Silibinin,a novel chemokine receptor type 4 antagonist,inhibits chemokine ligand 12-induced migration in breast cancer cells

PurposeC-X-C chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in cancer invasion and migration; therefore, CXCR4 antagonist can serve as an anti-cancer drug by preventing tumor metastasis. This study aimed to identify the CXCR4 antagonists that could reduce and/or inhibit tumor metastasis from natural products.Methods and resultsAccording to the molecular docking screening, we reported here silibinin as a novel CXCR4 antagonist. Biochemical characterization showed that silibinin blocked chemokine ligand 12 (CXCL12)-induced CXCR4 internalization by competitive binding to CXCR4, therefore inhibiting downstream intracellular signaling. In human breast cancer cells MDA-MB-231, which expresses high levels of CXCR4, inhibition of CXCL12-induced chemomigration can be found under silibinin treatment. Overexpression of CXCL12 sensitized MDA-MB-231 cells to the inhibition of silibinin, which was abolished by CXCR4 knockdown. The inhibition of silibinin was also observed in MCF-7/CXCR4 cells rather than MCF-7 cells that express low level of CXCR4.ConclusionsOur work demonstrated that silibinin is a novel CXCR4 antagonist that may have potential therapeutic use for prevention of tumor metastasis.     

The chemokine decoy receptor M3 blocks CC chemokine ligand 2 and CXC chemokine ligand 13 function in vivo

Chemokines and their receptors play a key role in immune homeostasis regulating leukocyte migration, differentiation, and function. Viruses have acquired and optimized molecules that interact with the chemokine system. These virus-encoded molecules promote cell entry, facilitate dissemination of infected cells, and enable the virus to evade the immune response. One such molecule in the murine gammaherpesvirus 68 genome is the M3 gene, which encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and blocks chemokine signaling in vitro. To test the hypothesis that M3 directly interferes with diverse chemokines in vivo, we examined the interaction of M3 with CCL2 and CXCL13 expressed in the pancreas of transgenic mice. CCL2 expression in the pancreas promoted recruitment of monocytes and dendritic cells; CXCL13 promoted recruitment of B and T lymphocytes. Coexpression of M3 in the pancreas blocked cellular recruitment induced by both CCL2 and CXCL13. These results define M3 as multichemokine blocker and demonstrate its use as a powerful tool to analyze chemokine biology.     

Liver-expressed chemokine/CC chemokine ligand 16 attracts eosinophils by interacting with histamine H4 receptor

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine that is constitutively expressed by the liver parenchymal cells and present in the normal plasma at high concentrations. Previous studies have shown that CCL16 is a low-affinity ligand for CCR1, CCR2, CCR5, and CCR8 and attracts monocytes and T cells. Recently, a novel histamine receptor termed type 4 (H4) has been identified and shown to be selectively expressed by eosinophils and mast cells. In this study, we demonstrated that CCL16 induced pertussis toxin-sensitive calcium mobilization and chemotaxis in murine L1.2 cells expressing H4 but not those expressing histamine receptor type 1 (H1) or type 2 (H2). CCL16 bound to H4 with a K(d) of 17 nM. By RT-PCR, human and mouse eosinophils express H4 but not H3. Accordingly, CCL16 induced efficient migratory responses in human and mouse eosinophils. Furthermore, the responses of human and mouse eosinophils to CCL16 were effectively suppressed by thioperamide, an antagonist for H3 and H4. Intravenous injection of CCL16 into mice induced a rapid mobilization of eosinophils from bone marrow to peripheral blood, which was also suppressed by thioperamide. Collectively, CCL16 is a novel functional ligand for H4 and may have a role in trafficking of eosinophils.     

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.     

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.     

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19 nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.     

Cxcl12/Cxcr4 chemokine signaling is required for placode assembly and sensory axon pathfinding in the zebrafish olfactory system

Positioning neurons in the right places and wiring axons to the appropriate targets are essential events for establishment of neural circuits. In the zebrafish olfactory system, precursors of olfactory sensory neurons (OSNs) assemble into a compact cluster to form the olfactory placode. Subsequently, OSNs differentiate and extend their axons to the presumptive olfactory bulb with high precision. In this study, we aim to elucidate the molecular mechanism underlying these two developmental processes. cxcr4b, encoding a chemokine receptor, is expressed in the migrating olfactory placodal precursors, and cxcl12a (SDF-1a), encoding a ligand for Cxcr4b, is expressed in the abutting anterior neural plate. The expression of cxcr4b persists in the olfactory placode at the initial phase of OSN axon pathfinding. At this time, cxcl12a is expressed along the placode-telencephalon border and at the anterior tip of the telencephalon, prefiguring the route and target of OSN axons, respectively. Interfering with Cxcl12a/Cxcr4b signaling perturbs the assembly of the olfactory placode, resulting in the appearance of ventrally displaced olfactory neurons. Moreover, OSN axons frequently fail to exit the olfactory placode and accumulate near the placode-telencephalon border in the absence of Cxcr4b-mediated signaling. These data indicate that chemokine signaling contributes to both the olfactory placode assembly and the OSN axon pathfinding in zebrafish.     

Direct interaction of STAT4 with the IL-12 receptor.

Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4. Direct interaction of the SH2 domain of STAT4 with a phosphotyrosine residue in the IL-12 receptor has been proposed to be required for the subsequent STAT4 phosphorylation. The IL-12 receptor beta2 subunit contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosphopeptides were synthesized according to the amino acid sequences surrounding each of these tyrosine residues. Only the phosphopeptide containing pTyr800 strongly bound to STAT4 in a cell-free binding assay. When this phosphopeptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 phosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elucidate which amino acids surrounding the pTyr800 residue are critical for STAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the binding. These results demonstrate that only a small region of the IL-12 receptor is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 signal transduction for treatment of autoimmune diseases.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

CC chemokine ligand 2 and its receptor regulate mucosal production of IL-12 and TGF-beta in high dose oral tolerance

Oral tolerance is the result of a complex immunoregulatory strategy used by the gut and its associated lymphoid tissues to render the peripheral immune system unresponsive to nonpathogenic proteins, such as food or commensal bacteria. The mechanism of oral tolerance induction and maintenance is not well understood. We have previously shown that the chemokine, CC chemokine ligand 2 (CCL2), is important for the induction and maintenance of oral tolerance. To address the role CCL2 plays in oral tolerance, we used both CCL2(-/-) and CCR2(-/-) mice. Cells from the spleen, mesenteric lymph nodes, and peripheral lymph nodes of CCL2(-/-) and CCR2(-/-) mice fed high doses of OVA showed robust proliferative responses compared with cells from Ag-fed wild-type mice. CCL2(-/-) and CCR2(-/-) mice also produced high amounts of Th1 cytokines such as IL-2 and IFN-gamma and very low amounts of IL-4 and IL-10. The ability of APCs from the gut of CCL2(-/-) and CCR2(-/-) OVA-fed mice to stimulate an indicator T cell line was evaluated. APCs from the Peyer's patch of OVA-fed knockout animals could induce a T cell response measured by an increase in proliferation and generation of IL-12 and IFN-gamma with a concomitant reduction of TGF-beta compared with wild-type controls that did not induce a Th1 response. These data indicate that CCL2 and signaling through its receptor CCR2 is critical for the induction of oral tolerance by regulating Ag presentation leading to a disruption in the balance of inflammatory and regulatory cytokines.     

The role of CXC chemokine ligand 4/CXC chemokine receptor 3-B in breast cancer progression

Chemokines and their receptors participate in the development of cancers by enhancing tumor cell proliferation, angiogenesis, invasion, metastasis and penetration of tumor immune cells. It remains unclear whether CXC chemokine ligand 4 (CXCL4)/CXC chemokine receptor 3-B (CXCR3-B) can be used as an independent molecular marker for establishing prognosis for breast cancer patients. We evaluated CXCL4 and CXCR3-B expression in 114 breast cancer tissues and 30 matched noncancerous tissues using immunohistochemistry and western blot, and determined the correlation between their expression and clinicopathologic findings. We observed that breast cancer tissues express CXCL4 strongly and CXCR3-B weakly compared to noncancerous tissues. Strong CXCL4 expression was detected in 94.7% and weak CXCR3-B expression was detected in 78.9% of the tissues. Therefore, CXCL4/CXCR3-B might play a crucial role in breast cancer progression. We found no significant correlation between CXCL4 and age, tumor stage, tumor grade or TNM stage. CXCR3-B was associated significantly with tumor grade. Moreover, the Chi-square test of association showed that the expression of CXCL4/CXCR3-B might be an independent prognostic marker for breast cancer. Therefore, we suggest that CXCR3-B is an indicator of poor prognosis and may also be a chemotherapeutic target.     

Molecular cloning of a novel human CC chemokine (Eotaxin-3) that is a functional ligand of CC chemokine receptor 3.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.     

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.     

A simplified model of the binding interaction between stromal cell-derived factor-1 chemokine and CXC chemokine receptor 4

We have synthesised the CXC chemokine receptor 4 (29-39) peptide, CXCR4[29-39]. This peptide is located in the N-terminal region of the receptor and is likely to be involved in the docking step of the receptor interaction with its natural ligand stromal cell-derived factor-1 chemokine, SDF-1. Preliminary experiments, performed in the presence of micellar detergents to model a membrane-like environment, show that the (1-17) segment of SDF-1 binds to CXCR4[29-39].