Molecular dynamics simulations on SDF-1alpha: binding with CXCR4 receptor

Insights into the interacting mode of CXCR4 with SDF-1alpha are crucial in understanding the structural and functional characteristics of CXCR4 receptor. In this paper a computational pipeline, integrating protein structure prediction, molecular dynamics simulations, automated molecular docking, and Brownian dynamics simulations were employed to investigate the dynamic and energetic aspects of CXCR4 associating with SDF-1alpha. The entire simulation revealed the surface distribution feature of electrostatic potentials and conformational "open-close" process of the receptor. The possible binding conformation of CXCR4 was identified, and the CXCR4-SDF-1alpha binding complex was generated. Arg188-Glu277 salt bridge plays an important role for both the extracellular domain conformational change and SDF-1alpha binding. Two binding sites were mapped at the extracellular domain (Site 1) and inside the transmembrane domain (Site 2), which are composed of conserved residues. Sites 1 and 2 contribute approximately 60% and 40% to the binding affinity with SDF-1alpha, respectively. The binding model is in agreement with most of the experimental data. Transmembrane VI has more significant motion in the harmonious conformational transition of CXCR4 during SDF-1alpha binding, which may be possibly associated with signal transduction. Based on the modeling and simulation, a binding mechanism hypothesis between CXCR4 and SDF-1alpha and its relationship to the signal transduction has been proposed.     

Induction of 15-lipoxygenase-1 impairs expression of HIV-1 receptors CD4 and CXCR4 in monocytic cells

The lipoxygenase pathway of immunocompetent cells has been related to HIV infection and interleukins-4 and -13 have been described as major regulators of this metabolic route. To explore whether 15-lipoxygenase1 expression might impact the responsiveness of monocytic cells for HIV we induced expression of this enzyme by two independent ways (stable transfection of U937 cells and culturing of blood monocytes in vitro in the presence of granulocyte/monocyte colony stimulating factor and interleukin 4) and assayed the cellular content of the two HIV-1 receptors CD4 and CXCR4 (CD184) by real time RT-PCR and fluorescence-activated cell sorting. Wild-type U937 cells express CD4 and CXCR4 at high levels and expression was not altered when the cells were transfected with control plasmids. In contrast, expression of these proteins was strongly reduced when the cells were stably transfected with 15-lipoxygenase1. Similar effects were observed when blood monocytes were cultured in the presence of granulocyte/monocyte colony stimulating factor and interleukin-4. Under these conditions strong overexpression of 15-lipoxygenase1 was paralleled by downregulation of CD4 and CXCR4. Since these surface proteins are essential for the binding of T-tropic HIV-1 strains expression of 15-lipoxygenase1 may confer resistance against HIV infection to monocytic cells.     

趋化因子SDF-1及受体CXCR4研究进展

趋化因子(chemokine)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、艾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。     

An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

Inflammatory angiogenesis and vascular remodeling play key roles in the chronic inflammatory skin disease psoriasis, but little is known about the molecular mediators of vascular activation. Based on the reported elevated mRNA levels of the angiogenic chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in psoriasis, we investigated the relevance of the SDF-1/CXCR4 axis in two experimental models of chronic psoriasis-like skin inflammation. The cutaneous expression of both SDF-1 and CXCR4 was upregulated in the inflamed skin of K14-VEGF-A transgenic mice and in imiquimod-induced skin inflammation, with expression of CXCR4 by blood vessels and macrophages. Treatment with the CXCR4 antagonist AMD3100 potently inhibited skin inflammation in both models, associated with reduced inflammatory angiogenesis and inflammatory cell accumulation, including dermal CD4⁺ cells and intraepidermal CD8⁺ T cells. Similar anti-inflammatory effects were seen after treatment with a neutralizing anti-SDF-1 antibody. In vitro, inhibition of CXCR4 blocked SDF-1-induced chemotaxis of CD11b⁺ splenocytes, in agreement with the reduced number of macrophages after in vivo CXCR4 blockade. Our results reveal an important role of the SDF-1/CXCR4 axis in skin inflammation and inflammatory angiogenesis, and they indicate that inhibition of the SDF-1/CXCR4 axis might serve as a novel therapeutic strategy for chronic inflammatory skin diseases.     

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.     

SDF-1/CXCR4 轴在间充质干细胞归巢机制中的研究进展

间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种具有多向分化潜能的成体干细胞,其具有分泌营养物质和调节炎症反应的能力,虽然间充质干细胞在组织修复、重塑和免疫调节方面已得到临床运用,但MSCs趋化和归巢的机制仍不清楚。基质细胞衍生因子-1(stromal cell-derived factor 1,SDF-1)和其趋化因子受体4(C-X-C chemokine receptor 4,CXCR4)在介导MSCs的分化、迁移和归巢中起着至关重要的作用,若能深入探讨、明确其在归巢中的作用,期望给间充质干细胞在临床的应用开辟新的应用前景。     

Leukocyte elastase negatively regulates Stromal cell-derived factor-1 (SDF-1)/CXCR4 binding and functions by amino-terminal processing of SDF-1 and CXCR4

Activation of CXCR4 by the CXC chemokine stromal cell-derived factor-1 (SDF-1) requires interaction of the amino-terminal domains of both molecules. We report that proteinases released from either mononucleated blood cells or polymorphonuclear neutrophils degranulated by inflammatory stimuli generate an SDF-1 fragment that is deleted from amino-terminal residues Lys(1)-Pro(2)-Val(3), as characterized by mass spectrometry analysis. The proteolyzed chemokine fails to induce agonistic functions and is unable to prevent the fusogenic capacity of CXCR4-tropic human immunodeficiency viruses. Furthermore, we observed that exposure of CXCR4-expressing cells to leukocyte proteinases results in the proteolysis of the extracellular amino-terminal domain of the receptor, as assessed by flow cytometry analysis and electrophoretic separation of immunoprecipitated CXCR4. Blockade of SDF-1 and CXCR4 proteolysis by the specific leukocyte elastase inhibitor, N-methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl ketone, identified elastase as the major enzyme among leukocyte-secreted proteinases that accounts for inactivation of both SDF-1 and CXCR4. Indeed, purified leukocyte elastase generated in either SDF-1 or CXCR4 a pattern of cleavage indistinguishable from that observed with leukocyte-secreted proteinases. Our findings suggest that elastase-mediated proteolysis of SDF-1/CXCR4 is part of a mechanism regulating their biological functions in both homeostatic and pathologic processes.     

Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.     

皮肤损伤后SDF-1分泌量的变化及其受体CXCR4在表皮组织中分布表达的实验研究

目的:研究皮肤受损后不同时间点伤口液和皮肤组织中基质细胞衍生因子-1(SDF-1)的含量变化,同时验证SDF-1受体CXCR4在表皮组织内的分布.方法:分别在伤后即刻、12小时、24小时、36小时、48小时、60小时、72小时、96小时留取Ⅱ°烧伤患者的大疱液,用ELISA法检测伤口液中SDF-1的含量;用免疫组织化学染色的方法检测伤后1天、3天、7天创缘表皮内SDF-1的表达水平和其受体CXCR4在表皮组织上的分布.结果:用ELLSA法检测发现在受伤后几小时内大疱液中SDF-1的含量开始升高,1天后达到最高水平,伤后的3天SDF-1分泌量逐渐下降,而免疫组织化学染色结果显示创缘表皮层和真皮层上有散在的SDF-1分泌,且分泌量随创面愈合时间的推移逐渐增多;表皮细胞表达有CXCR4,且越靠近表皮基底层细胞膜的阳性越强.结论:SDF-1在皮肤损伤后表达量的增多可能对皮肤组织创伤修复起一定的调节作用.     

Comodulation of CXCR4 and CD26 in human lymphocytes

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.     

SDF-1和及其受体CXCR4的结构与功能

近年基质细胞衍生因子 1(SDF 1)及其受体CXCR4的构效关系与相互作用机制研究进展很快 .研究证实 ,SDF 1N末端 (Nt)氨基酸残基是与CXCR4相互作用的关键区域 .SDF 1的 β链与蛋白聚糖 (GAG)作用而调节SDF 1的功能 ,C端α螺旋有助于维持SDF 1的活性构象 ;CXCR4Nt、ECL2和 (或 )ECL3对于SDF 1和HIVgp12 0对CXCR4的识别和激活都很重要 ,但在识别序列上存在部分交叉重叠 .SDF 1 CXCR4与肿瘤转移密切相关 ,本文还就SDF 1与CXCR4在肿瘤治疗方面的应用进行了讨论 .     

Genetic variants of SDF-1 and CXCR4 genes in endometrial carcinoma

In this study, we aimed to investigate a possible association between the Stromal cell-derived factor-1 (SDF-1) and CXCR4 polymorphisms and the risk of developing endometrial carcinoma. SDF-1 3??A and CXCR4 gene polymorphisms was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism in 139 healthy individuals and 113 patients with endometrial carcinoma. In our study groups SDF-1 3??A AA genotype frequency was higher in patients that of controls and individuals who had AA genotype showed a 2.6-fold increased risk for endometrial cancer. The carriers of CXCR4 T allele were higher in patients compared with controls and individuals who had TT genotype had a 2.5-fold high risk for endometrial carcinoma. Our finding suggest that there was no significant association between the (SDF-1) and CXCR4 polymorphisms and endometrium cancer risk. Further studies in a larger population are needed to better elucidate the role of (SDF-1) and CXCR4 gene polymorphisms in the risk of endometrial carcinogenesis. To the best of our knowledge, this is the first study to show such an association.     

N-linked glycan-dependent interaction of CD63 with CXCR4 at the Golgi apparatus induces downregulation of CXCR4

Efficient downregulation of CXCR4 cell surface expression by introduction of the CD63 gene has previously been reported by us. In the present study, it was found that CD63 and its mutant efficiently interact with CXCR4 in live cells and that CD63-induced downregulation and interaction are significantly abrogated by the N- linked glycosylation inhibitor, TM. Furthermore, the downregulation and interaction were clearly attenuated by alternation of all three N- linked glycosylation sites in CD63. Either CD63 or CD63ΔN formed a complex with CXCR4 at the Golgi apparatus and the late endosomes, while CD63 GD mutants lost the ability to form a complex with CXCR4 exclusively at the Golgi apparatus. These findings suggest that CD63 interacts with CXCR4 through the N- linked glycans-portion of the CD63 protein and that the complex induces direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. At the Golgi apparatus, there may be lysosome protein (CD63)-associated machinery that influences trafficking of other membrane proteins.     

基质细胞衍生因子-1及其受体CXCR4与肿瘤的生物学行为

基质细胞衍生因子-1(stromal cell derived factor-1, SDF-1)是由基质细胞持续产生并分布广泛的趋化因子,CXCR4则为SDF-1的高度特异性受体.最近研究显示,SDF-1/CXCR4生物轴除了调节肿瘤的侵袭转移能力外,还与多种肿瘤的生物学行为关系密切.本文主要介绍SDF-1/CXCR4的结构与功能、SDF-1/CXCR4与肿痛生物学行为的关系,探讨以SDF-1/CXCR4生物轴为靶点的肿瘤治疗前景.     

Blockade of SDF-1/CXCR4 reduces adhesion-mediated chemoresistance of multiple myeloma cells via interacting with interleukin-6

Resistance to chemotherapy represents a major cause for treatment failure in multiple myeloma (MM). Herein, this study was conducted to explore the effect of SDF-1/CXCR4 and interleukin-6 (IL-6) in MM cell adhesion-mediated chemoresistance. Enzyme-linked immunosorbent assay was applied to detect expressions of SDF-1α and IL-6 in MM patients and healthy controls. RPMI-8226 cells and isolated bone marrow stromal cells (BMSCs) were stimulated using recombinant SDF-1α and IL-6. Effect of cocultured BMSCs and RPMI-8226 cells on chemosensitivity and apoptosis of RPMI-8226 cells was analyzed. Effect of doxorubicin on the adhesion rate of RPMl-8226 cells to BMSCs was analyzed by calcitonin test. Effect of SDF-1α-induced upregulation of IL-6 on chemotherapeutic resistance and apoptosis of RPMI-8226 cells in adhesion state was analyzed. Cell adhesion model was treated with recombinant protein SDF-1α and phosphoinositide 3-kinase (P13K) inhibitor Wortmarmin. The levels of P13K and protein kinase B (AKT) and its phosphorylation as well as the expression of IL-6 were analyzed. SDF-1α was positively correlated with IL-6. Recombinant human SDF-1α increased IL-6 expression and induced IL-6 secretion in a time- and dose-dependent manner in BMSCs, which was inhibited by IL-6 and SDF-1α neutralizing antibodies. Coculture of MM cells with BMSCs increased the drug resistance and inhibited the apoptosis on MM cells. SDF-1α-induced IL-6 upregulation mediates chemoresistance and apoptosis of RPMI-8226 cells in adhesion state. SDF-1α may up-regulate the expression of IL-6 by activating the P13K/AKT signaling pathway. SDF-1/CXCR4 may up-regulate the expression of IL-6 through the activation of the P13K/AKT signaling pathway, thereby affecting the chemoresistance mediated by adhesion in MM cells.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null) mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.     

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

Introduction

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

Methods

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.

Results

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

Conclusions

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.