAPK- andNF-�B-dependent manners.

Activation of DDR1b up-regulates trans-activity of NF-�B indifferentiated THP-1 cells

To examine whether activation of DDR1b increases NF-�B trans-activity in macrophages, we performed a luciferase assay by trans-fecting differentiated DDR1b-overexpressing THP-1 cells withreporter constructs containing the sequences of NF-�B bindingsites of two different NF-�B-dependent gene promoters, the humanIg �-chain (NF-�B-Luc) or MCP-1 (pGLM-ENH) gene promoter.

As shown in Fig. 4A, there was no increase in luciferase activity inresponse to PMA, collagen, or PMA plus collagen in mock-trans-duced cells. However, in DDR1b-overexpressing cells, luciferaseactivity was markedly increased in response to PMA plus collagen,and it was dose-dependently inhibited by SB203580. CAPE alsocompletely inhibited the increased luciferase activity induced withPMA plus collagen. Almost identical results were obtained withpGLM-ENH, as shown in Fig. 4B. There was no increase in lu-ciferase activity when pGLM-MA1MA2 that contained mutationsin the NF-�B sequences was used. These results indicated thatcollagen activation of DDR1b increases NF-�B activity in differ-entiated DDR1b-overexpressing THP-1 cells in a p38 MAPK-de-pendent manner.

DDR1b-mediated degradation of I�B is independent of p38 MAPK

We next analyzed the mechanisms of DDR1b-mediated NF-�Bactivation using differentiated DDR1b-overexpressing THP-1cells. As shown in Fig. 5A, there was no change in the level ofI�B� in differentiated mock-transduced cells in response to colla-gen. However, in differentiated DDR1b-overexpressing cells, thelevel of I�B� rapidly decreased in response to collagen, and thenreturned to the original level. This is typical for the kinetics of I�Bdegradation. Although SB203580 inhibited the trans-activation ofNF-�B-dependent gene promoters as shown in Fig. 4, it had noeffect on DDR1b-mediated I�B degradation (Fig. 5B). Collagenactivation of DDR1b also induced nuclear translocation of NF-�B,and SB203580 had no effect, as determined by EMSA (Fig. 5C).These results indicated that DDR1b-mediated I�B degradation andsubsequent translocation of NF-�B were independent of p38 MAPK.

DDR1b-mediated degradation of I�B is regulated by Shc,TRAF6, and Act1

The juxtamembrane domain of DDR1b contains the LXNPXYmotif that corresponds to the consensus binding motif for the Shcphosphotyrosine binding domain (30, 31). As described above, we

FIGURE 1. Up-regulation of cytokine and chemokine mRNA expression by differentiated DDR1b-overexpressing THP-1 cells in response to collagen.The kinetics of IL-1�, IL-8, MIP-1�, and MCP-1 mRNA expression in each cell line after PMA treatment on collagen-coated plates were examined byNorthern blotting. The blots were hybridized with 32P-labeled IL-1�, IL-8, MIP-1�, MCP-1, or �-actin cDNA probe. Representative results of twoindividual experiments with similar results are shown.

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previously demonstrated that the activation of DDR1b induced therecruitment of Shc to the LXNPXY sequence and it was necessaryfor the activation of p38 MAPK in differentiated THP-1 cells (13).To test the involvement of Shc recruitment in DDR1b-mediatedI�B degradation, we transfected parental THP-1 cells with thewild-type or a mutant DDR1b cDNA in which tyrosine at the res-idue 513 in the LXNPXY motif is substituted to phenylalanine(Y513F) (13), and then examined the degradation of I�B degra-dation in these cells. As shown in Fig. 6A, collagen activation ofthe wild-type DDR1b caused the degradation of I�B (Fig. 6A,lanes 1 and 2). However, in Y513F-transfected cells, collagen ac-tivation did not cause I�B degradation (Fig. 6A, lanes 3 and 4),indicating that DDR1b-mediated I�B degradation is regulatedthrough the recruitment of Shc.

We have recently demonstrated that TRAF6 is a signaling mol-ecule downstream of Shc and regulates DDR1b-mediated p38MAPK activation (14). Therefore, we evaluated the role of TRAF6in DDR1b-mediated I�B degradation by overexpressing DN-TRAF6 in DDR1b-overexpressing THP-1 cells (Fig. 6B). Asshown in Fig. 6C, overexpression of DN-TRAF6 almost com-

pletely abrogated collagen-induced I�B degradation in these cells(Fig. 6C, lanes 3 and 4), indicating that TRAF6 also plays a criticalrole in DDR1b-mediated I�B degradation in differentiated THP-1cells.

Previous studies demonstrated that TAK1 is a downstream sig-naling molecule of TRAF6 and is involved in the activation ofNF-�B during IL-1R or TLR4 signaling (32, 33). TAK1 is acti-vated by its binding to TAB1 (34, 35); however, in our previousstudy the protein complex formed in response to the activation ofDDR1b contained TAB1�, a splicing variant of TAB1 lacking thedomain necessary for TAK1 binding (16), resulting in the absence

FIGURE 2. Collagen-mediated up-regulation of IL-1�, IL-8, MIP-1�,and MCP-1 release by DDR1b-overexpressing differentiated THP-1 cells.The release of IL-1�, IL-8, MIP-1�, and MCP-1 after a 48-h incubation ofeach cell line in the presence of 10 nM PMA on collagen-coated plates wasquantified by ELISA. Statistical analysis was performed by Bonferroni/Dunn with one-way factorial ANOVA. �, p � 0.001, n � 3. ��, p � 0.051,���, p � 0.01, n � 3. FIGURE 3. DDR1-mediated up-regulation of IL-1�, IL-8, MIP-1�, and

MCP-1 release by human primary macrophages. The release of IL-1�,IL-8, MIP-1�, and MCP-1 after a 48-h incubation of GM macrophages inthe presence of 513 Ab (5 �g/ml), normal mouse IgM (5 �g/ml), or LPS(1 �g/ml) was quantified by ELISA. To evaluate the effect of collagen,plates were coated by bovine type I collagen. Anti-�1 integrin blocking Ab(10 �g/ml), SB203580 (10 �M), CAPE (10 �g/ml), or DMSO was addedat the beginning of the culture to evaluate the effect of each inhibitor.Statistical analysis was performed by Bonferroni/Dunn with one-way fac-torial ANOVA. �, p � 0.001, n � 3; ��, p � 0.01, n � 3.

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of TAK1 in this protein complex (14). Therefore, it is highly un-likely that TAK1 is involved in DDR1b-mediated NF-�B activa-tion. Recently, Kanamori et al. (36) detected the capacity of aNF-�B activator, Act1, to bind to TRAF6, and proposed thatTRAF6 functions as an anchor for Act1 in unstimulated cells andthat activation of cells with IL-1 releases Act1, resulting in directbinding of Act1 to I�B kinase (IKK)� and subsequent activation ofIKKs (36). As shown in Fig. 7A, Act1 was equally expressed inboth differentiated mock and DDR1b-overexpressing THP-1 cells.To explore a potential involvement of Act1 in DDR1b-mediatedI�B degradation, we immunoprecipitated TRAF6 from the lysatesof collagen-activated, differentiated mock or DDR1b-overexpress-

ing THP-1 cells, and the association of Act1 with TRAF6 wasevaluated by Western blotting with anti-Act1 Ab. As we expected,Act1 was coimmunoprecipitated with TRAF6 from the lysates ofnonactivated differentiated DDR1b-overexpressing THP-1 cells(Fig. 7B, lanes 1 and 7) and also from collagen-activated differ-entiated mock-transduced cells (Fig. 7B, lanes 2 and 6). However,Act1 was no longer coimmunoprecipitated with TRAF6 from thelysates of differentiated DDR1b-overexpressing cells 30 min aftercollagen activation (Fig. 7B, lanes 9–12). Almost identical resultswere obtained when anti-Act1 Ab was used for immunoprecipita-tion (Fig. 7C). These results indicated that Act1 and TRAF6 as-sociate in nonactivated cells, but the activation of DDR1b inducesthe dissociation of the TRAF6-Act1 complex.

To identify the functional role of Act1, we inhibited the expres-sion of Act1 in differentiated DDR1b-overexpressing THP-1 cellsby RNA interference. Transfection of the cells with siRNA re-duced the level of Act1 by �90%, but it did not change the levelof DDR1 or the control protein actin (Fig. 7D, lane 2). As shownin Fig. 7E, Act1 was no longer coimmunoprecipitated with TRAF6

FIGURE 4. Increased NF-�B trans-activity in collagen-activated PMA-treated DDR1b-overexpressing THP-1 cells. A, NF-�B trans-activity wasevaluated in mock-transduced or DDR1b-overexpressing THP-1 cellstreated as indicated using the luciferase construct containing the NF-�Bbinding site of the human Ig �-chain gene (NF-�B-Luc). Cells were pre-treated for 30 min with SB203580 (10 �M), PD98059 (10 �M), CAPE (10�g/ml), or DMSO to evaluate the effect of each inhibitor. B, NF-�B trans-activity was evaluated in DDR1b-overexpressing THP-1 cells treated asindicated, using the luciferase construct containing the two upstreamNF-�B binding sites and the GC box of the human MCP-1 gene (pGLM-ENH). The pGLM-MA1MA2 containing mutations in the two NF-�B se-quences was also used. Cells were pretreated for 30 min with SB203580(10 �M), PD98059 (10 �M), or DMSO to evaluate the effect of eachinhibitor. Statistical analysis was performed by Bonferroni/Dunn with one-way factorial ANOVA. �, p � 0.001, n � 3; ��, p � 0.01, n � 3.

FIGURE 5. Collagen-induced I�B� degradation and NF-�B nucleartranslocation in PMA-treated DDR1b-overexpressing THP-1 cells. A, Col-lagen-induced degradation of I�B� in differentiated mock or DDR1b-over-expressing THP-1 cells was evaluated by Western blotting. B, DDR1b-overexpressing THP-1 cells were incubated with 10 nM PMA for 12 h,treated with SB203580 (10 �M) for 30 min, and then activated with col-lagen for 3 h. The degradation of I�B was evaluated by Western blotting.C, Nuclear translocation of NF-�B was evaluated by EMSA using a 32P-labeled DNA probe corresponding to the NF-�B site of the human Ig�-chain gene promoter. Cells were pretreated for 30 min with SB203580(10 �M) to evaluate its effect. Representative results of two to three indi-vidual experiments with similar results are shown.

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in the cells (lane 4). Interestingly, inhibition of Act1 expressionmarkedly reduced collagen-induced I�B degradation in differenti-ated DDR1b-overexpressing cells without affecting p38 MAPKactivation (Fig. 7F, lane 4). Thus, Act1 plays a critical role inDDR1b-mediated I�B degradation in differentiated THP-1 cells.

We also examined the association of Act1 with TRAF6 in GMmacrophages. As shown in Fig. 7H, Act1 was coimmunoprecipi-tated with TRAF6 in nonactivated GM macrophages (Fig. 7H, lane1). However, Act1 was no longer coimmunoprecipitated withTRAF6 30 min after activation of the cells with 513 Ab whenTRAF6 formed a protein complex with p38� MAPK (Fig. 7H,lanes 3–6). Identical results were obtained after activation of GMmacrophages with collagen (data not shown). These resultsstrongly suggest that Act1 regulates DDR1b-mediated degradationof I�B in primary macrophages.

DiscussionWe previously reported that the expression of two DDR1 isoforms,DDR1a and DDR1b, could be induced in monocytes (9), and ac-tivation of DDR1b facilitates their differentiation through activa-tion of the p38 MAPK pathway (13). Activation of p38 MAPK isdependent on the recruitment of the adaptor protein Shc to theLXNPXY motif in the juxtamembrane domain of this receptor(13), and it was due to the autophosphorylation of p38� MAPKmediated through the TRAF6/TAB1�/p38� cascade (14). In thepresent study, we examined the effects of DDR1b-collagen inter-action on the production of cytokines and chemokines by macro-phages using PMA-induced differentiated DDR1b-overexpressingTHP-1 cells and monocyte-derived human primary macrophages.Activation of DDR1b up-regulated the production of IL-1�, IL-8,MIP-1�, and MCP-1 in a p38 MAPK-dependent manner. Produc-tion of IL-8, MIP-1�, and MCP-1 was also dependent on NF-�B.DDR1b-mediated activation of NF-�B was regulated through Shc

and a novel signaling cascade involving TRAF6 and Act1. Thus,we have found that DDR1b plays a novel role in the production ofproinflammatory mediators, an important function of macro-phages, and that NF-�B is a novel target of the DDR1b signaling.

NF-�B is a ubiquitous, pleotropic, multisubunit transcriptionfactor activated in response to inflammatory and noninflammatoryexogenous stimuli. In most cells, NF-�B exists as an inactive het-erodimer, the predominant of which is composed of p50 and p65(Rel A) subunits, and is sequestered within the cytoplasm by as-sociation with the inhibitory protein, I�B. Interaction of I�B withthe NF-�B dimer prevents the nuclear translocation of NF-�B.Phosphorylation by IKKs leads to the ubiquitination and degrada-tion of I�B, resulting in the nuclear translocation of NF-�B andsubsequent activation of downstream target genes, including proin-flammatory cytokines and chemokines (37, 38). As describedabove, we have determined Shc and TRAF6 to be crucial upstreammolecules regulating the degradation of I�B during the DDR1bsignaling. Act1, also known as connection to I�B kinase andstress-activated protein kinase/c-Jun N-terminal kinase (CIKS),was originally cloned as a protein capable of binding to IKK�, theregulatory unit of IKK, and forced expression of Act1/CIKS in 293cells or HeLa cells induced constitutive activation of NF-�B in thecells (39, 40). A recent study by Kanamori et al. (36) demonstratedthat Act1 binds to TRAF6 and Act1-mediated NF-�B activationcan be inhibited by a DN-TRAF6, suggesting an involvement ofAct1 in IL-1R/TLR-mediated signaling. Therefore, we examinedthe role of Act1 in DDR1b-mediated I�B degradation. Act1 wasassociated with TRAF6 in nonactivated, differentiated mock-trans-duced or DDR1b-overexpressing THP-1 cells, as well as in non-activated primary macrophages; however, activation of DDR1bcaused dissociation of Act1 from TRAF6. Furthermore, inhibitionof Act1 expression by RNA interference markedly reducedDDR1b-mediated I�B degradation. Thus, we have demonstrated,for the first time, that Act1 is a physiological regulator of I�Bdegradation.

p38 MAPK regulates the transcription of many NF-�B-depen-dent genes (41, 42). In fact, inhibition of p38 MAPK withSB203580 almost completely abolished DDR1b-mediated IL-8,MIP-1�, and MCP-1 release by primary macrophages. SB203580also inhibited the trans-activation of the Ig �-chain or MCP-1 genepromoters in differentiated THP-1 cells. These results indicatedthat DDR1b-mediated release of these chemokines was dependenton p38 MAPK. However, inhibition of p38 MAPK had no effecton I�B degradation or nuclear translocation of NF-�B (Fig. 4, Band C). It has been shown that activation of NF-�B requires twoseparate mechanisms: the degradation of I�B and subsequent nu-clear translocation of NF-�B, and modification of the RelA/p65trans-activation subunit of NF-�B by p38 MAPK (43, 44). Thisprovides a mechanism whereby blocking p38 MAPK activity in-hibited DDR1b-mediated trans-activation of these two gene pro-moters without affecting the degradation of I�B.

Direct activation of DDR1 with agonistic anti-DDR1 Ab aug-mented LPS-induced IL-1�, IL-8, MIP-1�, and MCP-1 releasefrom GM macrophages, although it also induced low-level releaseof these proteins without LPS activation. This suggests that themain role of DDR1 signaling is not to induce strong responses butto amplify the effects by other stimuli, such as proinflammatorycytokines or bacterial products. Because collagen is abundant andpresent in almost all tissues, this may be a very important mech-anism to avoid unnecessary signaling through DDR1. An impor-tant question that remains to be answered is whether collagen indifferent forms equally activate DDR1. Normal tissues are com-posed of a fibrillar mesh of the ECM, including polymerized type

FIGURE 6. Role of Shc and TRAF6 in DDR1b-mediated I�B degra-dation. A, The wild-type or Y513F mutant of DDR1b was transfected intoparental THP-1 cells. Transfected cells were incubated in the presence of10 nM PMA and then activated with collagen for 3 h. Degradation of I�B�was evaluated by Western blotting. B, PMA-differentiated DDR1b-over-expressing THP-1 cells were transfected with FLAG-DN-TRAF6 expres-sion vector or control vector and the expression of FLAG-DN-TRAF6 wasexamined by Western blotting after immunoprecipitation with anti-FLAGAb. C, Each cell type was activated on collagen-coated plate for 3 h anddegradation of I�B� was evaluated by Western blotting. Representativeresults of two to three individual experiments with similar results are shown.

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I collagen. However, in inflammatory conditions, such as athero-sclerosis, polymerized collagen fibrils are absent from intermediatestages of lesion development that instead contain thin and disor-dered collagen fibers and collagen fragments. Several previous re-ports have indicated a differential role between monomeric andpolymerized collagen in arterial smooth muscle proliferation (45)and macrophage matrix metalloproteinase 9 production (46). Itwill be important to determine the optimal physical state of col-lagen for DDR1 binding and activation.

Our previous study demonstrated that DDR1b transduces sig-nals only in PMA-treated differentiated THP-1 cells, but not inundifferentiated THP-1 cells (13). In primary macrophages, a de-tectable level of Shc recruitment to DDR1b occurs after 3-dayincubation of monocytes with GM-CSF (W. Matsuyama and T.Yoshimura, unpublished data). Therefore, differentiation of THP-1cells or monocytes is necessary for signaling by DDR1b. PMA andGM-CSF are potent activators of THP-1 cells and monocytes, re-spectively, and these agents could influence the signaling in thesecells. However, we did not detect a significant basal level of eitherp38 MAPK phosphorylation or I�B degradation before DDR1 ac-tivation, indicating that the activation of p38 MAPK and NF-�Bdetected in this study was induced through activation of DDR1.The molecular mechanisms whereby DDR1b transduces signals onlyin differentiated THP-1 cells or macrophages remain unknown.

Macrophages are one of the major cell types infiltrating the sitesof chronic inflammatory diseases, such as atherosclerosis, pulmo-nary fibrosis, and rheumatoid arthritis, and release an array of cy-tokines and chemokines, and contribute to the development ofthese diseases. Our data presented here indicate that DDR1b-col-lagen interaction markedly amplifies macrophage production ofIL-8, MIP-1�, and MCP-1, providing a novel mechanism by whichmacrophages produce and release large amounts of these chemo-kines in a tissue microenvironment in the course of inflammatoryresponses. Intervention of DDR1b-collagen interaction or DDR1bsignaling may be useful to control the development of inflamma-tory diseases in which these proteins play an important role.

AcknowledgmentsWe are grateful to Dr. Joost J. Oppenheim for his invaluable comments.

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