4 páginas, 2 figuras -- PAGS nros. 383-386B-cell chronic lymphocytic leukemia (B-CLL) progression is frequently accompanied by clinical lymphadenopathy, and the CCL21 chemokine may play an important role in this process. Indeed, CCR7 (the CCL21 receptor), as well as matrix metalloproteinase-9 (MMP-9), are overexpressed in infiltrating B-CLL cells. We have studied whether MMP-9 is regulated by CCL21 and participates in CCL21-dependent migration. CCL21 significantly increased B-CLL MMP-9 production, measured by gelatin zymography. This was inhibited by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) activity or by cell transfection with CCR7-siRNA. Accordingly, CCL21/CCR7 interaction activated the ERK1/2/c-Fos pathway and increased MMP-9 mRNA. CCL21-driven B-CLL cell migration through Matrigel or human umbilical vein endothelial cells (HUVEC) was blocked by anti-CCR7 antibodies, CCR7-siRNA transfection, or the ERK1/2 inhibitor U0126, as well as by anti-MMP-9 antibodies or tissue inhibitor of metalloproteinase 1 (TIMP-1). These results strongly suggest that MMP-9 is involved in B-CLL nodal infiltration and expand the roles of MMP-9 and CCR7 in B-CLL progression. Both molecules could thus constitute therapeutic targets for this diseaseThis work was supported by grants PI060400 (to A.G.P.) and PI061637 (to M.J.T.) from the Ministerio de Sanidad y Consumo, and by the Fundación de Investigación Médica Mutua Madrileña (FMM; to A.G.P.). J.R.M. was supported by FMM and the Fundación Ramón ArecesPeer reviewe

Javier Redondo-Muñoz

Estefanía Ugarte-Berzal

María José Terol

Philippe E. Van den Steen

Mercedes Hernández del Cerro

Martin Roderfeld

Elke Roeb

Ghislain Opdenakker

José A. García-Marco

Angeles García-Pardo

Bambach, M.

Heppner, S.

Steinmetz, D.

Roters, F.

MPG.PuRe

Assessing and ensuring parameter identifiability for a physically-based strain hardening model for twinning-induced plasticity

Heppner, Stephan

Publikationsserver der RWTH Aachen University

Redondo-Muñoz, Javier

Terol, María José

García-Marco, José A.

García-Pardo, Angeles

Digital.CSIC

NEOPLASIABrief reportMatrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction viaextracellular signal-regulated kinase-1/2 signaling and is involved inCCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migrationJavier Redondo-Mun˜oz,1 Marı´a Jose´ Terol,2 Jose´ A. Garcı´a-Marco,3 and Angeles Garcı´a-Pardo11Departamento de Fisiopatologı´a Celular y Molecular, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas (CSIC),Madrid; 2Servicio de Hematologı´a y Medicina Oncolo´gica, Hospital Clı´nico, Valencia; and 3Servicio de Hematologı´a, Hospital Puerta de Hierro,Madrid, SpainB-cell chronic lymphocytic leukemia (B-CLL) progression is frequently accompa-nied by clinical lymphadenopathy, andthe CCL21 chemokine may play an impor-tant role in this process. Indeed, CCR7(the CCL21 receptor), as well as matrixmetalloproteinase-9 (MMP-9), are overex-pressed in infiltrating B-CLL cells. Wehave studied whether MMP-9 is regulatedby CCL21 and participates in CCL21-dependent migration. CCL21 significantlyincreased B-CLL MMP-9 production, mea-sured by gelatin zymography. This wasinhibited by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) activity orby cell transfection with CCR7-siRNA.Accordingly, CCL21/CCR7 interaction ac-tivated the ERK1/2/c-Fos pathway andincreased MMP-9 mRNA. CCL21-drivenB-CLL cell migration through Matrigel orhuman umbilical vein endothelial cells(HUVEC) was blocked by anti-CCR7 anti-bodies, CCR7-siRNA transfection, or theERK1/2 inhibitor U0126, as well as byanti-MMP-9 antibodies or tissue inhibitorof metalloproteinase 1 (TIMP-1). Theseresults strongly suggest that MMP-9 isinvolved in B-CLL nodal infiltration andexpand the roles of MMP-9 and CCR7 inB-CLL progression. Both molecules couldthus constitute therapeutic targets forthis disease. (Blood. 2008;111:383-386)© 2008 by The American Society of HematologyIntroductionAs B-cell chronic lymphocytic leukemia (B-CLL) progresses,malignant cells extravasate and infiltrate lymphoid tissues.1,2CCR7, the receptor for the CCL19 and CCL21 chemokines,3,4plays an important role in B-CLL cell entry into lymph nodes, andCCR7 overexpression correlates with the presence of lymphad-enopathy.5,6 Another important contributor to B-CLL cell migrationmay be matrix metalloproteinase-9 (MMP-9),7-9 which is producedby B-CLL cells10-12 and overexpressed when these cells infiltratetissues with a diffuse pattern.11We showed recently that MMP-9 is up-regulated by 41integrin or CXCR4 (the CXCL12 chemokine receptor) engage-ment12 and plays a crucial role in B-CLL cell migration throughbasement membranes and human umbilical vein endothelialcells (HUVEC).12 To further understand the role of MMP-9 inB-CLL progression, we have now studied whether MMP-9 isregulated by CCR7 ligation and/or involved in CCR7-dependentmigration.MethodsApproval was obtained from the review boards of the Hospital Clı´nicoUniversitario, Valencia, and the Hospital Universitario Puerta de Hierro,Madrid, for these studies. Informed consent was obtained in accordancewith the Declaration of Helsinki.Patients and cellsCD5 B-lymphocytes were purified from peripheral blood samples from6 patients with B-CLL as reported previously.12 HUVECs were providedby Dr M. L. Botella (Centro de Investigaciones Biolo´gicas, Madrid, Spain)and cultured as described previously.12Additional materials and methods can be found in Document S1(available on the Blood website; see the Supplemental Materials link at thetop of the online article).Results and discussionTo determine whether MMP-9 was regulated by CCR7 ligation,B-CLL cells were incubated with or without CCL21; after24 hours, the conditioned media were analyzed by gelatin zymogra-phy. Figure 1A shows that CCL21 significantly increased (P  .01)MMP-9 secretion compared with untreated cells. As previouslyreported for B-CLL cells,11,12 MMP-9 was present as a 92-kDaform. The CCL21 effect was completely blocked by inhibitingCCR7 or ERK1/2 signaling with pertussis toxin (PT) or U0126,respectively, while inhibiting phosphatidylinositol 3-kinase (PI3-K), p38 mitogen-activated protein kinase (MAPK), protein kinaseC, or Src kinases, had no effect (Figure 1A). To study whetherCCL21 activated ERK1/2, cells were incubated with CCL21 and,at various times, they were lysed and lysates analyzed by Westernblotting. CCL21 induced ERK1/2 phosphorylation, which peakedSubmitted August 14, 2007; accepted September 18, 2007. Prepublishedonline as Blood First Edition Paper, September 21, 2007; DOI 10.1182/blood-2007-08-107300.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.© 2008 by The American Society of Hematology383BLOOD, 1 JANUARY 2008  VOLUME 111, NUMBER 1For personal use only.on June 8, 2016. by guest  www.bloodjournal.orgFrom after 10 minutes of exposure (Figure 1B) and persisted for at least24 hours (not shown). This effect was specifically inhibited by PTand U0126 (Figure 1B). Further analysis of these samples revealedthat CCL21 also activated the ERK1/2 effector c-fos13 (Figure 1C),suggesting that CCL21/CCR7 may regulate MMP-9 at variouslevels including gene transcription. Indeed, CCL21 significantlyincreased MMP-9 mRNA levels measured after 6 to 16 hours ofexposure, then declining after 24 hours (Figure S1).To further establish that the CCL21/CCR7 interaction wasresponsible for MMP-9 up-regulation, we studied the effect ofCCL21 on siRNA transfected cells. Transfection with controlsiRNA did not affect CCR7 surface expression (Figure 1D) orMMP-9 induction by CCL21 (Figure 1E), indicating that MMP-9production was not impaired by the procedure. However, CCR7gene silencing significantly inhibited CCR7 expression (Figure1D), and CCL21-induced MMP-9 up-regulation (Figure 1E),confirming the specificity of the effect. Moreover, CXCL12,another chemokine that up-regulates MMP-9 upon binding toCXCR4,12 enhanced MMP-9 levels on CCR7 siRNA-cells (Figure1E), indicating that both axes, CCL21/CCR7 and CXCL12/CXCR4, functioned independently from each other. Indeed, simul-taneous treatment with CCL21 and CXCL12 did not have anadditive effect on MMP-9 production (Figure 1E). This probablyreflects the physiologic situation in which the 2 chemokines areassociated with different tissues4 yet may provide a continuousstimulus for MMP-9 production.We next studied whether MMP-9 was involved in CCL21-induced cell migration. B-CLL cells migrated through Matrigelor HUVEC in response to CCL21, and this was completelyinhibited by PT, anti-CCR7 antibodies (Abs), or CCR7 siRNAtransfection (Figure 2A,B). U0126 also blocked cell migrationin both systems, whereas PI3-K or p38 MAPK inhibitors had nosignificant effect. It is noteworthy that anti-MMP-9 Abs or theMMP-9–specific inhibitor known as tissue inhibitor of metallo-proteinase 1 (TIMP-1), but not control anti-MT1-MMP Abs,inhibited cell migration (Figure 2A,B). To determine whetherMMP-9 enzymatic activity was involved in this effect, weincubated the conditioned media of untreated or CCL21-treatedB-CLL cells with FITC-gelatin and measured the fluorescenceafter 24 hours. Incubation with the conditioned media ofuntreated cells had a minor effect, but the conditioned media ofCCL21-treated cells significantly decreased the fluorescence(Figure 2C). This was reversed by anti-MMP-9 Abs or TIMP-1,whereas these compounds had no effect in the absence ofconditioned media (Figure 2C). These results indicated thatsecreted MMP-9 had catalytic activity, that this activity wasclearly increased upon CCL21 treatment (which enhancesMMP-9 production), and that its role in cell migration probablyFigure 1. CCL21 up-regulates MMP-9 via ERK1/2signaling. (A) B-CLL cells were treated or not with1000 ng/mL CCL21 in the presence or absence of theindicated inhibitors (PT, 200 ng/mL; Wortmannin (Wmn),30 nmol/L; the rest, 5 mol/L). After 24 hours, theconditioned media was analyzed by gelatin zymography.(B) Western blots of lysates of B-CLL cells treated or not(10 minutes) with CCL21, and with or without the indi-cated inhibitors. (C) Reverse transcription-polymerasechain reaction analysis of c-fos in the same samplesshown in (B). Vertical lines in (A) and (C) have beeninserted to indicate a repositioned gel lane. (D) Flowcytometric analysis of CCR7 expression in untransfected(continuous lines), control siRNA- (dotted lines) or CCR7siRNA-transfected (shaded areas) cells. (E) Gelatinzymographic analysis of the conditioned media of untrans-fected or siRNA-transfected cells treated or not withCXCL12 or CCL21 for 24 hours. Error bars representSD. *P  .05; **P  .01.384 REDONDO-MUN˜ OZ et al BLOOD, 1 JANUARY 2008  VOLUME 111, NUMBER 1For personal use only.on June 8, 2016. by guest  www.bloodjournal.orgFrom involves the degradation of the matrix and/or basement mem-brane. Despite this functional assay, the mature 85-kDa MMP-9form was not detected in the B-CLL conditioned media. Similarobservations have been reported previously for other celltypes.14 Although the reason for this remains unclear, onepossibility is that mature MMP-9 is produced at levels that areundetectable yet sufficient for focalized activity. MMP-9 activa-tion most likely occurs at the pericellular space, where severalproteases, including plasmin, may be present. MMP-9 couldalso be activated by nonproteolytic mechanisms (conforma-tional changes, oxidative modifications)14 thus resulting in anactive 92-kDa enzyme.Our results therefore establish a novel role for the CCL21/CCR7 axis as a MMP-9 regulator and clearly show that MMP-9 isan important mediator in CCL21-driven cell migration. CCL21 isexpressed in high endothelial venules, a major route for lympho-cyte homing to lymphoid tissues,15-17 and lack of CCR7 impairsB-cell entry into lymph nodes.18 Besides its role in B-CLL nodalinfiltration, CCR7 may also induce B-CLL cell survival,19 thuscontributing in various ways to B-CLL progression. Our presentfindings expand the CCR7 roles in B-CLL and strongly suggest thatMMP-9 is involved in B-CLL lymph node infiltration. MMP-9and/or CCR7 could therefore constitute targets for treatment ofthis malignancy.AcknowledgmentsWe thank Mercedes Herna´ndez del Cerro for excellent technicalassistance.This work was supported by grants PI060400 (to A.G.P.) andPI061637 (to M.J.T.) from the Ministerio de Sanidad y Consumo,and by the Fundacio´n de Investigacio´n Me´dica Mutua Madrilen˜a(FMM; to A.G.P.). J.R.M. was supported by FMM and theFundacio´n Ramo´n Areces.AuthorshipContribution: J.R-M. performed research and designed someexperiments. M.J.T. and J.A.G-M. contributed patient samples.A.G-P. designed and supervised research and wrote the manuscript.Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.Correspondence: Angeles Garcı´a-Pardo, Departamento de Fi-siopatologı´a Celular y Molecular, Centro de Investigaciones Bio-lo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; e-mail:agarciapardo@cib.csic.es.References1. Zent CS, Kay NE. Advances in the understandingof biology and prognosis in chronic lymphocyticleukemia. Curr Oncol Rep. 2004;6:348-354.2. Rai KR. Pathophysiologic mechanisms of chroniclymphocytic leukemia and their application totherapy. Exp Hematol. 2007;35:134-136.3. Kakinuma T, Hwang ST. Chemokines, chemokinereceptors, and cancer metastasis. J Leukoc Biol.2006;79:639-651.4. Zlotnik A. Chemokines and cancer. Int J Cancer.2006;119:2026-2029.5. Till KJ, Lin K, Zuzel M, Cawley JC. The chemokinereceptor CCR7 and 4 integrin are important for mi-gration of chronic lymphocytic leukemia cells intolymph nodes. Blood. 2002;99:2977-2984.6. Lo´pez-Giral S, Quintana NE, Cabrerizo M, et al.Chemokine receptors that mediate B cell homingto secondary lymphoid tissues are highly ex-pressed in B cell chronic lymphocytic leukemiaand non-Hodgkin lymphomas with widespreadnodular dissemination. J Leukoc Biol. 2004;76:462-471.7. Egeblad M, Werb Z. New functions for the matrixmetalloproteinases in cancer progression. NatRev Cancer. 2002;2:161-176.8. Deryugina EI, Quigley JP. Matrix metalloprotein-ases and tumor metastasis. Cancer MetastasisRev. 2006;25:9-34.9. Stamenkovic I. Extracellular matrix remodelling:the role of matrix metalloproteinases. J Pathol.2003;200:448-464.10. Bauvois B, Dumont J, Mathiot C, Kolb JP. Pro-duction of matrix metalloproteinase-9 in earlyFigure 2. Role of MMP-9 in CCL21/CCR7-driven B-CLL cell migration. B-CLLcells with or without previous incubation with the indicated inhibitors or transfectedwith siRNAs were added to Transwell filters coated with Matrigel (A) or HUVEC (B).CCL21 (1000 ng/mL) was added to the medium in the bottom chamber, except for thecontrol. After 24 hours, migrated cells were counted by flow cytometry. Valuesrepresent the percentage of total cells added. (C) The conditioned media of untreatedor CCL21-treated B-CLL cells was added to FITC-gelatin-coated wells in thepresence or absence of the indicated inhibitors (TIMP-1, 5 nmol/L; Abs, 10 g/mL).TIMP-1 or anti-MMP-9 Ab alone were also added to the wells. After 24 hours, thefluorescence was quantitated. Values are the average of 2 different patients withduplicate determinations. *P  .05; **P  .01.CCL21/CCR7 INTERACTION REGULATES MMP-9 IN B-CLL 385BLOOD, 1 JANUARY 2008  VOLUME 111, NUMBER 1For personal use only.on June 8, 2016. by guest  www.bloodjournal.orgFrom stage B-CLL: suppression by interferons. Leuke-mia. 2002;16:791-798.11. Kamiguti AS, Lee ES, Till KJ, et al. The role ofmatrix metalloproteinase-9 in the pathogenesis ofchronic lymphocytic leukemia. Br J Haematol.2004;125:128-140.12. Redondo-Mun˜oz J, Escobar-Dı´az E, SamaniegoR, et al. MMP-9 in B-cell chronic lymphocytic leu-kemia is up-regulated by 41 integrin or CXCR4engagement via distinct signaling pathways, lo-calizes to podosomes, and is involved in cell inva-sion and migration. Blood. 2006;108:3143-3151.13. Murphy LO, Smith S, Chen R-H, et al. Molecularinterpretation of ERK signal duration by immedi-ate early gene products. Nature Cell Biol. 2002;4:556-564.14. Fridman R, Toth M, Chvyrkova I, et al. Cell sur-face association of matrix metalloproteinase-9(gelatinase B). Cancer Metastasis Rev. 2003;22:153-166.15. Gunn MD, Tangemann K, Tam C, et al. A chemo-kine expressed in lymphoid high endothelialvenules promotes the adhesion and chemotaxisof naive T lymphocytes. Proc Natl Acad Sci U S A.1998;95:258-263.16. Nagira M, Imai T, Yoshida R, et al. A lymphocyte-specific CC chemokine, seconary lymphoid tissuechemokine (SLC), is a highly efficient chemoat-tractant for B cells and activated T cells. Eur J Im-munol. 1998;28:1516-1523.17. Okada T, Ngo VN, Ekland EH, et al. Chemokinerequirements for B cell entry to lymph nodes andPeyers patches. J Exp Med. 2002;196:65-75.18. Fo¨rster R, Schubel A, Breitfeld D, et al. CCR7coordinates the primary immune response by es-tablishing functional microenvironments in sec-ondary lymphoid organs. Cell. 1999;99:23-33.19. Alfonso-Pe´rez M, Lo´pez-Giral S, Quintana NE, etal. Anti-CCR7 monoclonal antibodies as a noveltool for the treatment of chronic lymphocytic leu-kemia. J Leuk Biol. 2006;79:1157-1165.386 REDONDO-MUN˜ OZ et al BLOOD, 1 JANUARY 2008  VOLUME 111, NUMBER 1For personal use only.on June 8, 2016. by guest  www.bloodjournal.orgFrom online September 21, 2007 originally publisheddoi:10.1182/blood-2007-08-1073002008 111: 383-386  Javier Redondo-Muñoz, María José Terol, José A. García-Marco and Angeles García-Pardo migrationCCL21-driven B-cell chronic lymphocytic leukemia cell invasion andvia extracellular signal-regulated kinase-1/2 signaling and is involved in Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction http://www.bloodjournal.org/content/111/1/383.full.htmlUpdated information and services can be found at: (1930 articles)Signal Transduction     (4212 articles)Neoplasia     (1086 articles)Gene Expression     (564 articles)Chemokines, Cytokines, and Interleukins     (790 articles)Cell Adhesion and Motility     (1879 articles)Brief Reports    Articles on similar topics can be found in the following Blood collectionshttp://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:  Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American SocietyFor personal use only.on June 8, 2016. by guest  www.bloodjournal.orgFrom 

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