SDF-1及其受体CXCR4在结直肠癌组织中的表达及与PCNA的关系

目的:研究结直肠癌组织中基质细胞衍生因子(SDF-1)及其受体(CXCR4)的表达与结直肠癌生物学行为以及与核增殖抗原(PCNA)间的关系,以探讨SDF-1/CXCR4的表达以及PCNA在结直肠癌侵袭、转移中的生物学意义.方法:在结直肠癌组织芯片上,应用免疫组织化学Envision法检测SDF-1/CXCR4和PCNA的表达水平,分析SDF-1、CXCR4和PCNA在结直肠癌的表达与患者淋巴结转移状态、肿瘤分化程度的关系及其相互关系.结果:SDF-1的阳性表达率为60.2%(77/128),CXCR4的阳性表达率为43.8%(56/128).结直肠癌组织SDF-1及CXCR4的阳性表达率均高于癌旁正常黏膜组织(P<0.01).PCNA的阳性表达率为44.5%(57/128).SDF-1/CXCR4和PCNA与患者淋巴结转移状态、肿瘤分化程度相关(均P<0.05),SDF-1/CXCR4与PCNA之间呈正相关(r=0.084,P=0.005;r=0.087,P=0.030).结论:同时检测结直肠癌组织中的SDF-1/CXCR4和PCNA的表达对判断肿瘤的恶性程度和估计预后有一定意义.     

基质细胞衍生因子-1/CXCR4轴与肝脏疾病

基质细胞衍生因子-1(SDF-1)/CXCR4轴及其介导的细胞信号转导通路在肝脏疾病中的作用是国内外研究的热点.研究发现SDF-1/ CXCR4信号转导途径与肝脏再生、炎症、肝硬化以及肿瘤等疾病有关,但其具体机制尚未完全清楚.在细胞微环境中,SDF-1/CXCR4相互作用促进肝癌细胞生长,增强肿瘤的迁移、浸润以及转移能力.本文就SDF-1/CXCR4通路在肝再生、炎症、肿瘤疾病中的病理特征和致病机制的研究进展作一综述.     

Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway

Migration toward pathology is the first critical step in stem cell engagement during regeneration. Neural stem cells (NSCs) migrate through the parenchyma along nonstereotypical routes in a precise directed manner across great distances to injury sites in the CNS, where they might engage niches harboring local transiently expressed reparative signals. The molecular mechanisms for NSC mobilization have not been identified. Because NSCs seem to home similarly to pathologic sites derived from disparate etiologies, we hypothesized that the inflammatory response itself, a characteristic common to all, guides the behavior of potentially reparative cells. As proof of concept, we show that human NSCs migrate in vivo (including from the contralateral hemisphere) toward an infarcted area (a representative CNS injury), where local astrocytes and endothelium up-regulate the inflammatory chemoattractant stromal cell-derived factor 1alpha (SDF-1alpha). NSCs express CXC chemokine receptor 4 (CXCR4), the cognate receptor for SDF-1alpha. Exposure of SDF-1alpha to quiescent NSCs enhances proliferation, promotes chain migration and transmigration, and activates intracellular molecular pathways mediating engagement. CXCR4 blockade abrogates their pathology-directed chain migration, a developmentally relevant mode of tangential migration that, if recapitulated, could explain homing along nonstereotypical paths. Our data implicate SDF-1alpha/CXCR4, representative of the inflammatory milieu characterizing many pathologies, as a pathway that activates NSC molecular programs during injury and suggest that inflammation may be viewed not simply as playing an adverse role but also as providing stimuli that recruit cells with a regenerative homeostasis-promoting capacity. CXCR4 expression within germinal zones suggests that NSC homing after injury and migration during development may invoke similar mechanisms.     

Expression of human immunodeficiency virus coreceptors CXC chemokine receptor 4 and CC chemokine receptor 5 on monocytes is down-regulated during human endotoxemia

Lipopolysaccharide (LPS) can inhibit human immunodeficiency virus (HIV) infection in monocytes in vitro. To test the hypothesis that an LPS effect on CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), known coreceptors for HIV, contributes to this effect, 8 healthy men were intravenously injected with Escherichia coli LPS (4 ng/kg), and monocyte CXCR4 and CCR5 expression was monitored by fluorescence-activated cell sorter analysis. LPS induced a decrease in the fraction of peripheral blood monocytes expressing CXCR4 and CCR5, reaching a nadir after 2 h (both P<.001 vs. baseline). In whole blood in vitro, not only LPS but also lipoarabinomannan (a cell wall component of Mycobacterium tuberculosis) and lipoteichoic acid (a cell wall component of Staphylococcus aureus) down-regulated the expression of CXCR4 and CCR5 on monocytes (all P<.05). Exposure of monocytes to (myco)bacterial agents may render them relatively resistant to infection with HIV by an effect on HIV coreceptors.     

A chemokine, SDF-1/PBSF, and its receptor, CXC chemokine receptor 4, as mediators of hematopoiesis

A chemokine, stromal cell-derived factor/pre-B-cell growth-stimulating factor (SDF-1/PBSF), and its primary physiologic receptor, CXC chemokine receptor 4 (CXCR4), are essential for B-lymphocyte production and colonization of bone marrow by myeloid lineage progenitors during embryogenesis. Moreover, CXCR4 is an autonomous cell essential for long-term lymphoid and myeloid reconstitution in adult bone marrow. Upregulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.     

Rheumatoid fibroblast‐like synoviocytes overexpress the chemokine stromal cell–derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue

Objective

A characteristic feature of the inflammatory infiltrate in rheumatoid arthritis is the segregation of CD4 and CD8 T lymphocyte subsets into distinct microdomains within the inflamed synovium. The aim of this study was to test the hypothesis that chemokines in general and stromal cell–derived factor 1 (SDF‐1; CXCL12) in particular are responsible for generating this distinctive microcompartmentalization.

Methods

We examined how synovial CD4/CD8 T cell subsets interacted in coculture assays with fibroblasts derived from chronic inflammatory synovial lesions and normal synovial tissue as well as from fetal lung and adult skin. We used the ability of T cells to migrate beneath fibroblasts (a process called pseudoemperipolesis) as an in vitro marker of T cell accumulation within synovial tissue.

Results

Rheumatoid fibroblast‐like synoviocytes (FLS) displayed a unique ability to support high levels of CD4 and CD8 T cell pseudoemperipolesis. Nonrheumatoid FLS as well as fetal lung fibroblasts supported low levels of pseudoemperipolesis, while skin‐derived fibroblasts were unable to do so. CD8 T cells migrated under fibroblasts more efficiently and at a higher velocity than CD4 T cells, a feature that was intrinsic to CD8 T cells. Rheumatoid fibroblasts constitutively produced high levels of SDF‐1 (CXCL12), which was functionally important, since blocking studies showed reductions in T cell pseudoemperipolesis to levels seen in nonrheumatoid FLS. Rheumatoid fibroblasts also constitutively produced high levels of vascular cell adhesion molecule 1 (VCAM‐1; CD106), but this did not contribute to T cell pseudoemperipolesis, unlike the case for B cells, which require SDF‐1 (CXCL12)–CXCR4 and CD49d–VCAM‐1 (CD106) interactions. Importantly, only combinations of rheumatoid FLS and rheumatoid‐derived synovial fluid T cells supported pseudoemperipolesis when examined ex vivo, confirming the in vivo relevance of these findings.

Conclusion

These studies demonstrate that features intrinsic to both fibroblasts (the production of SDF‐1) and CD8/CD4 T cells (the expression of CXCR4) are responsible for the characteristic pattern of T lymphocyte accumulation seen in the rheumatoid synovium. These findings suggest that the SDF‐1/CXCR4 ligand/receptor pair is likely to play an important functional role in T lymphocyte accumulation and positioning within the rheumatoid synovium.

     

Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: Molecular modeling and experimental validation

Chemokines and their receptors regulate cell migration during development, immune system function, and in inflammatory diseases, making them important therapeutic targets. Nevertheless, the structural basis of receptor:chemokine interaction is poorly understood. Adding to the complexity of the problem is the persistently dimeric behavior of receptors observed in cell-based studies, which in combination with structural and mutagenesis data, suggest several possibilities for receptor:chemokine complex stoichiometry. In this study, a combination of computational, functional, and biophysical approaches was used to elucidate the stoichiometry and geometry of the interaction between the CXC-type chemokine receptor 4 (CXCR4) and its ligand CXCL12. First, relevance and feasibility of a 2:1 stoichiometry hypothesis was probed using functional complementation experiments with multiple pairs of complementary nonfunctional CXCR4 mutants. Next, the importance of dimers of WT CXCR4 was explored using the strategy of dimer dilution, where WT receptor dimerization is disrupted by increasing expression of nonfunctional CXCR4 mutants. The results of these experiments were supportive of a 1:1 stoichiometry, although the latter could not simultaneously reconcile existing structural and mutagenesis data. To resolve the contradiction, cysteine trapping experiments were used to derive residue proximity constraints that enabled construction of a validated 1:1 receptor:chemokine model, consistent with the paradigmatic two-site hypothesis of receptor activation. The observation of a 1:1 stoichiometry is in line with accumulating evidence supporting monomers as minimal functional units of G protein-coupled receptors, and suggests transmission of conformational changes across the dimer interface as the most probable mechanism of altered signaling by receptor heterodimers.The chemokine receptor CXCR4 regulates cell migration during many developmental processes (1, 2). Along with CCR5, it serves as one of the principal coreceptors for HIV entry into leukocytes (3), and is one of the most important chemokine receptors involved in cancer metastasis (4). Stromal-cell derived factor 1 (SDF-1 or CXCL12) was its only known ligand until recently, when CXCR4 was also shown to bind CXCL14 (5) and extracellular ubiquitin (6). Although structures of CXCR4 (7) and CCR5 (8) have been solved with synthetic antagonists, the structural basis for the interaction of CXCR4 (or any other chemokine receptor) with their natural ligands has yet to be determined. Numerous mutagenesis and NMR studies indicate that receptor:chemokine interactions involve two distinct sites (9–12), which has led to a two-site hypothesis of receptor activation (13). The so-called chemokine recognition site 1 (CRS1) (14) includes the N terminus of the receptor interacting with the globular core of the chemokine, whereas chemokine recognition site 2 (CRS2), located within the transmembrane (TM) domain pocket of the receptor, accommodates the flexible N terminus of the chemokine. Mutations in CRS1 typically reduce the binding affinity of the chemokine, whereas CRS2 is critical not only for binding but also for chemokine-induced activation (9, 10, 12, 15–20). Similarly, mutations to the core domain of the chemokine generally affect receptor-binding affinity, but truncations or modifications of as little as one amino acid in the N-terminal “signaling” domain frequently alter both ligand binding and pharmacology.The two-site model has been envisioned in the context of a monomeric receptor. However, like many other G protein-coupled receptors (GPCRs) (21), CXCR4 has been shown to dimerize in cell membranes. Evidence supporting CXCR4 dimerization includes immunoprecipitation (22), bioluminescence and fluorescence resonance energy transfer [BRET (23) and FRET (24), respectively], fluorescence and luminescence complementation assays (25), and bivalent ligands (26). Dimerization of a WT CXCR4 with a C-terminally truncated mutant causing the “warts, hypogammaglobulinemia, infections and myelokathexis” (WHIM) syndrome has been implicated in its resistance to desensitization and enhanced signaling in heterozygous WHIM patients (27). CXCR4 has also been shown to heterodimerize with other chemokine receptors and with GPCRs outside the chemokine family (28–32), with consequences including transinhibition of ligand binding (28) and changes in G protein and β-arrestin coupling (30, 33, 34). These observations establish the dimeric nature of CXCR4; however, the functional role of CXCR4 dimers has yet to be elucidated.In agreement with its persistently dimeric behavior, CXCR4 formed structurally similar parallel dimers in five crystal structures (7), despite being solved in different space groups and with different synthetic ligands. The cell-based and structure-based observations of CXCR4 dimers raised the key question as to whether CXCL12 binds to a single receptor subunit or to both subunits of the dimer, in a manner consistent with the two-site model. Several possible stoichiometries of the complex were suggested (7, 35, 36); among them, a 1:1 receptor:chemokine stoichiometry, a 2:1 stoichiometry with one chemokine molecule simultaneously binding to both subunits of a CXCR4 dimer, and a 2:2 stoichiometry with a chemokine dimer binding to the CXCR4 dimer. With respect to the latter, although CXCL12 dimers bind and act as partial agonists of CXCR4 (37), full agonist signaling requires a monomeric chemokine (37, 38). Consequently, the distinction between a 1:1 and a 2:1 receptor:chemokine stoichiometry is the most relevant question, and constituted the focus of the present study.Our initial molecular modeling efforts encompassed the available structural information in the form of (i) the NMR structure of a cross-linked CXCL12 dimer in complex with an N-terminal peptide of CXCR4 (residues M1-K38) (39), and (ii) the X-ray structures of full-length CXCR4 (7). The former structure contains components of the CRS1 interaction (Fig. 1A), whereas the latter contains the receptor side of the CRS2 interaction. Although the crystallization constructs used in the CXCR4 X-ray study contained the intact N terminus of the receptor, only residues P27–S319 could be detected in the electron density; thus, the overlap between the NMR and X-ray structures was limited to residues P27–K38. Modeling demonstrated that a 2:1 receptor:chemokine model with decoupled CRS1 and CRS2 best accommodated the structural and mutagenesis data. In this model, the globular core of the chemokine interacts with the CRS1 of one receptor subunit and the N-terminal residues of the chemokine reach into CRS2 of its dimeric partner (Fig. 1B). In addition to being spatially consistent, this model provides a direct explanation for the negative cooperativity in chemokine binding that is frequently observed with receptor heterodimers (28, 40), and with the notion that CXCL12 triggers CXCR4 dimerization (41), stabilizes preformed dimers (42), or induces conformational changes within the dimers (23). The model is also consistent with the original two-site hypothesis of receptor activation. On the other hand, a 1:1 model, in which CXCL12 interacts with CRS1 and CRS2 of the same receptor subunit, required significant deviations from the CRS1 component of the CXCR4:CXCL12 interaction suggested by the NMR structure (39) to orient the CXCL12 N-terminal signaling domain toward the receptor binding pocket (Fig. 1 C and D).Open in a separate windowFig. 1.Molecular models and experimental designs used in the present study. (A) NMR structure of CXCL12 (skin mesh) in complex with the N terminus of CXCR4 (residues M1–K38, ribbon) (39). Chemokine N terminus (green) and N-loop (blue) correspond to the expected interactions in CRS2 and CRS1, respectively. Receptor residues K25–R30 are shown as spheres, labeled, and colored in order from blue to red. CRS1 residue proximities observed in the NMR structure and maintained throughout the docking simulations include the interaction of CXCR4 K25 (blue sphere) with CXCL12 S16, while the subsequent receptor residues up to R30 (red sphere) are directed away from the chemokine N-loop (blue surface) toward the chemokine C-terminal helix; these proximities are shown as thin black lines. (B) A hybrid 2:1 model of the receptor:chemokine interaction accommodates both NMR proximity restraints (black lines) and the mutagenesis data. (C) A hydrid 1:1 model that accommodates NMR proximity restraints (black lines) is inconsistent with mutagenesis and with the two-site interaction hypothesis, because the N terminus of the chemokine invariably points away from the receptor CRS2. (D) A 1:1 model consistent with the two-site interaction hypothesis contradicts NMR proximity restraints, as receptor residues K25–R30 are directed along the chemokine N-loop toward its N terminus. (E–G) Conceptual designs of the functional complementation (E), dimer dilution (F), and cysteine trapping (G) experiments used in this study to probe the receptor:chemokine stoichiometry and geometry hypotheses.Three strategies were devised to elucidate the stoichiometry of the receptor:chemokine interaction. The first approach was based on functional complementation and designed to specifically probe the relevance of the 2:1 hypothesis. Functional complementation provides one of the strongest arguments for the existence and physiological role of GPCR dimers. In this type of experiment, different aspects of receptor function are restored by coexpression of two mutants of the receptor in question, each of which is incapable of producing the functional response when expressed alone (Fig. 1E). Functional rescue through dimerization has been demonstrated for several GPCRs. For example, domain swapping of histamine H1 receptor dimers reconstituted functional receptors from nonfunctional mutant components (43), and a related mechanism led to reconstitution of functional muscarinic and adrenergic receptors from receptor chimeras (44). Similarly, the binding site in the angiotensin II receptor was successfully reconstituted (45), and the function of the luteinizing hormone receptor was rescued by coexpression of two nonfunctional mutants (46). In the present study, the functional complementation strategy was used to probe the possibility of simultaneous interaction of CXCL12 with two CXCR4 monomers in the dimer.Another strategy for exploring the role of dimers in general, and the stoichiometry of GPCR interactions with ligands and effectors in particular, is based on dimer dilution. In this approach, functional responses or binding events that are dependent on GPCR dimers are reduced or completely ablated by introducing increasing amounts of a mutant that is capable of dimerizing with the WT receptor but incapable of mediating the functional or binding response (Fig. 1F). The mutant receptors compete with WT receptors for dimer formation and lead to an increase in the surface density of WT/mutant dimers, with a simultaneous decrease in WT/WT dimers (47). In contrast, if dimers are unnecessary for the functional response or binding event, one should see no change with increasing concentration of mutant; thus, this approach distinguishes 1:1 vs. 2:1 interactions. There are important caveats associated with this strategy, as increasing expression of mutant receptors may interfere not only with formation of WT/WT dimers, but also with the expression of WT receptors because of expression competition. In this study, a modified dimer dilution strategy that addressed these problems was designed.The above strategies are based on functional readouts and provide indirect evidence in favor of, or against, the different stoichiometries. We therefore complemented them by cysteine trapping studies, where pairs of cysteine mutations are introduced at different positions in the ligand and in the receptor, and spontaneous formation of disulfide bonds is monitored. These studies provide direct spatial proximity restraints that can be combined with modeling to determine the stoichiometry and geometry of the receptor:chemokine complex (Fig. 1G).The results of all three complementary experimental strategies were supportive of a 1:1 and not a 2:1 receptor:chemokine stoichiometry. These results also informed further molecular modeling efforts, which led to construction of an experimentally validated model of the CXCR4:CXCL12 complex. The model elucidates key features of the receptor:chemokine interaction and may facilitate further structure-function studies to understand the molecular basis for CXCR4:CXCL12 signaling.     

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.