CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1beta or TNF-alpha. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1alpha (SDF-1alpha) indicated that the CXCR4 receptors were functional. Incubation with SDF-1alpha resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene alpha. RPE cells also migrated in response to SDF-1alpha. As SDF-1alpha expression by RPE cells was detected constitutively, we postulate that SDF-1-CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.     

Hypoxia enhances CXCR4 expression favoring microglia migration via HIF-1alpha activation

Migration toward pathological area is the first critical step in microglia engagement during the central nervous system (CNS) injury, although the molecular mechanisms underlying microglia mobilization have not been fully understood. Here, we report that hypoxia promotes stromal cell-derived factor-1α (SDF-1α) induced microglia migration by inducing the CXC chemokine receptor 4 (CXCR4) expression. Exposure to hypoxia significantly enhanced CXCR4 expression levels in N9 microglia cell. Then, cell migration induced by SDF-1, a CXCR4-specific ligand, was observed accelerated. Blockade of hypoxia inducible factor-1α (HIF-1α) activation by inhibitors of phosphoinositide-3-kinase (PI3K)/Akt signaling pathway abrogated both of hypoxia-induced CXCR4 up-regulation and cell-migration acceleration. These results point to a crucial role of Hypoxia-HIF-1α-CXCR4 pathway during microglia migration.     

Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is characterized by the accumulation of CD4(+) memory T cells in the inflamed synovium. To address the mechanism, we analyzed chemokine receptor expression and found that the frequency of CXC chemokine receptor (CXCR)4 expressing synovial tissue CD4(+) memory T cells was significantly elevated. CXCR4 expression could be enhanced by IL-15, whereas stromal cell-derived factor (SDF)-1, the ligand of CXCR4, was expressed in the RA synovium and could be increased by CD40 stimulation. SDF-1 stimulated migration of rheumatoid synovial T cells and also inhibited activation-induced apoptosis of T cells. These results indicate that SDF-1-CXCR4 interactions play important roles in CD4(+) memory T cell accumulation in the RA synovium, and emphasize the role of stromal cells in regulating rheumatoid inflammation.     

The chemokine SDF-1/CXCL12 regulates the migration of melanocyte progenitors in mouse hair follicles

Mouse skin melanocytes originate from the neural crest and subsequently invade the epidermis and migrate into the hair follicles (HF) where they proliferate and differentiate. Here we demonstrate a role for the chemokine SDF-1/CXCL12 and its receptor CXCR4 in regulating the migration and positioning of melanoblasts during HF formation and cycling. CXCR4 expression by melanoblasts was upregulated during the anagen phase of the HF cycle. CXCR4-expressing cells in the HF also expressed the stem cell markers nestin and LEX, the neural crest marker SOX10 and the cell proliferation marker PCNA. SDF-1 was widely expressed along the path taken by migrating CXCR4-expressing cells in the outer root sheath (ORS), suggesting that SDF-1-mediated signaling might be required for the migration of CXCR4 cells. Skin sections from CXCR4-deficient mice, and skin explants treated with the CXCR4 antagonist AMD3100, contained melanoblasts abnormally concentrated in the epidermis, consistent with a defect in their migration. SDF-1 acted as a chemoattractant for FACS-sorted cells isolated from the anagen skin of CXCR4–EGFP transgenic mice in vitro, and AMD3100 inhibited the SDF-1-induced migratory response. Together, these data demonstrate an important role for SDF-1/CXCR4 signaling in directing the migration and positioning of melanoblasts in the HF.     

Leukocyte-endothelium interaction promotes SDF-1-dependent polarization of CXCR4

Chemokine-driven migration is accompanied by polarization of the cell body and of the intracellular signaling machinery. The extent to which chemokine receptors polarize during chemotaxis is currently unclear. To analyze the distribution of the chemokine receptor CXCR4 during SDF-1 (CXCL12)-induced chemotaxis, we retrovirally expressed a CXCR4-GFP fusion protein in the CXCR4-deficient human hematopoietic progenitor cell line KG1a. This KG1a CXCR4-GFP cell line showed full restoration of SDF-1 responsiveness in assays detecting activation of ERK1/2 phosphorylation, actin polymerization, adhesion to endothelium under conditions of physiological flow, and (transendothelial) chemotaxis. When adhered to cytokine-activated endothelium in the absence of SDF-1, CXCR4 did not localize to the leading edge of the cell but was uniformly distributed over the plasma membrane. In contrast, when SDF-1 was immobilized on cytokine-activated endothelium, the CXCR4-GFP receptors that were present on the cell surface markedly redistributed to the leading edge of migrating cells. In addition, CXCR4-GFP co-localized with lipid rafts in the leading edge of SDF-1-stimulated cells, at the sites of contact with the endothelial surface. Inhibition of lipid raft formation prevents SDF-1-dependent migration, internalization of CXCR4, and polarization to the leading edge of CXCR4, indicating that CXCR4 surface expression and signaling requires lipid rafts. These data show that SDF-1, immobilized on activated human endothelium, induces polarization of CXCR4 to the leading edge of migrating cells, revealing co-operativity between chemokine and substrate in the control of cell migration.     

Clearing the path for germ cells

The chemokine SDF-1a and its receptor CXCR4b guide germ cell migration in zebrafish by activating downstream signaling events. Boldajipour et al. (2008) now report that a second SDF-1a receptor, CXCR7, is also required for guided migration but does not function as a signaling receptor, and instead sequesters SDF-1a. These results highlight the importance of ligand clearance during guided cell migration.     

DNA-binding specificity and embryological function of Xom (Xvent-2)

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.     

Long-chain glucosylceramides crosstalk with LYN mediates endometrial cell migration

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYN^pTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYN^pTyr396 axis in endometriosis.     

趋化因子SD-1及其受体CXCR4在卵巢癌中的研究进展

基质细胞衍生因子-1(Stromal cell derived factor-1,SDF-1)是CXC趋化因子家族的重要成员,系统命名为CXCL12,能与它的唯一受体CXC趋化因子受体-4(CXC chemokine receptor-4,CXCR4)形成CXCL12-CXCR4生物学轴,CXCL12-CXCR4生物学轴在肿瘤生长、侵袭、转移过程中发生重要作用。到目前为止,已发现CXCL12-CXCR4在卵巢癌、胰腺癌、肝癌等多种肿瘤组织中表达。然而,国内目前还没有关于CXCL12-CXCR4与卵巢癌关系的相关综述,本文将从趋化因子CXCL12及其受体CXCR4,CXCL12/CXCR4轴与卵巢癌细胞系实验研究,CXCL12-CXCR4轴与卵巢癌的临床研究,CXCL12/CXCR4与卵巢癌预后,CXCL12/CXCR4与卵巢癌治疗展望等五个方面对CXCL12-CXCR4生物轴与卵巢癌的关系,及其在卵巢癌治疗中的应用展开综述。     

Persistent induction of the chemokine receptor CXCR4 by TGF-beta 1 on synovial T cells contributes to their accumulation within the rheumatoid synovium

Chemokines and their receptors determine the distribution of leukocytes within tissues in health and disease. We have studied the role of the constitutive chemokine receptor CXCR4 and its ligand, stromal-derived factor-1 (SDF-1) in the perivascular accumulation of T cells in rheumatoid arthritis. We show that synovial T cells, which are primed CD45RO+CD45RBdull cells and consequently not expected to express constitutive chemokine receptors, have high levels of the chemokine receptor CXCR4. Sustained expression of CXCR4 was maintained on synovial T cells by specific factors present within the synovial microenvironment. Extensive screening revealed that TGF-beta isoforms induce the expression of CXCR4 on CD4 T cells in vitro. Depletion studies using synovial fluid confirmed an important role for TGF-beta1 in the induction of CXCR4 expression in vivo. The only known ligand for CXCR4 is SDF-1. We found SDF-1 on synovial endothelial cells and showed that SDF-1 was able to induce strong integrin-mediated adhesion of synovial fluid T cells to fibronectin and ICAM-1, confirming that CXCR4 expressed on synovial T cells was functional. These results suggest that the persistent induction of CXCR4 on synovial T cells by TGF-beta1 leads to their active, SDF-1-mediated retention in a perivascular distribution within the rheumatoid synovium.     

The role of CXCR7 on the adhesion, proliferation and angiogenesis of endothelial progenitor cells

Previous studies confirmed that stromal cell-derived factor 1 (SDF-1) was a principal regulator of retention, migration and mobilization of haematopoietic stem cells and endothelial progenitor cells (EPCs) during steady-state homeostasis and injury. CXC chemokine receptor 4 (CXCR4) has been considered as the unique receptor of SDF-1 and as the only mediator of SDF-1-induced biological effects for many years. However, recent studies found that SDF-1 could bind to not only CXCR4 but also CXC chemokine receptor 7 (CXCR7). The evidence that SDF-1 binds to the CXCR7 raises a concern how to distinguish the potential contribution of the SDF-1/CXCR7 pathway from SDF-1/CXCR4 pathway in all the processes that were previously attributed to SDF-1/CXCR4. In this study, the role of CXCR7 in EPCs was investigated in vitro. RT-PCR, Western blot and flow cytometry assay demonstrate that both CXCR4 and CXCR7 were expressed highly in EPCs. The adhesion of EPCs induced by SDF-1 was inhibited by blocking either CXCR4 or CXCR7 with their antibodies or antagonists. SDF-1 regulated the migration of EPCs via CXCR4 but not CXCR7. However, the transendothelial migration of EPCs was inhibited by either blocking of CXCR4 or CXCR7. Both CXCR7 and CXCR4 are essential for the tube formation of EPCs induced by SDF-1. These results suggested that both CXCR7 and CXCR4 are important for EPCs in response to SDF-1, indicating that CXCR7 may be another potential target molecule for angiogenesis-dependent diseases.     

Interaction of ligand-receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis

Interaction of ligand-receptor systems between stromal-cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) is closely involved in the organ specificity of cancer metastasis. We hypothesized that SDF-1-CXCR4 ligand-receptor system plays an important role in prostate cancer metastasis. To test this hypothesis, expression level of SDF-1 and CXCR4 was analyzed in prostate cancer (PC) cell lines (LNCaP, PC3, and DU145) and normal prostate epithelial cell line (PrEC). We also performed migration assay and MTT assay to investigate the chemotactic effect and growth-promoting effect of SDF-1 on DU145 and PC3 cells, respectively. Furthermore, we performed immunohistochemical analysis of CXCR4 expression in tissues from 35 cases of human prostate cancer. CXCR4 expression was detected in all three prostate cancer cell lines, but not in PrECs. SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner, and anti-CXCR4 antibody inhibited this chemotactic effect. However, SDF-1 itself did not significantly stimulate the cell growth rate of prostate cancer cell lines. Positive CXCR4 protein was found in 20 out of 35 clinical PC samples (57.1%). Three patients with lung metastasis showed definitely positive CXCR4 immunostaining. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0.05). Furthermore, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P = 0.017). These findings suggest that the interaction between SDF-1 and CXCR4 ligand-receptor system is involved in the process of PC metastasis by the activation of cancer cell migration. This is the first report to investigate the role of interaction of ligand-receptor systems between SDF-1 and CXCR4 in prostate cancer metastasis.     

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

The chemokine SDF-1alpha suppresses fibronectin-mediated in vitro lymphocytes adhesion

Stromal cell-derived factor (SDF-1) is a CXC chemokine that selectively activates the CXCR4 chemokine receptor. Fibronectin is an intracellular matrix component that binds integrin and mediates cell-matrix adhesion. Activation of the integrin receptor can occur in two ways: by ligand binding (outside-in signaling), and in response to intracellular events (inside-out signaling). In the current study we showed that SDF-1a inhibited adhesion of T lymphocyte Jurkat cells resulting from binding high concentrations of fibronectin as well as that of THP-1 monocytes. The effect of SDF-1a on fibronectin-mediated adhesion was partly reversed by the CXCR4 receptor antagonist T140. Our results suggest that an SDF-1/ CXCR4 signal pathway modulates fibronectin-mediated lymphocytes adhesion.     

Ischemic brain extract increases SDF-1 expression in astrocytes through the CXCR2/miR-223/miR-27b pathway

Ischemic cerebral stroke is one of the leading global causes of mortality and morbidity. Ischemic preconditioning (IPC) refers to a sublethal ischemia and resulting in tolerance to subsequent severe ischemic injury. Although several pathways are reportedly involved in IPC-mediated neuroprotection, the functional role of astrocytes is not fully understood. Stromal cell-derived factor-1 (SDF-1), a CXC chemokine produced mainly in astrocytes, is a ligand for chemokine receptor CXCR4. SDF-1 is reported to play a critical role in neuroprotection after stroke by mediating the migration of neuronal progenitor cells. We hypothesized that stimuli derived from ischemic brain were involved in the protective effects of IPC. To investigate this hypothesis, the mechanism in which ischemic brain extract (IBE) induced SDF-1 expression was investigated in C6 astrocytoma cells. IBE treatment of C6 cells increased SDF-1 expression compared to that in untreated or normal brain extract (NBE)-treated cells by downregulating SDF-1 targeting miRNA, miR-27b. MiR-223 was inversely upregulated in IBE-treated cells; overexpression of miR-223 decreased the expression of miR-27b by suppressing IKKα expression. Analysis of cytokine array data revealed an IBE associated enhanced expression of CINC-1 (CXCL1) and LIX1 (CXCL5). Knockdown or inhibition of their receptor, CXCR2, abolished IBE-mediated increased expression of SDF-1. These results were confirmed in primary cultured astrocytes. Taken together, the data demonstrate that IBE-elicited signals increase SDF-1 expression through the CXCR2/miR-223/miR-27b pathway in C6 astrocytoma cells and primary astrocytes, supporting the view that increased expression of SDF-1 by ischemic insults is a possible mechanism underlying therapeutic application of IPC.     

SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4

Human colonic epithelial cells express CXCR4, the sole cognate receptor for the chemokine stromal cell-derived factor (SDF)-1/CXC chemokine ligand (CXCL) 12. The aim of this study was to define the mechanism and functional consequences of signaling intestinal epithelial cells through the CXCR4 chemokine receptor. CXCR4, but not SDF-1/CXCL12, was constitutively expressed by T84, HT-29, HT-29/-18C1, and Caco-2 human colon epithelial cell lines. Studies using T84 cells showed that CXCR4 was G protein-coupled in intestinal epithelial cells. Moreover, stimulation of T84 cells with SDF-1/CXCL12 inhibited cAMP production in response to the adenylyl cyclase activator forskolin, and this inhibition was abrogated by either anti-CXCR4 antibody or receptor desensitization. Studies with pertussis toxin suggested that SDF-1/CXCL12 activated negative regulation of cAMP production through G(i)alpha subunits coupled to CXCR4. Consistent with the inhibition of forskolin-stimulated cAMP production, SDF-1/CXCL12 also inhibited forskolin-induced ion transport in voltage-clamped polarized T84 cells. Taken together, these data indicate that epithelial CXCR4 can transduce functional signals in human intestinal epithelial cells that modulate important cAMP-mediated cellular functions.     

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.     

Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways

Human mesenchymal stem cells (hMSCs) have been used for cell-based therapies in degenerative disease and as vehicles for delivering therapeutic genes to sites of injury and tumors. Recently, umbilical cord blood (UCB) was identified as a source for MSCs, and human UCB-derived MSCs (hUCB-MSCs) can serve as an alternative source of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, migration signaling pathways required for homing and recruitment of hUCB-MSCs are not fully understood. Stromal cell-derived factor-1 (SDF-1), a ligand for the CXCR4 chemokine receptor, plays a pivotal role in mobilization and homing of stem cells and modulates different biological responses in various stem cells. In this study, expression of CXCR4 in hUCB-MSCs was studied by western blot analysis and the functional role of SDF-1 was assessed. SDF-1 induced the migration of hUCB-MSCs in a dose-dependent manner. The induced migration was inhibited by the CXCR4-specific peptide antagonist (AMD3100) and by inhibitors of phosphoinositide 3-kinase (LY294002), mitogen-activated protein kinase/extracellular signal related kinase (PD98059) and p38MAPK inhibitor (SB203580). hUCB-MSCs treated with SDF-1 displayed increased phosphorylation of Akt, ERK and p38, which were inhibited by AMD3100. Small-interfering RNA-mediated knock-down of Akt, ERK and p38 blocked SDF-1 induced hUCB-MSC migration. In addition, SDF-1-induced actin polymerization was also blocked by these inhibitors. Taken together, these results demonstrate that Akt, ERK and p38 signal transduction pathways may be involved in SDF-1-mediated migration of hUCB-MSCs.