C-X-C Chemokine Receptor 4 Ligand Research Articles

FOXO1 regulates developmental lymphangiogenesis by upregulating CXCR4 in the mouse-tail dermis.

Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.

Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.

Multifaceted C-X-C Chemokine Receptor 4 (CXCR4) Inhibition Interferes with Anti–Vascular Endothelial Growth Factor Therapy–Induced Glioma Dissemination

Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.1.1. Brain tissue was analyzed for tumor volume, invasiveness, hypoxia, vascular density, proliferation, apoptosis, GSCs, and MGCs. Glioma cells were evaluated for CXCR4 expression and polymorphism and POL5551's effects on CXCR4 ligand binding, cell viability, and migration. No CXCR4mutations were identified. POL5551 inhibited CXCR4 binding to its ligand, stromal cell-derived factor-1α, and reduced hypoxia- and stromal cell-derived factor-1α-mediated migration dose-dependently but minimally affected cell viability. In vivo, B20-4.1.1 increased hypoxic foci and invasiveness, as seen in GBM patients receiving anti-VEGF therapy. Combination of POL5551 and B20-4.1.1 reduced both glioma invasiveness by 16% to 39% and vascular density compared to B20-4.1.1 alone in both glioma models. Reduced populations of GSCs and MGCs were also seen in CT-2A tumors. POL5551 concentrations, evaluated by mass spectrometry, were higher in tumors than in neighboring brain tissues, likely accounting for the results. Inhibition of CXCR4-regulated tumoral, stem cell, and immune mechanisms by adjunctive CXCR4 antagonists may help overcome antiangiogenic therapy resistance, benefiting GBM patients.

CXCL12/SDF-1α induces migration via SRC-mediated CXCR4-EGFR cross-talk in gastric cancer cells.

Metastasis is the primary cause of mortality in patients with advanced gastric carcinoma, and multiple signaling pathways promote the development of this condition. Stromal cell-derived factor-1 (SDF-1α/CXCL12), the main ligand for CXC chemokine receptor-4 (CXCR4), serves an important role in gastric cancer cell migration. Previous studies have demonstrated that CXCL12 could also stimulate the secretion of epidermal growth factor receptor (EGFR) ligands, including amphiregulin and heparin-binding epidermal growth factor-like growth factor, from gastric cancer cells, resulting in an increase in the ability of migration. However, it remains to be elucidated whether CXCL12 activates EGFR intracellular signaling and therefore stimulates migration in gastric cancer. The present study demonstrated that three gastric cancer cell lines, SGC-7901, MGC-803 and BGC-823, all expressed CXCR4. The increased chemotactic migratory ability stimulated by CXCL12 was effectively abrogated by the CXCR4 antagonist, AMD3100. Furthermore, a rapid phosphorylation of Akt/extracellular signal-regulated kinase (ERK)/EGFR was demonstrated to be involved in CXCL12/CXCR4-induced gastric cancer cell migration. Knockdown of EGFR gene or the use of a monoclonal antibody against EGFR (C225) blocked the activation of ERK/Akt and partially prevented the ability of migration induced by CXCL12, which indicated that EGFR signaling is located downstream of CXCL12. In addition, it was also revealed that the activation of non-receptor tyrosine kinase c-steroid receptor co-activator (SRC) and the formation of the SRC/EGFR heterodimer are promoted by CXCL12, whereas the SRC inhibitor, PP2, blocks the SRC/EGFR heterodimer and the activation of EGFR, as well as CXCR4-meditated migration induced by CXCL12. The present results indicated that SRC mediates a potential CXCR4-EGFR cross-talk, and thereby utilizes the EGFR-Akt/ERK axis to promote cellular migration. The present study provided a novel insight into the underlying regulatory mechanisms of the CXCL12/CXCR4 pathway in gastric cancer cell migration.

Abstract 365: Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis

Abstract Metastatic prostate cancer cells preferentially home to bone, and skeletal metastases are present in approximately 90% of patients upon autopsy. Our lab was the first to show that prostate cancer cells compete with hematopoietic stem cells (HSCs) for space within the endosteal niche. The HSC niche is located on the surface of endosteal osteoblasts, where HSCs are kept in a slow-cycling, protective state. The niche also provides prostate cancer cells with a chemoprotective environment in which they can remain dormant for an extended period of time, even decades. C-X-C chemokine receptor 4 (CXCR4) is required for HSC recruitment to the HSC niche. Osteoblasts secrete stromal cell derived factor 1 (SDF-1, also known as CXCL12), the ligand for CXCR4, which attracts HSCs to the niche via a chemotactic gradient. CXCR4 is also expressed by disseminated prostate cancer cells and is required for prostate cancer cells homing to bone. HSCs can be mobilized from the niche into the bloodstream physiologically via infection or stress and pharmacologically via proteins like granulocyte-colony stimulating factor (G-CSF) or small molecule inhibitors like AMD3100 (plerixafor). AMD3100, which antagonizes CXCR4, is capable of mobilizing both HSCs and prostate cancer cells from the niche. When combined with AMD3100, docetaxel (the standard of care for prostate cancer) is more effective at killing tumor cells than when given as a single agent. This indicates that mobilization of disseminated tumor cells (DTCs) from the endosteal niche by targeting the SDF-1/CXCR4 axis might cause these cells to be more sensitive to chemotherapy in patients. This strategy would theoretically kill DTCs, which, when they become metastatic lesions, lead to most of the morbidity and mortality that affects prostate cancer patients. We are engaged in a Phase 0 clinical trial in metastatic prostate cancer patients to test this strategy in human patients. To complement the human trial, we are testing several CXCR4 inhibitors combined with G-CSF and docetaxel. We are able to detect CTCs in human patients, as well as CTCs and DTCs in mice via several techniques, and will use this technology as a readout for the clinical trial, as well as the preclinical studies. Citation Format: Kenneth C. Valkenburg, Kenneth J. Pienta. Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2015-365

Matrix metalloproteinase 10 contributes to hepatocarcinogenesis in a novel crosstalk with the stromal derived factor 1/C-X-C chemokine receptor 4 axis.

Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10-deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C-X-C chemokine receptor-4 (CXCR4), was reduced in DEN-induced MMP10-deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10-stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal-derived factor-1 (SDF1), through the extracellular signal-regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter. MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model.

Involvement of the nuclear factor-κB signaling pathway in the regulation of CXC chemokine receptor-4 expression in neuroblastoma cells induced by tumor necrosis factor-α

Metastasis is a hallmark of malignant neuroblastoma and is the main reason for therapeutic failure and recurrence of the tumor. The CXC chemokine receptor-4 (CXCR4), a Gi protein-coupled receptor for the ligand CXCL12/stromal cell-derived factor-1α (SDF-1α), is expressed in various types of tumor. This receptor mediates the homing of tumor cells to specific organs that express the ligand, CXCL12, for this receptor and plays an important role in tumor growth, invasion, metastasis and angiogenesis. In the present study, the inflammatory cytokine, tumor necrosis factor-α (TNF-α) upregulated CXCR4 expression in neuroblastoma cells and increased migration to the CXCR4 ligand SDF-1α. In addition, this effect was dependent upon NF-κB transcriptional activity, as blocking the NF-κB pathway with pyrrolidinedithiocarbamic acid ammonium salt suppressed TNF-α-induced upregulation of CXCR4 expression and reduced the migration towards the CXCR4 ligand, SDF-1α. Treating neuroblastoma cells with TNF-α resulted in the activation of nuclear factor-kappa B (NF-κB) and subsequently, the translocation of NF-κB from the cytoplasm to the nucleus. Using immunohistochemistry, NF-κB and CXCR4 were significantly correlated with each other (P=0.0052, Fisher’s exact test) in a cohort of neuroblastoma samples (n=80). The present study indicates that the inflammatory cytokine, TNF-α, partially functions through the NF-κB signaling pathway to upregulate CXCR4 expression to foster neuroblastoma cell metastasis. These findings indicate that effective inhibition of neuroblastoma metastasis should be directed against the inflammatory cytokine-induced NF-κB/CXCR4/SDF-1α signaling pathway.

A Locked, Dimeric CXCL12 Variant Effectively Inhibits Pulmonary Metastasis of CXCR4-Expressing Melanoma Cells Due to Enhanced Serum Stability

The CXC chemokine receptor-4 (CXCR4) plays a critical role in cancer by positively regulating cancer cell metastasis and survival. We previously showed that high concentrations of the CXCR4 ligand, wild-type CXCL12 (wtCXCL12), could inhibit colorectal cancer metastasis in vivo, and we have hypothesized that wtCXCL12 dimerizes at high concentration to become a potent antagonist of CXCR4. To address this hypothesis, we engineered a covalently locked, dimeric variant of CXCL12 (CXCL122). Herein, we show that CXCL122 can not only inhibit implantation of lung metastasis of CXCR4-B16-F10 melanoma cells more effectively than AMD3100, but that CXCL122 also blocks the growth of established pulmonary tumors. To identify a basis for the in vivo efficacy of CXCL122, we conducted Western blot analysis and ELISA analyses, which revealed that CXCL122 was stable for at least 12 hours in serum, whereas wtCXCL12 was quickly degraded. CXCL122 also maintained its antagonist properties in in vitro chemotaxis assays for up to 24 hours in serum, whereas wtCXCL12 was ineffective after 6 hours. Heat-inactivation of serum prolonged the stability and function of wtCXCL12 by more than 6 hours, suggesting enzymatic degradation as a possible mechanism for wtCXCL12 inactivation. In vitro analysis of amino-terminal cleavage by enzymes dipeptidylpeptidase IV (DPPIV/CD26) and matrix metalloproteinase-2 (MMP-2) resulted in 25-fold and 2-fold slower degradation rates, respectively, of CXCL122 compared with wtCXCL12. In summary, our results suggest CXCL122 possesses greater potential as an antimetastatic drug as compared with AMD3100 or wtCXCL12, potentially due to enhanced serum stability in the presence of N-terminal degrading enzymes.

Pancreatic adenocarcinoma upregulated factor, a novel endothelial activator, promotes angiogenesis and vascular permeability

Pancreatic adenocarcinoma upregulated factor (PAUF) was recently reported to be a metastasis factor for pancreatic cancer cells. Here, we demonstrate a novel role for PAUF as a potent endothelial activator, promoting both angiogenesis and vascular permeability. Overexpression of PAUF in a mouse pancreatic cancer model resulted in increased tumor vascularity. Recombinant PAUF (rPAUF) enhanced proliferation, migration and capillary-like tube formation of human endothelial cells (ECs), consistently with increased neovascularization in vivo. rPAUF also increased endothelial permeability through the disruption of vascular endothelial-cadherin-facilitated cell-cell junctions in vitro and induced vascular leakage in mouse skin. These effects were attenuated upon treatment with an antibody against PAUF. Moreover, PAUF evoked a time- and dose-dependent activation of extracellular signal-regulated kinase (ERK)1/2, AKT and endothelial NO synthase (eNOS) in ECs, which are closely linked to rPAUF-induced angiogenesis. Finally, rPAUF upregulated the expression of C-X-C chemokine receptor 4 (CXCR4) in ECs and potentiated the in vitro and in vivo EC angiogenic responses to stromal cell-derived factor-1 (SDF-1), a ligand for CXCR4. Taken together, these data demonstrate that PAUF has a novel function in promoting angiogenesis and vascular permeability. Our findings suggest new possibilities for PAUF's role in the pathogenesis of 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.

Brain-Derived Neurotrophic Factor Inhibits Human Immunodeficiency Virus-1/gp120-Mediated Cerebellar Granule Cell Death by Preventing gp120 Internalization

The human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 has been implicated in the pathogenesis of HIV-1 dementia. Thus, inhibition of gp120 activity could reduce HIV toxicity in the brain. We have used primary cultures of rat cerebellar granule cells to examine mechanisms whereby gp120 causes cell death and to characterize neuroprotective agents. gp120 induced a time- and concentration-dependent apoptotic cell death, which was caspase-3-mediated but caspase-1 independent, and was totally blocked by the irreversible caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone. Caspase-3 activation was observed only in neurons that internalize gp120, indicating that internalization is key to gp120 toxicity. Because brain-derived neurotrophic factor (BDNF) prevents caspase-3-mediated neuronal cell death, we examined whether BDNF could prevent gp120-mediated apoptosis. Preincubation of neurons with BDNF before the addition of gp120 reduced caspase-3 activation, and consequently rescued 80% of neurons from apoptosis. Most importantly, BDNF reduced the levels of CXC chemokine receptor-4 (CXCR4), a receptor that mediates HIV-1 gp120-induced apoptosis. This effect correlated with the ability of BDNF to reduce gp120 internalization and apoptosis. Moreover, BDNF blocked the neurotoxic effect of stromal-derived factor-1alpha, a natural ligand for CXCR4, further establishing a correlation between neuroprotection and downregulation of CXCR4. We propose that BDNF may be a valid therapy to slow down the progression of HIV/gp120-mediated neurotoxicity.

CXC Chemokine Receptor 4 Expression and Function in Human Astroglioma Cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.

Chemokine stromal cell–derived factor 1α activates basophils by means of CXCR4

Background: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell–derived factor 1α (SDF-1α) is the only known ligand for CXCR4. To date, the CXCR4 expression and function of SDF-1α in basophils are unknown. Objective: The purpose of this study was to investigate the expression of CXCR4 and functions of SDF-1α in basophils and to characterize the role of the CXCR4-SDF-1α receptor ligand pair in the allergic inflammation. Methods: Basophil purification, flow cytometry, real-time quantitative RT-PCR assay, Northern blotting, intracellular free Ca2+ change, chemotaxis assay, and histamine release assay were used. Results: CXCR4 is abundantly expressed on peripheral blood resting basophils (91%). Likewise, CXCR4 messenger (m)RNA is expressed in resting basophils (3.2 × 103 copies per 2 × 102 cells). The existence of CXCR4 mRNA was also confirmed in basophils by means of Northern blot analysis. SDF-1α induces an increase in intracellular free Ca2+ in basophils. SDF-1α activates basophils to chemotaxis (chemotactic index = 3.8) and histamine release (36% of total content) through CXCR4 on the cells. The chemokines SDF-1α, eotaxin, RANTES, monocyte chemoattractant protein (MCP) 1, and macrophage inflammatory protein (MIP) 1α have been demonstrated at different potencies in induction of chemotaxis (eotaxin > SDF-1α > RANTES ≅ MCP-1 >> MIP-1α) and histamine release (MCP-1 ≅ SDF-1α > eotaxin > RANTES > MIP-1α). The optimal concentration seen for SDF-1α effects (chemotaxis and histamine release) on basophils was 100 ng/mL. Conclusion: These results indicate that the CXCR4-SDF-1α receptor ligand pair may be important for the recruitment and activation of the basophils, which is a characteristic effector cell of the allergic inflammation. (J Allergy Clin Immunol 2000;106:313-20.)

Functional expression of chemokine receptor CXCR4 on human epithelial cells.

Chemokines and their receptors play an important role in the process of leucocyte recruitment at sites of inflammation. However, recent evidence suggests that these proteins can also regulate non-leucocyte cell functions such as angiogenesis, migration and proliferation. We have investigated the expression of the CXC chemokine receptor 4 (CXCR4) on primary cultures of type II alveolar epithelial cells, their transformed counterpart, the A549 cell line and also on other epithelial cell lines from various tissues. We found that all epithelial cell types tested express mRNA for CXCR4. Flow cytometric analysis and immunocytochemical staining shows that CXCR4 chemokine receptor is abundantly expressed on the surface of A549 epithelial cells. Furthermore, A549 cells responded to the CXCR4 ligand, stromal-derived factor-1alpha (SDF-1alpha) with a rapid and robust calcium mobilization and not to other CXC chemokines, suggesting that CXCR4 is functionally active and is able to couple to G-protein signalling mechanisms. A549 cells did not proliferate in response to either SDF-1alpha or interleukin-8 (IL-8) CXC chemokines. These findings may have important implications for epithelial physiology and pathology.

Inhibition of Tyrosine Kinase Activation Blocks the Down-Regulation of CXC Chemokine Receptor 4 by HIV-1 gp120 in CD4+ T Cells

Because the binding of HIV-1 envelope to CD4 initiates a configurational change in glycoprotein 120 (gp120), enabling it to interact with fusion coreceptors, we investigated how this process interferes with the expression and function of CXC chemokine receptor 4 (CXCR4) in CD4+ T lymphocytes. A recombinant gp120 (MN), after preincubation with CD4+ T lymphocytes, significantly inhibited the binding and chemotaxis of the cells in response to the CXCR4 ligand stromal cell-derived factor-1alpha (SDF-1alpha), accompanied by a markedly reduced surface expression of CXCR4. gp120, but not SDF-1alpha, induced rapid tyrosine phosphorylation of src-like kinase p56lck in CD4+ T cells, whereas both gp120 and SDF-1alpha caused phosphorylation of the CXCR4. The tyrosine kinase inhibitor herbimycin A abolished the phosphorylation of p56lck and CXCR4 induced by gp120 in association with maintenance of normal expression of cell surface CXCR4 and a migratory response to SDF-1alpha. Thus, a CD4-associated signaling molecule(s) including p56lck is activated by gp120 and is required for the down-regulation of CXCR4.

Progressive and Persistent Downregulation of Surface CXCR4 in CD4+ T Cells Infected With Human Herpesvirus 7

We have previously shown that infection of CD4+ T lymphocytes with the T-lymphotropic human herpesvirus 7 (HHV-7) downregulates surface CD4, which represents the high-affinity receptor for HHV-7. In this study, we report that HHV-7 infection also causes a progressive loss of the surface CXC-chemokine receptor 4 (CXCR4) in CD4+ T cells, accompanied by a reduced intracellular Ca2+ flux and chemotaxis in response to stromal cell-derived factor-1 (SDF-1), the specific CXCR4 ligand. Moreover, CXCR4 is downregulated from the surface of HHV-7–infected T cells independently of CD4. Because intracellular CXCR4 antigen and mRNA levels are unaffected in productively HHV-7–infected cells, the downregulation of CXCR4 apparently does not involve a transcritional block. Since CXCR4 functions in association with CD4 to permit entry of several human immunodeficiency virus (HIV) isolates, the potential of HHV-7 to persistently downregulate the surface expression of CXCR4 may provide novel strategies for limiting HIV infection.

Cutting Edge: CXCR4 Is a Functional Coreceptor for Infection of Human Macrophages by CXCR4-Dependent Primary HIV-1 Isolates

Abstract The identification of HIV-1 coreceptors has provided a molecular basis for the tropism of different HIV-1 strains. CXC chemokine receptor-4 (CXCR4) mediates the entry of both primary and T cell line-adapted (TCLA) syncytia-inducing strains. Although macrophages (Mφ) express CXCR4, this coreceptor is assumed to be nonfunctional for HIV-1 infection. We addressed this apparent paradox by infecting human monocyte-derived Mφ with primary and TCLA isolates that were rigorously characterized for coreceptor usage and by adding the natural CXCR4 ligand, stem cell differentiation factor-1, to specifically block CXCR4-mediated entry. Our results show that primary HIV-1 isolates that selectively use CXCR4 productively infected both normal and C-C chemokine receptor-5-null Mφ. By contrast, Mφ supported the entry of CXCR4-dependent TCLA strains with variable efficiency but were not productively infected. Thus, the tropism of HIV isolates results from complex virus/host cell interactions both at the entry and postentry levels.

Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1α

The chemokine stromal-cell-derived factor-1α (SDF-1α) chemoattracts lymphocytes and CD34 + haematopoietic progenitors and is the ligand for CXCR4 (CXC chemokine receptor 4), the main co-receptor for T-tropic HIV-1 strains. SDF-1α was NH 2-terminally cleaved to SDF-1α(3-68) by dipeptidyl-peptidase IV (CD26/DPP IV), which is present in blood in soluble and membrane-bound form. SDF-1α(3-68) lost both lymphocyte chemotactic and CXCR4-signaling properties. However, SDF-1α(3-68) still desensitized the SDF-1α(1-68)-induced Ca 2+ response. In contrast to CD26/DPP IV-processed RANTES(3-68), SDF-1α(3-68) had diminished potency to inhibit HIV-1 infection. Thus, CD26/DPP IV impairs the inflammatory and haematopoietic potency of chemokines but plays a dual role in AIDS.