Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo, with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker-treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow haematopoietic stem cell population. Importantly, although the haematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3-blocker-treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the haematopoietic system.

CXC chemokine receptor 7 ameliorates renal fibrosis by inhibiting ß-catenin signaling and epithelial-to-mesenchymal transition in tubular epithelial cells.

Renal fibrosis is a common feature of various chronic kidney diseases. However, the underlying mechanism remains poorly understood. The CXC chemokine receptor (CXCR) family plays a role in renal fibrosis; however, the detailed mechanisms have not been elucidated. In this study, we investigated the potential role of CXCR7 in mediating renal fibrosis. CXCR7 expression is decreased in unilateral ischemia-reperfusion injury (UIRI) and unilateral ureteral obstruction mouse models. Furthermore, CXCR7 was specifically expressed primarily in the Lotus Tetragonolobus Lectin-expressing segment of tubules, was slightly expressed in the peanut agglutinin-expressing segment, and was barely expressed in the Dolichos biflorus agglutinin-expressing segment. Administration of pFlag-CXCR7, an overexpression plasmid for CXCR7, significantly inhibited the activation of ß-catenin signaling and protected against the progression of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a UIRI mouse model. Using cultured HKC-8 cells, we found that CXCR7 significantly downregulated the expression of active ß-catenin and fibrosis-related markers, including fibronectin, Collagen I, and α-SMA. Furthermore, CXCR7 significantly attenuated TGF-ß1-induced changes in ß-catenin signaling, EMT and fibrosis. These results suggest that CXCR7 plays a crucial role in inhibiting the activation of ß-catenin signaling and the progression of EMT and renal fibrosis. Thus, CXCR7 could be a novel therapeutic target for renal fibrosis.

An emerging paradigm of CXCL12 involvement in the metastatic cascade.

The chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF1), has emerged as a pivotal regulator in the intricate molecular networks driving cancer progression. As an influential factor in the tumor microenvironment, CXCL12 plays a multifaceted role that spans beyond its traditional role as a chemokine inducing invasion and metastasis. Indeed, CXCL12 has been assigned functions related to epithelial-to-mesenchymal transition, cancer cell stemness, angiogenesis, and immunosuppression, all of which are currently viewed as specialized biological programs contributing to the "metastatic cascade" among other cancer hallmarks. Its interaction with its cognate receptor, CXCR4, initiates a cascade of events that not only shapes the metastatic potential of tumor cells but also defines the niches within the secondary organs that support metastatic colonization. Given the profound implications of CXCL12 in the metastatic cascade, understanding its mechanistic underpinnings is of paramount importance for the targeted elimination of rate-limiting steps in the metastatic process. This review aims to provide a comprehensive overview of the current knowledge surrounding the role of CXCL12 in cancer metastasis, especially its molecular interactions rationalizing its potential as a therapeutic target.

G protein­mediated EGFR transactivation is a common mechanism through which the CXCL12 receptors, CXCR4 and CXCR7, control human cancer cell migration.

C­X­C motif chemokine 12 (CXCL12) promotes metastasis of several tumors by affecting cell migration and invasion via its receptors, C­X­C chemokine receptor type (CXCR)4 and CXCR7. Current therapeutic approaches focus on the selective inactivation of either CXCR4 or CXCR7 in patients with cancer. Alternative strategies may emerge from the analysis of downstream events that mediate the migratory effects of CXCL12 in cancer cells. While CXCR4 activates cell signaling through both G proteins and arrestins, CXCR7 is believed to preferentially signal through arrestins. The present study analyzed the CXCL12­dependent chemotaxis of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells, in which either the activity of G proteins, EGFR or Src kinase was inhibited pharmacologically or the expression of arrestins was inhibited by RNA interference. The results demonstrated that CXCL12­induced migration of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells was attenuated by the Gαi/o­inhibitor pertussis toxin (PTX), but was unaffected by small interfering RNA­mediated gene silencing of ß­arrestin1/2. In particular, the sensitivity of DLD­1 migration to PTX was unexpected, as it is solely dependent on the non­classical chemokine receptor, CXCR7. Furthermore, chemotactic responses to CXCL12 were additionally prevented by inhibiting EGFR activity via AG1478 and Src kinase activity via Src inhibitor­1. In conclusion, the results of the present study suggest that G protein­ and Src­dependent transactivation of EGFR is a common mechanism through which CXCL12­bound CXCR4 and/or CXCR7 control cancer cell migration and metastasis. These findings highlight EGFR as a potential therapeutic target that interferes with CXCL12­induced cancer expansion.

[Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors].

OBJECTIVE: To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. METHODS: Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). RESULTS: Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). CONCLUSION: Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

G protein activation via chemokine (C-X-C motif) receptor 4 and α1b -adrenoceptor is ligand and heteromer-dependent.

It is unknown whether heteromerization between chemokine (C-X-C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b -adrenoceptor (α1b -AR) influences effects of the CXCR4/ACKR3 agonist chemokine (C-X-C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist-promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4-mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit ß-arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero-trimerize with α1b -AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from hetero-oligomers comprising CXCR4. CXCL12 enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from CXCR4:α1b -AR heterodimers and reduces phenylephrine-stimulated α1b -AR-promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand-dependent functions of the receptor partners.

[Correlation between macrophage chemotaxis and disease severity in patients with knee osteoarthritis].

OBJECTIVE: To investigate the enhancement of macrophage chemotaxis in patients with knee osteoarthritis (KOA) and its correlation with the disease severity. METHODS: Eighty patients with KOA admitted from July 2019 to June 2022 were enrolled as the observation group and divided into 29 cases of moderate group, 30 cases of severe group and 21 cases of extremely severe group. At the same time, 30 healthy subjects were included as the control group. The gene expressions of NF-κB, CXC chemokine receptor 7 (CXCR7) and CXC chemokine ligand 12 (CXCL12) in macrophages of each group were analyzed. Visual analogue scale(VAS) was used to evaluate the degree of joint pain. Joint function was evaluated by knee Joint Society Scoring system(KSS). Finally, data analysis was carried out. RESULTS: The expression levels of NF-κB, CXCR7 and CXCL12 in moderate group, severe group and extreme recombination group were higher than those in control group. The VAS, the expression of NF-κB, CXCR7 and CXCL12 in the severe group and the extreme recombination group were higher than those in the moderate group, whereas KSS was lower than that in the moderate group. The VAS, expression levels of NF-κB, CXCR7 and CXCL12 in the extremely severe group were higher than those in the severe group, and KSS was lower than that in the severe group (all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with VAS score, but negatively correlated with KSS(all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with the severity of disease. After excluding the influence of traditional factors (gender, age and disease duration), multiple linear regression analysis further showed that the expression levels of NF-κB, CXCR7 and CXCL12 were still positively correlated with the severity of disease(all P<0.01). CONCLUSION: The chemotaxis of macrophages in patients with KOA increased with the aggravation of the disease, and was related to the degree of pain and function impairment.

Chemokine receptor CXCR7 activates Aurora Kinase A and promotes neuroendocrine prostate cancer growth.

CXCR7 is an atypical chemokine receptor that recruits ß-arrestin (ARRB2) and internalizes into clathrin-coated intracellular vesicles where the complex acts as a scaffold for cytoplasmic kinase assembly and signal transduction. Here, we report that CXCR7 was elevated in the majority of prostate cancer (PCa) cases with neuroendocrine features (NEPC). CXCR7 markedly induced mitotic spindle and cell cycle gene expression. Mechanistically, we identified Aurora Kinase A (AURKA), a key regulator of mitosis, as a novel target that was bound and activated by the CXCR7-ARRB2 complex. CXCR7 interacted with proteins associated with microtubules and golgi, and, as such, the CXCR7-ARRB2-containing vesicles trafficked along the microtubules to the pericentrosomal golgi apparatus, where the complex interacted with AURKA. Accordingly, CXCR7 promoted PCa cell proliferation and tumor growth, which was mitigated by AURKA inhibition. In summary, our study reveals a critical role of CXCR7-ARRB2 in interacting and activating AURKA, which can be targeted by AURKA inhibitors to benefit a subset of patients with NEPC.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

BACKGROUND: Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. METHODS: Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. RESULTS: Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. CONCLUSIONS: We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

CXC Chemokine Receptor Type 4 Antagonistic Gold Nanorods Induce Specific Immune Responses and Long-Term Immune Memory to Combat Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is highly challenging in its treatment because of the lack of the targeted markers. TNBC patients are not able to acquire benefits from endocrine therapy and targeted therapy except for chemotherapy. CXCR4 is highly expressed on TNBC cells that mediated the tumor cell metastasis as well as proliferation by the response of its ligand CXCL12, therefore holding promising potential of a candidate target for the treatment. In this work, a novel conjugate of CXCR4 antagonist peptide E5 and gold nanorods was fabricated (AuNRs-E5), which was applied to murine breast cancer tumor cells and an animal model, aiming to induce endoplasmic reticulum stress by endoplasmic reticulum-targeted photothermal immunological effects. Results showed that AuNRs-E5 could induce much more generation of damage-related molecular patterns in 4T1 cells under laser irradiation than AuNRs, which significantly promoted the maturation of dendritic cells and stimulated systematic anti-tumor immune responses by enhancing the infiltration of CD8+T cells into the tumor and tumor-draining lymph node, downregulating the regulatory T lymphocytes, and upregulating M1 macrophages in tumors, reversing the microenvironment from "cold" tumors to "hot" tumors. The administration of AuNRs-E5 with laser irradiation not only inhibited the tumor growth significantly but also exerted specific long immune responses to the triple-negative breast cancer tumor cells, which led to the prolonged survival of the mice and the specific immunological memory.

Molecular characterization, structural and expression analysis of twelve CXC chemokines and eight CXC chemokine receptors in largemouth bass (Micropterus salmoides).

The chemokine-receptor system plays important roles in the leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. In the present study, the sequence features, structures and expression patterns of twelve CXC chemokine ligands (CXCL8a.1, CXCL8a.2, CXCL8b.1, CXCL8b.2, CXCL12a, CXCL12b, CXCL13.1, CXCL13.2, CXCL14, CXCL18a, CXCL18b and CXCL19) and eight CXC chemokine receptors (CXCR1, CXCR2, CXCR3.1, CXCR3.2, CXCR3.3, CXCR4a, CXCR4b and CXCR5) of largemouth bass (Micropterus salmoides) were analyzed. All the CXCLs and CXCRs of largemouth bass shared high sequence identities with their teleost counterparts and possessed conserved motifs and structures of CXCLs and CXCRs family. Realtime qPCR revealed that these CXCLs and CXCRs were ubiquitously expressed in all examined tissues, with high expression levels in the immune-related tissues (spleen, head kidney, and gill). Following lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) stimulations, most of these CXCLs and CXCRs were significantly up-regulated in spleen. In addition, the potential interacted molecules of these CXCLs and CXCRs were analyzed by protein-protein interaction network analysis. To the best of our knowledge, this is the first study that in detail analyzes the CXCLs and CXCRs of largemouth bass. Our results provide valuable basis for study the function and mechanism of chemokine-receptor system in largemouth bass.

An expression and function analysis of the CXCR4/SDF-1 signalling axis during pituitary gland development.

The chemokine SDF-1 (CXCL12) and its receptor CXCR4 control several processes during embryonic development such as the regulation of stem cell proliferation, differentiation, and migration. However, the role of this pathway in the formation of the pituitary gland is not understood. We sought to characterise the expression patterns of CXCR4, SDF-1 and CXCR7 at different stages of pituitary gland development. Our expression profiling revealed that SDF-1 is expressed in progenitor-rich regions of the pituitary anterior lobe, that CXCR4 and CXCR7 have opposite expression domains and that CXCR4 expression is conserved between mice and human embryos. We then assessed the importance of this signalling pathway in the development and function of the murine pituitary gland through conditional deletion of CXCR4 in embryonic pituitary progenitors. Successful and specific ablation of CXCR4 expression in embryonic pituitary progenitors did not lead to observable embryonic nor postnatal defects but allowed the identification of stromal CXCR4+ cells not derived from HESX1+ progenitors. Further analysis of constitutive SDF-1, CXCR7 and CXCR4 mutants of the pathway indicates that CXCR4 expression in HESX1+ cells and their descendants is not essential for normal pituitary development in mice.

CXCR7 as a novel therapeutic target for advanced prostate cancer.

Chemokines and their cognate receptors comprise an intricate signaling network that becomes high-jacked by cancer cells for uncontrollable tumor growth and dissemination. ACKR3 (Atypical Chemokine Receptor 3), traditionally called CXCR7, is up-regulated in many cancers, including advanced prostate cancer, and represents promising targets for therapeutic intervention. Unlike typical G protein-coupled receptors such as CXCR4, CXCR7, once bound by its cognate ligand CXCL12, initiates the recruitment of ß-arrestin instead of G proteins, and results in rapid internalization and degradation of CXCL12, functioning as a scavenger receptor. However, recent evidence suggests that CXCR7 may be more than a scavenger or auxiliary receptor of CXCR4 and that it may play essential roles in regulating cancer progression, some of which are independent of CXCR4 and its ligands, such as CXCL12. Constitutively active CXCR7 binds to ß-arrestin. This protein complex internalizes to form a scaffold for assembling and activating various cytoplasmic kinases necessary for cell survival and tumor growth. Here we review and discuss the up-to-date knowledge on CXCR7 regulation and function and how this new understanding guides the development of CXCR7 inhibitors, focusing on prostate cancer.

Exploring the CXCR4/CXCR7/CXCL12 Axis in Primary Desmoid Tumors.

BACKGROUND: Desmoid tumors have an extremely variable natural history. The uncertainty behind desmoid behavior reflects the complexity, which subtends its development and non-linear advancement. Apart from Wnt- ßcatenin mutation, estrogen receptors, and COX-2 overexpression, little is known about the ability of desmoids to grow and recur while being unable to metastasize. Several tumors have been shown to express the CXCR4/CXCR7/CXCL12 axis, whose functions are essential for tumoral development. AIMS: This study aimed to investigate the expression of the CXCR4/CXCR7/CXCL12 axis in primary desmoid tumors and discuss the potential role of this key-signaling as an antiangiogenic therapeutic strategy. METHODS: In this study, 3 µm-thick consecutive sections from each formalin-fixed and paraffin-embedded tissue block were treated with mouse monoclonal antibodies developed against CD34, CXCR4, CXCR7, and CXCL12. RESULTS: Two distinct vessel populations: CXCR4+ and CXCR4- vessels, have been found. Similarly, chemokine receptor CXCR7 expression in the entire desmoid tumor series positively stained a portion of tumor-associated vessels, identifying two distinct subpopulations of vessels: CXCR7+ and CXCR7- vessels. All 8 neoplastic tissue samples expressed CXCL12. Immunohistochemical positivity was identified in both stromal and endothelial vascular cells. Compared to CXCR4 and CXCR7, the vast majority of tumor-associated vessels were found to express this chemokine. CONCLUSION: It is the first time, as per our knowledge, that CXCR4/CXCR7/CXCL12 axis expression has been identified in a desmoid type-fibromatosis series. CXCL12 expression by neoplastic cells, together with CXCR4 and CXCR7 expression by a subgroup of tumor-associated vessels, was detected in all desmoid tumor tissue samples examined. Since chemokines are known contributors to neovascularization, CXCR4/CXCR7/CXCL12 axis may play a role in angiogenesis in this soft-tissue tumor histotype, thereby supporting its growth.

Chemokine receptor 7 mediates miRNA-182 to regulate cerebral ischemia/reperfusion injury in rats.

AIMS: Chemokine receptor 7 (CXCR7) exerts protective effects on the brain. MicroRNAs (miRNAs) are involved in cerebral ischemia/reperfusion (I/R) injury, but their involvement in CXCR7-mediated brain protection is unknown. In this study, we investigated the role of miRNAs in CXCR7-mediated brain protection. METHODS: CXCR7 levels in peripheral blood samples from patients with acute ischemic stroke (AIS) and ischemic penumbra area brain tissues from middle cerebral artery occlusion (MCAO) rats after recanalization were measured. An miRNA microarray analysis was performed to examine the expression of miRNAs caused by CXCR7 knockdown in ischemic penumbra area brain tissue in middle cerebral artery occlusion-reperfusion rats and to predict corresponding downstream target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed the most enriched pathways. A dual-luciferase reporter assay confirmed the direct regulation of miR-182 on the target gene TCF7L2. The correlation between TCF7L2 and CXCR7/miR-182 was verified using rescue assays. RESULTS: CXCR7 expression was upregulated in MCAO rats and mechanical thrombectomy patients with AIS compared to that in controls. The motor and sensory functions of MCAO rats with CXCR7 knockdown further decreased, and the infarct volume and cerebral edema increased. miRNA microarray data showed that seven miRNAs were differentially expressed after shRNA-CXCR7 treatment. The dual-luciferase reporter assay confirmed that miR-182 directly targeted the TCF7L2 gene. Rescue assays confirmed that TCF7L2 is downstream of CXCR7/miR-182. KEGG pathway analysis showed that the Hippo pathway may be a key pathway in CXCR7 upregulation and plays a role in protecting the brain after interventional surgery. Animal experiments have shown that CXCR7-mediated cerebral I/R injury promotes the phosphorylation of key molecules YAP and TAZ in the Hippo pathway. CONCLUSION: CXCR7 protects against cerebral I/R injury, possibly via the miR-182/TCF7L2/Hippo pathway. These results indicate that CXCR7 affects cerebral ischemia-reperfusion injury through miRNA regulation and downstream pathways.

Structural snapshot of a ß-arrestin-biased receptor.

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to ß-arrestins (ßarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors / 中国实验血液学杂志

OBJECTIVE@#To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma.@*METHODS@#Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR).@*RESULTS@#Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05).@*CONCLUSION@#Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.