Role of the SDF-1 CXCR4 axis in the pathogenesis of lung injury and fibrosis

Seminars in Cancer Biology 20 (2010) 178–185Contents lists available at ScienceDirectSeminars in CancerBiologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s e m c a n c erReviewCan inhibition of the SDF-1/CXCR4axis eradicate acute leukemia?Sigal Tavor a ,∗,Isabelle Petit ba Institute of Hematology and Bone Marrow Transplantation,Sourasky Medical Center,Tel Aviv,Israel bINSERM U898,Faculty of Medicine,Av.Valombrose,Nice 06107,Francea r t i c l ei n f o Keywords:LSC AML ALL SDF-1CXCR4a b s t r a c tPoor prognosis of acute leukemia with current treatments is mainly due to the relapse of the disease fol-lowing chemotherapy.In the last decade,an emerging concept has proposed that the leukemia stem cells (LSCs)and their interactions with the BM microenvironment are the major cause of the acute leukemia relapse.Adhesion to the stromal niche is crucial for LSCs as it directly supports self-renewal,prolifera-tion,arrest of differentiation and protects from damaging chemo-agents.One of the key players in this crosstalk between leukemic cells and the BM stroma niche is the chemokine SDF-1.SDF-1regulates the process of homing and engraftment of LSCs into the BM and inhibition of its receptor CXCR4induces leukemic cell mobilization into the circulation.However,besides its chemotactic and adhesive functions,SDF-1is also a pleiotropic cytokine that regulates leukemic cell proliferation as well as their program of differentiation.CXCR4antagonists are used in combination with chemotherapy in preclinical and clin-ical studies,which demonstrate that blocking CXCR4is a novel promising approach of therapy.In this review,we focus on the multifaceted SDF-1/CXCR4axis in acute leukemia and discuss how targeting this pathway could provide potential interest to eradicate the LSCs.© 2010 Elsevier Ltd. All rights reserved.1.Leukemic stem cells:the reason for relapseLeukemic cells are heterogeneous in cell morphology,surface markers,genetic and epigenetic abnormalities,cell proliferation kinetics and response to therapy [1].An emerging model,which is well demonstrated in acute myelogenous leukemia (AML),is that cancer arises from malignant stem cells.Pioneer studies showed in vitro that although the leukemic blasts cells display heterogeneous features in the different subtypes of leukemia,the cell of origin is an early multipotential cell with limitless self-renewal potential [2,3].Cancer stem cells possess the capacity of self-renewal and to proliferate in order to generate a continuously growing tumor.In human AML,cancer stem cells were first defined by J.Dick’s group as the subpopulation of cells that have the capacity of initiating a human AML graft in recipient non-obese diabetic severe com-bined immunodeficiency (NOD/SCID)mice,and were termed SCID leukemia-initiating cells (SL-ICs)[4–6].SL-ICs are rare,on average one cell per 106AML cells,and most of them display CD34+CD38−cell surface phenotype similar to nor-mal hematopoietic stem cells (HSCs),supporting the idea that AML stem cells originated from normal HSCs [7].However,some cell∗Corresponding author at:Department of Hematology and Bone Marrow Trans-plantation,Tel Aviv Sourasky Medical Center,6Weizmann Str.,Tel Aviv 64239,Israel.Tel.:+97236973576;fax:+97236974452.E-mail address:tavorsigal@ (S.Tavor).surface markers are differently expressed on leukemic stem cells (LSCs)compared to their normal counterpart CD34+/CD38−cells,such as interleukine-3receptor alpha chain (IL-3R ␣or CD123)and CD47,which are overexpressed on their surface [8,9].Expression levels of those markers in AML stem cells have been associated with bad prognosis [10–12].LSCs have other unique features that distinguish them from their normal counterparts,such as increased expression of drug transporters,abnormal cholesterol metabolism,enhanced adhesion and deregulation of several intracellular sig-nals,leading to drug resistance and reduced apoptosis [13].While PTEN is expressed in HSCs and positively regulate HSC functions,in LSCs however,PTEN is frequently mutated and its loss of func-tion leads to increased PI3K-AKT-mTOR signaling,which mediates LSC survival and multi-drug resistance.Similarly,the Raf/MEK/ERK pathway is overexpressed in acute leukemias and involved in leukemia resistance to treatment [14].The NF ␬B transcription fac-tor is also selectively active in LSCs but not in HSCs [15].The existence of LSCs deriving from normal HSCs is not clearly demonstrated in acute lymphoblastic leukemia (ALL).The com-mitted lymphoid progenitor cell with an aberrant CD19+CD34+CD38−/low immunophenotype has recently been proposed as a candidate LSC population in ALL,but it is still a matter of contro-versy [16–18].There is some evidence that AML can also develop from genetic alterations that occur in committed hematopoietic progenitors.In murine AML models of MLL fusion protein,the leukemia-initiating cells have an immuophenotype of more dif-ferentiated myeloid progenitors.These cells acquired stem cell1044-579X/$–see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.semcancer.2010.07.001S.Tavor,I.Petit/Seminars in Cancer Biology20 (2010) 178–185179characteristics of self-renewal through the oncogenic transforma-tion of MLL-AF9,MOZ-TIF2or MLL-ELN[19–21].LSCs are not homogeneous and have wide variation in repopu-lation potential and self-renewal capacity.In AML there is a large proliferative fraction of cells and a small subpopulation of qui-escent LSCs,which can engraft into NOD/SCID mice and initiate leukemia.In contrary to normal HSCs,most quiescent G0LSCs can enter active cell cycle also without growth factors[22,23].A current concept suggests that these quiescent LSCs reside in the BM,evade the conventional chemotherapy,which targets prolif-erative cells,and are responsible for the high rate of relapse in acute leukemia.Better understanding of the LSC phenotype,the molecules which regulate their retention to the BM and their dissemination to the circulation will help us to selectively tar-get the LSCs and maybe succeed to prevent the relapse of the leukemia.2.The BM microenvironment:the sweet home of HSCs2.1.The BM HSC nichesThe BM is composed of a variety of different cells,which form complex and still poorly defined spatio-localized microenvi-ronments.These“niches”regulate normal HSC self-renewal and differentiation,quiescence and proliferation.The use of genetic murine animals and the development of in vivo imaging techniques have allowed a better insight of the HSC niche this past decade. Two distinct stem cell niches have originally been described and traditionally opposed:the osteoblastic niche(or endosteal)and the vascular niche.The osteoblastic niche is localized at the inner surface of the bone,and is defined by the presence of osteoblasts, osteoclasts and its proximal stomal cells.A large number of stud-ies convincingly demonstrate that the endosteal niche provides a quiescent microenvironment for long-term(LT)-HSCs through release of soluble factors(Stroma-cell derived factor-1(SDF-1), angiopoietin-1(Ang1),osteopontin)and cell contact interactions (Stem cell factor(SCF),N-cadherin)[24,25].In contrast,the vascular niche,defined by the presence of sinusoidal vascular endothe-lium,is considered important for maintaining a boundary between immature cells and peripheral blood circulation and promotes pro-liferation and differentiation of actively cycling short-term HSCs[26].The presence of LT-HSCs near sinusoids in the adult BM is stilla matter of debate.Spatial observations reveal that proliferating cells preferably re-localize in the central zone of the BM.Moreover, the presence of a large number of sinusoids near the endosteum has led to the hypothesis that osteoblastic and vascular niches may rather form one and unique niche near the endosteum in the trabec-ular zone,while proliferating or migrating progenitors are located within the central core of the BM,away from the endosteal fac-tors maintaining quiescent HSCs(reviewed in Ref.[27]).Adventitial reticular cells expressing high level of SDF-1cells are found in close association in both the osteoblastic and vascular niches,and may possibly serve as a transit pathway for migrating HSCs[28,29]. Finally,recent evidence has showed that cells from the nervous system are active components of the BM environment and regu-late HSC functions by releasing neurotransmitters[30,31].The HSC quiescent niche is characterized by its low concentration of O2and it is assumed that there is a low gradient of O2from less than1% near the bone to6%in the sinusoidal vasculature.Evidence of co-localization of HSCs with hypoxic area,mostly near the endosteum, strongly suggest that low O2,and its major downstream effector molecule hypoxia inducible factor1-alpha(HIF-1␣),play critical role in the HSC niche function,possibly by maintaining low cell cycle activity,protecting from deleterious reactive oxygen species (ROS)accumulation and regulating key components of the niche such as SDF-1,SCF,Notch1and vascular endothelial growth factor (VEGF)[32].Inside the HSC niches,stem cells are in contact with the stromal cells through multiple adhesive interactions[33].Beyond sim-ple retention,those adhesive connections also induce intracellular pathways activating stem cell function such as quiescence and self-renewal.The bidirectional molecular crosstalk between HSCs and the cells present in the niche can be compared to a“stem-cell-niche synapse”as proposed by A.Trumpp[34].Adhesion of HSCs is mediated by adhesive molecules such as very late antigen-4 (VLA-4),VLA-5,lymphocyte function-associated antigen-1(LFA-1)and the glycoprotein CD44expressed on HSCs,which bind to their respective ligands present in the ECM or at the surface of osteoblasts.These adhesion molecules are activated by cytokines present in the niche including SCF and SDF-1.Membrane-bound SCF expressed by osteoblasts binds to c-kit on HSCs and posi-tively regulates HSC retention and self-renewal as demonstrated by impaired hematopoiesis in membrane-bound SCF deficient mice [35]and by the dramatic effect of a c-kit blocking antibody,which induces rapid clearance of HSCs from the BM[36].Despite the lack of HSC defects in N-cadherin KO mice[37],other studies suggest that homotypic N-cadherin interactions are not only mediating anchorage of HSCs within the niche but also signals intracellularly and recruits p120-catenin to maintain quiescence and long-term self-renewal[38].Other signaling pathways such as Notch1,Wnt and sonic hedgehog(SHH)are also involved in maintaining the HSC pool.Osteopontin is a factor secreted by osteoblasts that sup-ports both HSC retention and long-term HSC function by preventing HSC proliferation through outside-in signaling after ligation with ␤1-integrins and CD44receptors expressed on HSC[39].Similarly, osteoblast-derived Ang1promotes both HSC adhesion and HSC qui-escence,through upregulation of N-cadherin and p21,an inhibitor of proliferation[40].It appears clear that adhesion of HSCs inside the niche does not only anchor the HSCs in the right location to be exposed to secreted factors but also control the quiescent state by activating intracellular signaling cascades,suggesting thatfine tuning of adhesive interactions might modulate HSC fate during homeostasis and stress.2.2.SDF-1and CXCR4:master regulators of the nicheThe chemokine SDF-1(CXCL12)is a major molecule govern-ing HSC maintenance,including retention,survival and migration in and out of the BM.SDF-1is highly expressed within the BM by a large number of cells including those found in HSC niches: osteoblasts,endothelial cells and reticular cells.SDF-1can bind to proteoglycans present in the ECM,facilitating presentation to cells expressing its receptor CXCR4.It is the most potent chemokine act-ing on stem and progenitor cells,which express CXCR4.As such, SDF-1regulates migration of HSCs during ontogeny and adult life [41–43].The high concentration of SDF-1in the BM compartment induces egress of circulating CXCR4+stem and progenitors out of the blood into the BM tissue,a process called“homing”[44].Higher concentration of SDF-1in the endosteal niche may induce proper lodging and subsequent engraftment,although no experimental evidence has shown yet a concentration gradient of SDF-1within the BM.SDF-1binding to CXCR4enhances adhesive properties of HSCs by inside-out signaling leading to activation of the integrins VLA-4,VLA-5,LFA-1and of the adhesion molecule CD44[45,46]. The critical function of SDF-1in retaining HSCs in their niche has been demonstrated by studies showing that disruption of SDF-1/CXCR4interactions,either by BM SDF-1reduction or by CXCR4 blockage,causes a rapid and massive mobilization of HSCs.BM SDF-1reduction is observed during natural circadian oscillations, which induce HSC egress by modulating SDF-1transcriptional lev-els[47].Treatment with the mobilizing agent granulocyte-colony180S.Tavor,I.Petit/Seminars in Cancer Biology20 (2010) 178–185stimulating factor(G-SCF)also decreases SDF-1in the BM but by proteolytic cleavage,together with the disruption of other adhe-sive contacts such as VLA-4/VCAM-1and c-kit/SCF.Direct blocking of SDF-1function by CXCR4antagonists is however sufficient to cause a rapid HSC mobilization.AMD3100,a CXCR4antagonist used in humans,can trigger mobilization within few hours[48].Accord-ingly,CXCR4deficient mice reveal reduced number of HSCs in the BM[28].Besides its chemotactic and adhesive functions,SDF-1is also a pleiotropic cytokine that positively regulates normal HSC pro-liferation as well as survival[49–51].In vitro,SDF-1stimulates circulating CD34+cell proliferation and in vivo increased pro-duction of SDF-1promotes myelopoiesis[52].In addition,low concentrations of SDF-1support G0/G1transition,sensitize cells to other cytokines and protect cells from apoptosis both in vitro and in vivo causing survival of LT-HSCs[50,53].SDF-1is there-fore an important survival factor for HSCs and progenitors,both in steady-state and stress conditions following damage to the HSC compartment[53,54].Finally,SDF-1signals to induce secretion of metalloproteinases such as MMP-9[55].CXCR4engagement switches on intracellular signaling pathways classically activated by G-protein coupled chemokine receptors.Activation of the PI3K/AKT,mitogen-activated protein kinase(MAPK)and PKC␨pathways has been demonstrated in SDF-1-induced effects on HSC, including adhesion and migration[56].The intensity and duration of SDF-1signaling are subtly modulated,mainly through CXCR4 phosphorylation and internalization[57].A novel receptor for SDF-1has been recently described and named CXCR7[58].CXCR7is expressed on glial cells,T cells,CD34+cells and several solid tumor cells.Its function is still unclear,it has scavenger activity towards SDF-1and the chemokine I-TAC but seems to play minor role in hematopoiesis[59].3.The LSC niche:a place to hide3.1.Role of the BM microenvironment in leukemia developmentThe BM is a permissive zone for cancer cell homing and sur-vival where solid tumor very frequently home and metastasize. In leukemia,the BM is the primary site of disease development. Lately,many studies began to define the role of the BM niche in the development of the leukemia.These studies put on view that the interactions with the BM microenvironment support the adhe-sion,proliferation,arrest of differentiation and chemoresistance of leukemic cells.Similarly to normal HSCs,LSCs strongly rely on adequate niches to support their self-renewal and proliferation. Indeed LSCs home and engraft in the same endosteal regions of the BM where HSCs are residing,therefore competing for the same microenvironment[60,61].LSCs closely resemble normal HSCs and it is not surprising tofind similarities in their interactions with the BM environment.The current model suggests that LSCs highjack the HSC niche and use it to sustain leukemia development but they may also modify it to their advantage.Indeed abnormalities have been observed in leukemic BM,such as increased acidity and hypoxia[62]coincid-ing with increased HIF-1␣expression in leukemic cells[63].SCF expression is elevated in the leukemic niche,both in ALL patients and in a xenograft model[64],and SDF-1levels are also elevated in leukemic patients[65,66].Finally,high VEGF expression caus-ing increased angiogenesis in the BM is observed in AML and ALL [67,68].Deregulation of such important constituents of the BM niche could explain one of the hallmarks of acute leukemia,the suppression of normal hematopoiesis.Indeed,in a SCID xenograft model of Pre-B ALL Nalm-6cells,the leukemic cell growth dis-rupted normal homing of normal CD34+HSCs in the BM niches.Normal CD34+cells could still migrate into tumor bed,attracted by the abnormal secretion of SCF but their number declined over time in this abnormal microenvironment,resembling what is observed in leukemic patients[64,69].Recent studies revealed that modifications within the BM microenvironment can per se trigger a leukemogenic transfor-mation of normal cells.This new concept further places the BM niche as the key regulator of normal stem cell functions and hematopoiesis.Genetic alterations causing loss of retinoblastoma protein or retinoic acid receptor in the BM microenvironment lead to myeloproliferative disorders[70].It was also shown that specific deletion of dicer1in osteoblastic precursors resulted in myelodys-plasia eventually leading to leukemogenesis and development of AML,proposing that deregulation of the BM environment is the initial event,which then favors clonal expansion of aberrant cells partly by increased proliferation[71].3.2.The SDF-1/CXCR4axis in acute leukemia:much more thanjust a traffic controllerAcute leukemia cells retained their responsiveness to SDF-1. Cell surface CXCR4is highly expressed on ALL B-cell progeni-tors whereas the intracellular CXCR4level is low.No correlation between the chemotactic response to SDF-1and the CXCR4expres-sion on B-ALL was found[72].In contrast to ALL,high expression of internal CXCR4was found in AML cells,including cells that do not express surface CXCR4[73].Moreover,external CXCR4expression is highly variable but correlates with SDF-1-induced intracellular free calcium mobilization and transendothelial migration in vitro [74,75].Importantly,high CXCR4expression in leukemic cells is an adverse predictor of overall survival and relapse-free survival in patients with AML[76,77].The CXCR4surface expression level is a dynamic process and is regulated by many factors.For example,under the condition of reduced partial pressure of oxygen at BM,CXCR4expression level on AML cells is upregulated and migration towards SDF-1is enhanced[78],suggesting that in hypoxic area of the BM niche the upregulation of CXCR4expression could enhance leukemic cell adhesion and signaling,favoring their retention to the microen-vironment.On the other hand,CXCR4level is downregulated by proteases like elastase that degrade both CXCR4and SDF-1in the process of mobilization[79].Since AML cells secrete abnormal high level of elastase,which decreases SDF-1and CXCR4expression,this proteolytic enzyme could facilitate the egress of the leukemic cells from the BM into the peripheral blood[80].Aberrant pathways deregulated or mutated in leukemia like Flt3-internal tandem duplication,which occurs in25%of AML and correlates with poor prognosis,or Pax5/TEL fusion protein,enhance SDF-1/CXCR4signaling through the upregulation of cell surface CXCR4[81–83].This effect seems to be mediated by elevated levels of the PIM1kinase,which positively controls cell surface reex-pression of CXCR4from internal pool through phosphorylation of CXCR4[84].Dynamics of cell surface CXCR4expression,resulting from internalization and reexpression from internal pool,therefore appears to be deregulated in acute leukemia,possibly in order to subtly adapt SDF-1signaling to the tumor needs[85].Reciprocally, SDF-1/CXCR4signaling activates several signaling cascades,which are often deregulated in leukemia compared to normal HSCs such as the PI3K/AKT,MAPK and NFkappaB pathways[56,86,87](Fig.1). Altogether,these observations indicate that in acute leukemia the SDF-1/CXCR4pathway is intimately linked to other aberrant signal-ing cascades,possibly synergizing together to efficiently promote leukemia maintenance.Several studies showed the important role of SDF-1/CXCR4axis in the regulation of ALL and AML cell homing and engraftment into the BM.Pretreatment of primary human leukemic cells with neu-S.Tavor,I.Petit/Seminars in Cancer Biology20 (2010) 178–185181Fig.1.SDF-1signaling in acute leukemia cells.Upon binding of SDF-1to CXCR4,several signaling cascades are activated,leading to various cellular functions in leukemic cells.Two major downstream pathways of CXCR4are PI3K and MAPK,whose activity is increased in leukemic cells.Activation of PI3K by SDF-1is essential for migration, through activation of PKC␨,as well as for transcriptional activities important for proliferation,survival and release of soluble factors(MMPs,VEGF and IL-8).Members of the Rho/rac/cdc42family are involved in controlling adhesion and motility,by activating downstream molecules such as the integrin VLA-4.These signaling pathways resemble the ones activated in normal HSCs to the exception of p38and NFkappaB,which are selectively activated in leukemic cells.Internalization and reexpression of CXCR4are modulated in Flt3-internal duplication(ITD)positive leukemic cells by mutated Flt3through upregulation of PIM1.tralizing CXCR4antibodies blocked their homing into the BM and spleen of transplanted NOD/SCID/B2mnull mice.Administration of anti-human CXCR4monoclonal antibodies to mice previously engrafted with primary human AML or ALL cells led to a dramatic decrease in the levels of leukemic cells[72,73,88].In vitro,SDF-1 mediates the transmigration of ALL blasts into layers of BMfibrob-lasts and enhances the adhesion of precursor B ALL cells via the␤1 integrins VLA-4and VLA-5[89,90].In vivo,the activation of VLA-4was most prominent in homing of precursor B-ALL.The small GTP binding proteins from Rho family(RhoA,Cdc42and Rac),the PKC signaling pathway and the P38MAPK signaling are activated in migration and homing of B-ALL cells[72,88,91](Fig.1).Similarly to its pleiotropic effects on normal HSCs,SDF-1is also a multi-functional cytokine in leukemic cells.In addition to controlling cell motility,SDF-1regulates cell survival,proliferation,cell cycle pro-gression and the ability of leukemic progenitor cells to form colony [92–94].Indeed thefirst study characterized SDF-1as a pre B-cell growth stimulating factor[95]and later SDF-1was found to support the survival of leukemic B cell precursors[96,97].A number of studies demonstrated that adhesion to the BM stroma is essential for the survival,proliferation and chemother-apy resistance of AML cells[98,99].On the other hand,the direct effect of SDF-1on AML cell survival is still a matter of debate.It was shown that inhibition of CXCR4decreases proliferation offlt3-mutated AML cells but notflt3-wild-type AML during co-culture on stromal cells[92,94].In absence of stroma,addition AMD3100 was found to inhibit the outgrowth of leukemia colony-forming units but not proliferation.However,we observed that prolong treatment with CXCR4antagonists could arrest proliferation and decrease survival of AML cells cultured in absence of stroma[73,93]. The different results emphasize that the survival of AML cells is more complex in vivo or in culture on BM stroma and is dependent on interactions with other growth factors and cytokines in the BM microenvironment.Cell surface elastase expression on AML cells is increased by SDF-1and mediates SDF-1-induced cell polarization,podia forma-tion,adhesion and in vivo homing of human AML cells to the BM of transplanted NOD/SCID/B2m null mice[80].These results suggest that SDF-1effects on AML cells could be partly mediated by cell sur-face elastase.Indeed,gene profile analysis showed that inhibition of the SDF-1/CXCR4axis or elastase upregulates genes controlling the process of differentiation in normal and leukemic cells,and downregulated genes that are overexpressed in AML and are impor-tant for the development of leukemia[93].In vitro,inhibition of CXCR4or elastase triggered changes that mimicked differentiation, including morphological changes and expression of myeloid differ-entiation antigens,indicating that SDF-1signaling could mediate arrest of differentiation in AML[93].SDF-1activates adhesion molecules like the integrin VLA-4and the hyaluronate receptor CD44,which are expressed on leukemic cells and mediate leukemia growth and chemoresistance.VLA-4 is crucial for leukemic chemoresistance and maintenance of BM minimal residual disease(MRD)[100,101].Combined therapy of VLA-4specific antibodies with chemotherapy abrogates MRD in a leukemia mice model.The interaction of VLA-4with stromal cells activates the PI3K/Akt pathways leading to increased expression of Bcl-2,an anti-apoptotic molecule.In AML patients,increased bind-ing of soluble VCAM-1via VLA-4was associated with longer overall survival[101].The activation of VLA-4through SDF-1signaling is important for cell adhesion and transendothelial migration of precursor-B ALL cells[45,88,89,102].By activating VLA-4,SDF-1 therefore plays a major role in LSC adhesion in the BM and chemore-sistance.The higher expression of the isoform CD44-6v on AML cells correlates with shorter overall survival[10].CD44supports AML cell proliferation,arrest of differentiation and retention in the BM [103].Targeted CD44antibody therapy reduces leukemia repop-ulation,cell dissemination as well as loss of the LSC immature phenotype[104].The observation that CD44ligation triggers dif-ferentiation of the immature leukemic cells was thefirst evidence that preventing adhesion of leukemic cells to the niche can over-come arrest of differentiation and induce cell maturation.Similarly, inhibition of the SDF-1/CXCR4pathway in vitro also causes a more mature phenotype of AML cells mimicking normal differentiation [93].CD44and SDF-1pathways are indeed linked since SDF-1regu-lates CD44-mediated adhesion of normal HSCs[46].Taken together,182S.Tavor,I.Petit /Seminars in Cancer Biology20 (2010) 178–185Fig.2.Sensitizing LSCs by inhibiting multiple SDF-1/CXCR4functions.Within the BM,LSCs are located in a protective niche,which confers them with chemoprotection through quiescence and adhesion.High levels of SDF-1near the endosteum controls firm retention of LSCs to the stroma through adhesion molecules and provide survival and anti-differentiation signals.Therapeutic strategies aiming to disrupt SDF-1/CXCR4interactions with CXCR4antagonists should have multiple consequences on the LSC niche:(1)loss of SDF-1-mediated adhesion will cause delocalization of LSCs out of their niche and away from its numerous protective signals;(2)loss of direct survival and proliferation signals activated by SDF-1;(3)increased differentiation due to the combined effects of inhibition of SDF-1-mediated arrest of differentiation and delocalization away from the niche;and (4)massive mobilization towards the blood circulation when using AMD3100.Each one of these effects should make the LSCs more vulnerable to chemotherapy agents (black arrow)suggesting that SDF-1/CXCR4inhibition combined with chemotherapy may be a promising strategy.these data reveal that the adhesion of the leukemic cells in the BM niche enhances their proliferation and their immature phenotype through direct stimulation by SDF-1and by network interactions with other key molecules.4.Novel therapies:targeting the LSCs and their niche by disrupting the SDF-1/CXCR4axisAlthough most of the leukemic patients initially respond to chemotherapy,disease free survival is achieved in only 10–40%of AML patients,mainly due to relapses.Apparently,the chemother-apy eliminates the bulk of rapidly dividing tumor cells,without affecting a small and undetectable number of cells able to re-initiate the tumor.Therefore the inability to eradicate the LSCs seems to be the cause of relapse.Translating to the clinical field,if we will eradicate the LSCs,will we be able to prevent the relapse of acute leukemia?One approach is by targeting specifically self-renewal or survival pathways necessary for LSCs but not normal HSCs such as PI3K/PTEN/AKT/mTOR,Ras/Raf/MEK/ERK or NFkap-paB [105].For example,the natural product parthenolide (PTL)and its analog,dimethylamino-parthenolide (DMAPT),are com-pounds that in preclinical studies have activity against LSCs and spare the HSCs.Molecular studies indicate that the activities of DMAPT include induction of oxidative stress responses,inhibition of NFkappaB,and activation of p53[106,107].Targeting specific cell surface markers is also an attractive therapy,indeed treatment with anti-IL-3R ␣neutralizing Abs impairs homing and engraftment of AML cells into NOD/SCID mice,activates the innate immunity of the mice and reduces leukemia progression [108].Based on the experimental evidence that adhesion to BM stroma mediates LSC quiescence and chemoresistance,one may consider targeting adhesion molecules,such as VLA-4and CD44inter-actions,as a novel therapeutic bined therapy of VLA-4specific antibodies with chemotherapy abrogates MRD in leukemia cell-injected mice [100],and targeted CD44antibody therapy reduces leukemia repopulation,cell dissemination as well as loss of the LSC immature phenotype [104].Moreover,enforcedcell cycling of LSCs with G-SCF treatment has for consequence a loss of adhesion and anchorage within the protective niche caus-ing higher sensitivity to chemotherapy [61].Combining G-CSF with chemotherapy therefore seems to target the powerful shielding tools of leukemias:quiescence and adhesion in the niche.How-ever,the use of myeloid growth factors (G-CSF or GM-CSF)in AML patients,has been applied in priming strategies with chemotherapy in multiple trials with variable outcome,possibly due to differences in patient groups [109].Another strategy would be to confront the leukemic cells by targeting its multi-functional regulator:the SDF-1/CXCR4axis.By controlling leukemic cell motility,adhesion,survival and dif-ferentiation,SDF-1signaling seems a target of choice for an anti-LSC strategy.As depicted in Fig.2,an anti-CXCR4treatment is expected to have multiple effects on LSC function.Blockade of SDF-1signaling should disrupt several adhesive interactions with the niche components,including VLA-4and CD44.This may induce a relocation of the LSCs outside the niche,causing a loss of quiescence and survival signals.Anti-CXCR4treatment also directly leads to the loss of survival/proliferation signals and triggers increased differentiation.Some anti-CXCR4antag-onists,such as AMD3100,are potent mobilizers,therefore they induce a massive egress of leukemic cells and LSCs.Anti-CXCR4treatment-induced delocalization,loss of quiescence,differenti-ated phenotype and mobilization should consequently sensitize the tumor cells to chemotherapy agents.Indeed recent studies in mice models showed that disruption of the interaction between AML cells and the marrow microenvironment with CXCR4antag-onists (AMD3100or AMD3465)induced mobilization of AML cells into the circulation,enhanced cell sensitization to chemotherapy or flt3inhibitor and prolonged survival of the animals [110,111].The synergistic effect of CXCR4antagonists with chemotherapy appears to be not only due to the accessibility of the mobilized leukemic cells,but also to the loss of adhesion-mediated chemore-sistance signals.By targeting different angles of LSC function,anti-CXCR4strategy appears as an excellent tool to target acute leukemia.。

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这些发现，为缺血性脑血管病的治疗带来了新的契机，研究 ＥＰＣｓ对缺血后血管再生的机制及治疗有重要的意义。ＥＰＣｓ来 源于骨髓干细胞，是分化成熟的细胞，在正常生理条件下外周血 中ＥＰＣｓ数量非常有限【２】。近年ＳＤＦ—ＩｌＣＸＣＲ４轴促进ＥＰＣｓ归 巢作用机制被广为关注，基质细胞衍生因子一ＩＯｔ（ＳＤＦ一１ｄ）在募 集和贮留骨髓源性趋化因子受体４（ＣＸＣＲ４）细胞到血管源小生 境支持缺血组织和肿瘤生长的血管形成的过程中起着重要的作 用。ＳＤＦ一１不仅通过连接脉管细胞上表达的ＣＸＣＲ４促进血管再 生。同时动员干细胞。加速缺血组织的血管形成，作为ＳＤＦ一１的 特异性受体ＣＸＣＲ４广泛表达于ＥＰＣｓ、造血细胞、淋巴细胞、内
２．１
目前认为ＳＤＦ—ｌ促进血管发生的机制是：缺血后，损伤组 织释放缺氧诱导因一１（ＨＩＦ．１）等刺激ＳＤＦ—ｌ（ｘ释放入血，改变骨 髓微环境，ＳＤＦ—ｌ仅与其受体ＣＸＣＲ４结合后，激活磷脂酰肌醇一 ３激酶（Ｐ１３Ｋ）／蛋白激酶Ｂ（ＰＫＢ／Ａｋｔ）信号途径，促进骨髓ＥＰＣｓ 释放并归巢到缺血组织参与血管生成［４］。
・１６３２・
中国中医急症２０１２年ｌＯ月第２１卷第１０期
ＪＥＴＣＭ．Ｏｃｔ．２０１２，Ｖ０１．２１，Ｎｏ．１０
ＳＤＦ一１／ＣＸＣＲ４轴与电针治疗缺血陛脑血管病水
孙宏毅
罗
勇△
（重庆医科大学，重庆４０００１０）
中图分类号：Ｒ７４３文献标志码：Ａ文章编号：１００４－７４５Ｘ（２０１２）１０—１６３２－０４ 【关键词】ＳＤＦ一１／ＣＸＣＲ４轴ＥＰＣｓ电针缺血性脑血管病 自证实人类出生后的外周血中存在能分化成为血管的内皮 祖细胞（ＥＰｃｓ）之后，也发现ＥＰＣｓ同样存在于骨髓、脐带血、胚 胎组织、脂肪组织等部位。新生血管中的内皮约有５％～２５％来源

基质细胞衍生因子-1(SDF-1)及其受体CXCR4在细胞中运输的调控机制的研究进展桂钰;葛健;夏瑞祥【摘要】基质细胞衍生因子-1(SDF-1)主要表达在骨髓的基质细胞及内皮细胞中,能够特异性地引起表达CXCR4的相关细胞的生理及病理反应.既往的研究表明,SDF-1及CXCR4在促进心脏,血管的稳态及造血干细胞的动员和分化中有着重要的作用,更进一步研究了二者在相关细胞中的表达,运输,相互作用的机制及其他影响因素等,进而对于二者在疾病的诊断,治疗中有了新的认识.在此基础上,该文对于SDF-1及其受体CXCR4在细胞中的表达及运输机制进行简要归纳和综述.【期刊名称】《安徽医药》【年(卷),期】2013(017)011【总页数】3页(P1836-1838)【关键词】SDF-1;CXCR4;Gα13;Rho;IL-21;CD63;造血干细胞;内皮细胞;淋巴细胞;细胞迁移;归巢;内化;转胞吞【作者】桂钰;葛健;夏瑞祥【作者单位】安徽医科大学第一附属医院血液内科,安徽,合肥,230022;安徽医科大学第一附属医院血液内科,安徽,合肥,230022;安徽医科大学第一附属医院血液内科,安徽,合肥,230022【正文语种】中文骨髓基质细胞和内皮细胞表达的SDF-1及其受体CXCR4之间的相互作用，参与造血的生成，胚胎发育及组织再生等，目前的研究主要集中在SDF-1及CXCR4的表达并分析其表达规律，调控机制，旨在能够提高和改善骨髓造血龛中细胞的增殖和迁移，进而对于改善骨髓造血干细胞的迁移和归巢起着重要作用。

然而，CXCR4的表达不仅存在于骨髓造血干细胞及造血龛中，一些细胞中的SDF-1和CXCR4的表达和运输，以及相关因子的介入，同样导致二者表达和运输的异常，调控机制的改变以及疾病的发生。

因而，对于二者在细胞表达及运输中的机制研究对于相关疾病的发生，诊断和治疗有着巨大的研究意义和临床应用意义。

1 SDF-1及受体SDF-1是一类具有趋化活性的细胞因子，属于CXC型趋化因子，由骨髓基质细胞及其他相关的间皮细胞和上皮细胞分泌，SDF-1的缺失会导致造血干细胞的动员，缺失，再植异常，它能够支持造血干细胞，间充质干细胞，淋巴细胞，内皮细胞等在骨髓中的作用［1］。

趋化因子SDF-1及受体CXCR4研究进展第23卷第1期2OO6年2月生物学杂志JOURNALOFBIOLOGYV o1.23No.1Feb,2006趋化因子SDF一1及受体CXCR4研究进展储子彦,陈晓萍,方晶晶(浙江工业大学生物与环境工程学院,杭州I310014)摘要:趋化因子(chenmklne)是一类一级结构相似,以对白细胞等多种细胞具有趋化定向运动作用为特征的小分子蛋白.功能研究表明,趋化因子在胚胎发育,血管生成,炎症,肿瘤,史滋病等机体多种生理和病理过程中发挥重要作用,部分趋化因子的衍生物或抑制物具有潜在的临床应用前景.不久的将来,趋化因子及其受体可能成为疾病治疗的分子靶点.关键词:趋化因子;SDF一1;CXCR4中图分类号:4文献标识码:A文章编号:1008—9632(2006)Oi一0011—03趋化因子是一类重要的免疫调节因子,直接引导自细胞,包括多种免疫活性细胞进行有方向性迁移,不仅能精确地调节免疫系统的反应,还对组织,器官形成,造血系统功能有调节作用.1趋化因子与受体的结构趋化因子分子结构中有4个保守的半胱氨酸,形成2对二硫键,可分成4个亚类:(1)CC亚类,2对二硫键间无其它氨基酸间隔;(2)CXC亚类,间隔1个氨基酸;(3)Cx3C亚类,间隔3个氨基酸;(4)C亚类,仅有1对二硫键.SDF一1(stromalcell—derivedfactor1)基质细胞来源因子,属于趋化因子CXC亚家族,编码区含267bp,编码89个氨基酸残基多肽.CXCR4为SDF一1受体,高度保守,a螺旋跨膜7次,由352个氨基酸组成,在人体内,编码基因位于人染色体2q21,有一个胞外N端,3个胞内环,3个胞外环和1个胞内C端,SDF—l与CXCR4的N端结合,并与CX—CR4和第二胞外环ECI_2(secondextracelluarloop)相互作用才能启动下游信号通路.2SDF一1/CXCR4生物学意义2.1与HW病毒感染的关系CXCR4为嗜T细胞性SI株辅助受体,能与CD4协同作用,参与CD4抗原与HW表面糖蛋白gpl20结合介导病毒吸附侵入的过程,CXCR4的N端结构参与病毒结合,有多个CXCR4结构域特别是第二细胞外环结构能与HⅣ相互作用.趋化因子与受体的结合能阻断受体与HⅣ的结合位点防治HⅣ进入细胞,同时趋化因子对受体的封闭和下调作用也成为一个有效的防治手段,故趋化因子及其衍生物是辅助受体拮抗剂主要成分之一.Fig1Representationof313modelforSDF—ld(NMR}围1SDF一1n的3D模型除了SDF一1的衍生物,还发现CXCR4的其他抑制剂,如:AMD一3100通过与CXCR4的第二膜外环的负电荷区域结合,成为迄今为止与CXCR4结合最有效的非肽类抑制剂.2.2与造血细胞的关系2.2.1SDF一1对造血于/祖细胞增殖和分化的影响Lataillade等【J采用无血清培养基在CD34细胞体外培养中发现,SDF—la对造血干细胞有刺激其增殖的作用,并且与其他造血细胞生长因子(HCF)如干细胞因子(SCF)及白介素一3(IL一3)有协调作用,它还能促进更多的CD34细胞进入细胞周期,使s期和C2期/M期细胞明显多于对照组.2.2.2SDF一1介导造血干/祖细胞的动员过程研究表明,G—SCF动员外周血时,多种蛋白水解酶作用于胞外基质,粘附因子,细胞因子及趋化因子解除细胞与细胞之间形成的紧密结合,促进造血干细胞收稿日期:2005—05—08;修回日期:2005—08—04作者简介:储子彦(1982一),女,汉族,本科生;通讯作者:陈晓萍(1961一),女.汉族,副教授,博士,研究方向为趋化因子作用的分子机制,E—mail:ch)66@.基金项目:国家人事部留学择优基金和浙江省教育厅基金资助项目,项目号~20040586第23卷第1期2006年2月生物学杂志JOURNALOFBIOLOGYV o1.23No.1Feb,2006跨越内皮细胞的转运[21,裂解SDF一1及HSC表面的CXCR4的N端,使HSC失去对SDF一1的趋化作用.骨髓动员的鼠中,骨髓SDF一1浓度会下降,直接导致体内丝氨酸蛋白酶的积累,后者直接引起骨髓造血干/祖细胞的动员[3l.Fig2Cartoonrepresentationof3DmodelforCXCR4receptor图2CXCR4的3D模型图2.2.3对造血干/祖细胞的归巢的影响2.2.3.1SDF一1/CXCR4介导造血干/祖细胞的趋化转移效应来源于骨髓,脐血,动员外周血中的CD34造血干细胞表面表达SDF一1受体CXCR4,而骨髓基质表达的SDF一1能特异对CXCR4产生趋化作用【.因此表达CXCR4的造血干细胞就能够沿着SDF一1的浓度梯度迁移实现归巢过程.CXCR4抗体孵育后的干细胞无法实现归巢,诱导CXCR4在CD34上的表达,能提高造血干l细胞移植成功率.Benb0ubke一5J在研究造血干细胞活动能力与SDF一1基因多态性之间相关性时发现SDF一13'A等基因存在时唯一能预测CD34细胞具有良好动员能力的因素.2.2.3.2SDF一1/CXCR4诱导CD34细胞穿越内层黏附于骨髓基质的作用对CD34细胞进行趋化活性试验发现,SDF一1能引起造血干/祖细胞迁移率增20~1[6].人的骨髓内皮细胞连续表达SDF一1,SDF—l吸附在内皮细胞表面的蛋白多糖上,结合CD34细胞表面的CXCR4捕获造血干/祖细胞,在黏附分子参与下. HSC与内皮细胞产生黏附,SDF—la可增强HSPC与骨12髓基质的黏附作用,这与SDF—la的聚糖和葡萄糖胺聚糖的特性有关.2.3SDF一1/CXCR4在免疫功能上的作用SDF一1/CXCR4是B细胞生长发育成熟的关键细胞因子,骨髓中前B细胞形成需要CXCR4,而胚胎期SDF一1缺乏会损害胎肝前B细胞的发育【.研究发现,SDF一1缺乏鼠的淋巴细胞和骨髓系细胞不能正常发育,由于SDF一1和CxcR4对T细胞,单核细胞的迁移及B淋巴细胞生成中的生物学效应,可能在免疫监视过程中也发挥重要生理功能,在炎症反应中,白细胞向炎症部位集中也与此有关.利用双室模型中研究发现SDF一1的趋化活性较其他趋化因子高1O倍,局部有放射状细胞积聚现象,并观察其趋化活性有浓度梯度依赖性,SDF一1是CD4T细胞活化的共刺激因子,在类风湿性关节炎病变的滑膜中有CD4T细胞的积聚现象,提示SDF一1在免疫及局部炎症过程中是一个重要的调节因子.2.4SDF一1/CXCR4维持胚胎发育敲除小鼠SDF一1基因的2个等位基因,小鼠出生后即死亡,有B细胞增殖,骨髓细胞发育,神经系统发育受阻以及室间隔缺损等缺陷,基因敲除CXCR4小鼠与基因敲除SDF一1小鼠有几乎相同的表现,说明SDF 一1/CXCR4在胚胎发育过程中具有非常重要的作用.2.5sDF一1/CXCR4与肿瘤转移的关系很多研究报告指出,CXCR4在肿瘤细胞高度表达,而SDF一1在某器官的高浓度表达代表肿瘤细胞首先转移的目的地,实验证明CXCR4的抗体有效地阻断了肿瘤细胞向肺部转移的过程,可见SDF一1/CXCR4在肿瘤细胞扩散转移中起到重要作用.CXCR4是肿瘤细胞上很常见的趋化因子受体,同时是其他治疗癌症中的调节因子,例如血管内皮生长因子(VEGF)能诱导CXCR4大量表达,因此ⅦCF抗体能通过抑制CXCR4表达,达到控制肿瘤细胞转移的作用.已发现AMD3100为CXCR4的特异性拮抗剂,通过抑制CXCR4/SDF一1的结合,抑制肿瘤细胞的定向转移,另外,在乳腺癌患者中雌激素受体(ER)表达明显增高,雌激素的真正标靶为SDF—l,能迅速诱导SDF—hx,SDF一1l3的产生,这种效应可被纯ER拮抗剂ICⅡ82780拮抗.3SDF—l/CxCR4的信号转导SDF—l与CXCR4结合,激活了CxCR4受体耦联的c蛋白,通过激活磷脂酰激醇一3激酶(PI3),丝裂原结合蛋白酶(mitogen—associatedproteinkinase,MAPK)和第23卷第1期2006年2月生物学杂志JOURNALOFBIOLOGYV0J.23No,lFeb,2006转录因子NF—KB,为SDF一1/CxCR4一PI3一~LAJPK—NF一皿信号通路,具体如下:(1)SDF一1刺激可使PI一3激酶P85亚单位及PLC一了酪氨酸磷酸化,PLC一可激活PKC,PKC进一步激化一系列局部黏附蛋白,从而介导细胞迁移.SDF一1诱导的细胞趋化可被PI一3激酶抑制剂wortmannin和PKC抑制剂GF109203X所阻断.(2)SDF一1刺激引起核转录因子NF—B活化,支持SDF一1/CXCR4在细胞,增殖及维持生存中发挥作用. (3)SDF一1刺激还经MEK(MAPKandERKkinase)激活P44/42ERK(ExtraceUnlarSignal—regulatedki—nase),但不能激活p38或JNKMA_PK(Mito—activated proteinkinase).SDF一1诱导的细胞也不能被MEK抑制剂PD98059阻断,考虑MEK可能在SDF一1/CXCR4诱导的细胞内信号转导中起调节作用.4结束语SDF一1和CXCR4的特异性结合及其所启动的下游信号通路在细胞的胚胎发育,造血,免疫及HIV治疗上具有重要的意义,对具体的特异性识别过程及调控环节的详尽研究,将有助于我们深入理解细胞问相互作用以及人体中正常的生理功能,并有助于人们开发各种抑制齐IJ运用于临床治疗.参考文献:[1]LatailladeJJ,ClayD,DupuyC,cta1.ChemoHneSDF一1enhanc~ clreulatlngCD34cellprolifemdoninsynergywithcy[0nespossi—bleroleinrogenitorsurvival[JJ.Blood,2000,95:756—768.[2]Va&yGG,Lidey0.Extracelhlarmatrixmot/ct/s,Cytokines,anden—zymes,dymmiceffectsonimmunecellbe|mviorandinflammation [J].JI_eukecBiol,2000,67:149—159.[3]LevesqueJP,HendyT,TakamatsuY,eta1.DisruptionoftheCX—CR4/CXCLRchemotacticinteractionduringhematopoictiestemcell mobilizationinducedbyG—SCForcyclophosphamidelJJ.JElla Invest,2003,111:187—196.[4]WrightDE,BowtnanEP,WagersAJ,eta1.Hematopoictic8[eIilceils areuniquelyselectiveintheirmigratoryresponsetochemokines[JJ. JExpMed,2002,195:1145～1154[5]BenboubkerL,WarierH,CationA,eta1.BrJHaematol,2001,ll3 (1):247.[6]NaiyerAL.JodY,AhnJ,eta1.Stromalderivedfactor一1induced clleImnesofcordbloodCD34cells(1ongtermcuhttreinitiatingcells)th~ue,hendothelialcellsismediatesbyE一~lecfin[J】.Blood,1999,94:柏11一柏19.[7]Fe~T,KawabataK,KawamotoH,eta1.TheearlieststagesofB celldevelopmentrequireachemokinestromalcell—derivedfactor/pm—B础growth—stimulatingfactor[J].Immunity,2001,15(2):323334.ProgressesofchemokineSDF一1andCXCR4CHUZi—yah,CHENXiao—ping,FANGJing—jing (ZhejiangUniversityofTechnology,BiologicalandEnvimnmentalEngineeringCollege,H angzhou310014,China)Abstract:Chemokineisagroupofsmallmolecularproteinswithsimilarilyprimarystructure andchemotaxisactivity.Re—cendyresearchshowedtheirimportantregulatingeffectsonembryonicdevelopment,vascul ogenesis,inflammation,tumor,AIDSandmanypathologicprocess.Thederivantofchemokineoritsinhibitorsshowdgoodclinical applicationprospect.Nearlyfuture, chemokinesandtheirreceptorswillbethetherapeuticmoleculartargettodiseases. Keywords:chemokine;stromalcell—derivedfactor一1;CXCreceptor4(上接7页)[17]张伟.滚筒式填料筛板萃取器反胶团法萃取蛋白质[J]化工,2000.12(增刊):227～231.[18]赵雪雁,佟晓冬,孙颜.StreamlineSP膨胀床纯化溶菌酶[J].过程工程2004,4(2):104—110[19]MamanoJGSandTsotsisTT.CatalyticMembranesandMembraaeReactors[M].Wiley—VCHVerlagGmbH,WeinheimPress,2002. ProgressesofresearchonthepurificationandseparationofthelysozymeFENGOuan.NGBin(AnhuiUniversityofTechnologyandScience,Wuhu241000,China)Abstract:Recentresearchprogressesonthepurificationandseparationofthelysozymewerereviewed.suchasion—ex—changechromatography,ultrafiltmtion,reversedmicellarextraction,affinity—basedbioseparationandsoon.Atthesadietime,de. velopmentprospectsofwaysofthepurificationandseparationofthelysozymewerediscusse d-Keywords:lysozyme;purification;separation13。