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Ray, Paramita, Sarah A. Lewin, Laura Anne Mihalko, Sasha-Cai Lesher-Perez, Shuichi Takayama, Kathryn E. Luker, and Gary D. Luker. "Secreted CXCL12 (SDF-1) forms dimers under physiological conditions." Biochemical Journal 442, no. 2 (February 13, 2012): 433–42. http://dx.doi.org/10.1042/bj20111341.
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Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.
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Gonzalez-Meljem, Jose Mario, Sarah Ivins, Cynthia Lilian Andoniadou, Paul Le Tissier, Peter Scambler, and Juan Pedro Martinez-Barbera. "An expression and function analysis of the CXCR4/SDF-1 signalling axis during pituitary gland development." PLOS ONE 18, no. 2 (February 17, 2023): e0280001. http://dx.doi.org/10.1371/journal.pone.0280001.
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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.
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Friedman, Daniel, Antony Long, Piers EM Patten, and Robbert Hoogeboom. "Identification of a Novel Proliferating Cell Fraction in Chronic Lymphocytic Leukaemia with High Expression of IgM and Chemokine Receptors." Blood 138, Supplement 1 (November 5, 2021): 3711. http://dx.doi.org/10.1182/blood-2021-153415.
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Abstract Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant CD5+ B cells in the peripheral blood (PB), secondary lymphoid tissues and bone marrow. Currently considered an incurable disease, B cell receptor (BCR) signalling plays a key role in the disease aetiology as evidenced by the therapeutic success of BCR signalling inhibitors such as ibrutinib. Previous studies using incorporation of 2H-labelling of DNA in vivo demonstrated sub-clonal heterogeneity in PB CLL cell fractions sorted based on reciprocal densities of chemokine C-X-C motif receptor 4 (CXCR4) and CD5. The CXCR4 loCD5 hi fraction was shown to be enriched in recently born proliferating cells while the CXCR4 hiCD5 lo fraction consists of resting, quiescent cells thought to reflect their migratory and BCR signalling histories in tissue. Whilst these proliferating/resting fractions have since been more closely examined, the remaining bulk PB CLL population has been left relatively unexplored leaving other therapeutically relevant cell fractions undetected. Here, we have comprehensively analysed the phenotype of subpopulations of PB cells from 11 CLL patients using flow cytometry to identify activated and proliferating cell fractions. CD19 +CD5 +cells were divided into 9 fractions based on CXCR4/CD5 densities and to permit comparisons between fractions, each cell fraction was defined as containing 1-2% of the total clonal CD19 +CD5 + population. Surprisingly, we detected enrichment for Ki67+ proliferating cells and high expression of AID in the cell fraction with highest expression levels of both CXCR4 and CD5 (CXCR4 hiCD5 hi), demonstrating that CXCR4 loCD5 hi cells are not the only proliferating fraction in the blood. Moreover, we could detect mitotic cells in the CXCR4 hiCD5 hi fraction using imaging flow cytometry of a nuclear stain. This CXCR4 hiCD5 hi fraction showed the highest surface expression levels of IgM, CD86, CCR7, CXCR3 and CXCR5 of all the fractions assessed (p<0.05), indicating they are highly activated and primed for migration to lymph nodes (LNs) for further activation and proliferation. Proliferation of CLL cells is highest in secondary lymphoid tissues, however the phenotype of proliferating cells in tissue is unknown. To examine the phenotype of proliferating CLL cells in LNs, we analysed a fine-needle aspirate obtained from an enlarged cervical node using flow cytometry and compared this to a matched PB sample. Flow cytometric gates set on the PB sample were used to define and quantify LN cell fractions. Expression levels of both Ki67 and surface IgM were highest in the CXCR4 hiCD5 hi fraction which was expanded to 20% of the CD19 +CD5 + population in the LN whilst CXCR4 loCD5 hi cells (accounting for 2% of the bulk LN population) expressed very low surface IgM and Ki67 levels, suggesting CXCR4 hiCD5 hi cells may be the most proliferative cells in CLL. The CXCR4 loCD5 hi cell fraction has been shown to be a key target of ibrutinib, however the impact of ibrutinib on the CXCR4 hiCD5 hi fraction is unknown. Administration of ibrutinib to PB CLL cells for 48hr in vitro resulted in selective targeted depletion of the CXCR4 loCD5 hi fraction, as evidenced by induction of apoptotic markers in this compartment; conversely, persistent cells after 48hr ibrutinib administration in vitro were exclusively of the CXCR4 hi phenotype. In conclusion, we have identified a potentially dangerous fraction of proliferating cells in the PB of CLL patients with high expression of CXCR4, CD5, IgM, CCR7, CXCR3 and CXCR5 open for both migration to tissue and reception of BCR signals. Furthermore, CXCR4 hiCD5 hi cells in the periphery may closely mirror tissue-resident activated cell phenotypes and may represent critical targets for therapeutic intervention, particularly in high-risk CLL patients refractory to BCR inhibitor therapies. Disclosures Patten: ROCHE: Research Funding; GILEAD SCIENCES: Honoraria, Research Funding; NOVARTIS: Honoraria; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.
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Barbieri, Federica, Stefano Thellung, Roberto Würth, Federico Gatto, Alessandro Corsaro, Valentina Villa, Mario Nizzari, Manuela Albertelli, Diego Ferone, and Tullio Florio. "Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System." International Journal of Endocrinology 2014 (2014): 1–16. http://dx.doi.org/10.1155/2014/753524.
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Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioningviaautocrine/paracrine mechanisms was also reported. Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.
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Weissleder, Christin, Maree J. Webster, and Cynthia Shannon Weickert. "M174. REDUCED CHEMOKINE SIGNALLING CAPACITY IS ASSOCIATED WITH INHIBITORY INTERNEURON DYSFUNCTION IN SUBCORTICAL BRAIN REGIONS IN SCHIZOPHRENIA AND BIPOLAR DISORDER." Schizophrenia Bulletin 46, Supplement_1 (April 2020): S202—S203. http://dx.doi.org/10.1093/schbul/sbaa030.486.
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Abstract Background The subependymal zone (SEZ) adjacent to the lateral ventricles represents the largest reservoir of postnatally-generated cortical and striatal inhibitory interneurons in the human brain. Expression of markers representing the generation of neuronal progenitors from neural stem cells is reduced in the adult SEZ in schizophrenia and bipolar disorder; however, underlying mechanisms and relationships to inhibitory interneuron dysfunction remain unknown. Stem cell maintenance, neuronal migration and cell survival are regulated by signaling of the CXC motif chemokine 12 (CXCL12) through CXC motif chemokine receptors 4 (CXCR4) and 7 (CXCR7), which are increasingly implicated in the pathophysiology of psychiatric disorders. Methods Post-mortem tissue was obtained from 33 schizophrenia, 32 bipolar disorder and 33 control cases from the Stanley Medical Research Institute. SEZ and caudate nucleus tissue was dissected from 60 µm sections for RNA isolation and cDNA synthesis. Gene expression of CXCL12, CXCR4 and CXCR7 were determined by quantitative polymerase chain reactions. Semi-partial correlations were performed to assess whether CXC chemokine family member mRNAs may correlate with markers of neural stem cells (PROM1, GFAPD), neuronal progenitors (SOX2, ASCL1) and inhibitory interneurons (CALB2, NPY) in the SEZ and caudate nucleus. Results In the SEZ, CXCL12 mRNA was decreased in schizophrenia compared to controls and bipolar disorder (14–24%, all p≤0.03). CXCR4 and CXCR7 mRNAs were both decreased in schizophrenia and bipolar disorder compared to controls (9–33%, all p≤0.05). CXCL12, CXCR4 and CXCR7 expression positively correlated with PROM1, GFAPD, SOX2 and ASCL1 mRNAs (0.28≥sr≤0.61). In the caudate nucleus, CXCL12 mRNA was decreased in schizophrenia and bipolar disorder compared to controls (19–26%, all p≤0.05). CXCR4 mRNA was decreased in schizophrenia compared to controls (20%, p=0.01), while CXCR7 expression did not significantly differ across diagnostic groups. CALB2 and NPY mRNAs were increased in bipolar disorder compared to controls (13–27%, all p≤0.05). CXCR4 expression positively correlated with CALB2 mRNA (sr=0.26), while CXCR7 expression negatively correlated with NPY mRNA (sr=0.26). Discussion These findings provide the first molecular evidence of decreased CXC chemokine family member expression in the SEZ and caudate nucleus in psychiatric disorders, with exacerbated deficits in schizophrenia compared to bipolar disorder. Dysregulated CXC chemokine family member expression may hamper neural stem cell maintenance and neuronal differentiation, which may contribute to inhibitory interneuron dysfunction in psychiatric disorders. Future work will determine the cellular localisation of CXCR4 and CXCR7 expression in the SEZ and caudate nucleus to disentangle the regulatory role of CXCL12 signalling in the generation, migration and survival of inhibitory interneurons in the human brain.
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Willett, Brian J., Karen Adema, Nikolaus Heveker, Anne Brelot, Laurent Picard, Marc Alizon, Julie D. Turner, et al. "The Second Extracellular Loop of CXCR4 Determines Its Function as a Receptor for Feline Immunodeficiency Virus." Journal of Virology 72, no. 8 (August 1, 1998): 6475–81. http://dx.doi.org/10.1128/jvi.72.8.6475-6481.1998.
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ABSTRACT The feline homolog of the α-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV.
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Arvidsson, Yvonne, Anders Bergström, Linda Arvidsson, Erik Kristiansson, Håkan Ahlman, and Ola Nilsson. "Hypoxia stimulates CXCR4 signalling in ileal carcinoids." Endocrine-Related Cancer 17, no. 2 (June 2010): 303–16. http://dx.doi.org/10.1677/erc-09-0085.
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Tumour hypoxia is associated with increased metastatic potential and resistance to radiotherapy and chemotherapy. Ileal carcinoids are usually metastatic at the time of diagnosis and respond poorly to chemotherapy. The aim of this study was to investigate the extent of hypoxia in ileal carcinoids and the response of tumour cells to induced hypoxia. Vascular endothelial growth factor (VEGF), carbonic anhydrase (CA-IX), hypoxia-inducible factor (HIF)-1α and HIF-2α were studied by immunohistochemistry in biopsies from 24 patients with ileal carcinoids. All hypoxic markers were shown to be highly expressed in localized areas of the tumours irrespective of tumour location or stage. However, HIF-2α expression was significantly higher in distant metastases compared to primary tumours in the same patient. Global gene expression profiling of GOT1 carcinoid cells revealed a marked response to hypoxia. Expression of genes related to epithelial-to-mesenchymal transition and development was altered including increased expression of the C-X-C chemokine receptor type 4 (CXCR4), an important regulator of invasive growth and metastasis formation. High expression of CXCR4 was confirmed by immunohistochemistry in tumour biopsies. Stimulation of GOT1 cells by the CXCR4 ligand, CXCL12 (stromal cell-derived factor 1 (SDF-1)), activated the mitogen-activated protein kinase (MAPK) p42/44 signalling pathway and increased tumour cell migration. We conclude that ileal carcinoids contain hypoxic areas expressing HIF-1α, HIF-2α and CXCR4. Signalling through the CXCL12–CXCR4 axis may contribute to the metastatic potential of ileal carcinoids. Targeting of HIFs and/or the CXCR4 signalling pathway may offer new therapeutic strategies for carcinoid tumour disease.
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Murphy, Philip T., Brendan p. Power, Patrick D. Thornton, and Judith H. Harmey. "Regulation of B-Cell Chronic Lymphocytic Leukaemia Cell Survival and Migration by the VEGF/SEMA3A Axis." Blood 112, no. 11 (November 16, 2008): 2083. http://dx.doi.org/10.1182/blood.v112.11.2083.2083.
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Abstract B-cell Chronic Lymphocytic Leukaemia (B-CLL) is characterized by the accumulation of B-CLL lymphocytes in the blood, marrow and secondary lymphoid tissues. B-CLL cells have a long survival owing to alterations in the normal pathways of apoptosis. In the marrow and lymphoid tissues CLL cells are in close contact with stromal cells that constitute distinct microenvironments. The secretion of the CXCR4 ligand, CXCL12, by stromal cells attracts B-CLL cells and provides protection from spontaneous or induced apoptosis. Studies in other cell types have shown VEGF signalling is involved in regulating CXCR4 expression levels. The aim of this study was to examine VEGF receptor expression and the role of VEGF signalling in cell survival and CXCR4 expression. Expression levels of CXCR4 and VEGF receptors, VEGFR-1 and -2, in CLL samples were determined by flow cytometry. Expression of the VEGFR co-receptor, Neuropilin-1 (NRP1), was examined by Western blot. Cell migration towards CXCL12 was assessed using CoStar Transwell chambers (5mm pore size). Informed consent was received from all patients. VEGFR1 and VEGFR2 positive cells in 22 patient samples ranged from 0–38% and 1.5–83% respectively. NRP1 expression was detected in all samples analysed thus far (n=6). The NRP1 ligand, SEMA3A, a competitive inhibitor of VEGF binding to NRP1, decreased CXCR4 expression in patient CLL cells (n=8, p<0.05). This decrease in CXCR4 levels correlated with reduced migration of SEMA3A treated CLL cells towards CXCL12 (88 +/− 12.7% of control levels, n=8, p<0.05). Treatment of CLL cells with the VEGFR signalling inhibitor SU5416 decreased CLL cell survival which correlated with VEGFR1 expression levels (n=16, R=0.745, p<0.01) but not with VEGFR2 expression levels. These results show that signalling through the VEGF co-receptor NRP1 plays an important role in regulating CXCR4 levels in CLL cells, as well as CLL cell migration along a CXCL12 gradient. Our results also suggest that VEGF signalling through VEGFR1 may be particularly important in regulating CLL cell survival. Thus, the VEGFR/NRP1 signalling pathway may represent an important therapeutic target in B-CLL.
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Tiveron, Marie-Catherine, and Harold Cremer. "CXCL12/CXCR4 signalling in neuronal cell migration." Current Opinion in Neurobiology 18, no. 3 (June 2008): 237–44. http://dx.doi.org/10.1016/j.conb.2008.06.004.
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Boujedidi, Hédia, Olivier Robert, Alexandre Bignon, Anne-Marie Cassard-Doulcier, Marie-Laure Renoud, Hélène Gary-Gouy, Patrice Hemon, et al. "CXCR4 dysfunction in non-alcoholic steatohepatitis in mice and patients." Clinical Science 128, no. 4 (October 17, 2014): 257–67. http://dx.doi.org/10.1042/cs20130833.
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The CXCL12/CXCR4 signalling pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of the chemokine CXCL12 to its main receptor CXCR4 is involved in this process.
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Torossian, Frederic, Aurélie Chabanon, Denis Clay, Bernadette Guerton, Adrienne Anginot, Richard Proust, Jean-Francois Ottavi, et al. "CXCR7 Functions Together with CXCR4 in SDF-1/CXCL12 Induced CD34+ Hematopoietic Progenitor Cell Cycling Through β-Arrestin 2-Dependent Akt Activation." Blood 120, no. 21 (November 16, 2012): 3448. http://dx.doi.org/10.1182/blood.v120.21.3448.3448.
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Abstract Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.
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Wright, Kathryn, Kumudika de Silva, Karren M. Plain, Auriol C. Purdie, Tamika A. Blair, Iain G. Duggin, Warwick J. Britton, and Stefan H. Oehlers. "Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis." PLOS Pathogens 17, no. 4 (April 7, 2021): e1009186. http://dx.doi.org/10.1371/journal.ppat.1009186.
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Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model ofMycobacterium marinuminfection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenicM.marinumand that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation ofcxcl12aandcxcr4bin infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown ofcxcl12aandcxcr4bexpression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.
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del Molino del Barrio, Irene, Georgina Wilkins, Annette Meeson, Simi Ali, and John Kirby. "Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12." International Journal of Molecular Sciences 19, no. 11 (November 14, 2018): 3592. http://dx.doi.org/10.3390/ijms19113592.
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Upon binding with the chemokine CXCL12, the chemokine receptor CXCR4 has been shown to promote breast cancer progression. This process, however, can be affected by the expression of the atypical chemokine receptor ACKR3. Given ACKR3’s ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differed from their lone expression in terms of their signalling pathways. We created single and double CXCR4 and/or ACKR3 Chinese hamster ovary (CHO) cell transfectants. ERK and Akt phosphorylation after CXCL12 stimulation was assessed and correlated with receptor internalization. Functional consequences in cell migration and proliferation were determined through wound healing assays and calcium flux. Initial experiments showed that CXCR4 and ACKR3 were upregulated in primary breast cancer and that CXCR4 and ACKR3 could form heterodimers in transfected CHO cells. This co-expression modified CXCR4’s Akt activation after CXCL12’s stimulation but not ERK phosphorylation (p < 0.05). To assess this signalling disparity, receptor internalization was assessed and it was observed that ACKR3 was recycled to the surface whilst CXCR4 was degraded (p < 0.01), a process that could be partially inhibited with a proteasome inhibitor (p < 0.01). Internalization was also assessed with the ACKR3 agonist VUF11207, which caused both CXCR4 and ACKR3 to be degraded after internalization (p < 0.05 and p < 0.001), highlighting its potential as a dual targeting drug. Interestingly, we observed that CXCR4 but not ACKR3, activated calcium flux after CXCL12 stimulation (p < 0.05) and its co-expression could increase cellular migration (p < 0.01). These findings suggest that both receptors can signal through ERK and Akt pathways but co-expression can alter their kinetics and internalization pathways.
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Schrader, A. J., O. Lechner, M. Templin, K. E. J. Dittmar, S. Machtens, M. Mengel, M. Probst-Kepper, et al. "CXCR4/CXCL12 expression and signalling in kidney cancer." British Journal of Cancer 86, no. 8 (April 2002): 1250–56. http://dx.doi.org/10.1038/sj.bjc.6600221.
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Kucia, Magda, Kacper Jankowski, Ryan Reca, Marcin Wysoczynski, Laura Bandura, Daniel J. Allendorf, Jin Zhang, Janina Ratajczak, and Mariusz Z. Ratajczak. "CXCR4–SDF-1 Signalling, Locomotion, Chemotaxis and Adhesion." Journal of Molecular Histology 35, no. 3 (March 2003): 233–45. http://dx.doi.org/10.1023/b:hijo.0000032355.66152.b8.
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Li, Nan, Yingying Wang, Haoyu Xu, Hexi Wang, Yingying Gao, and Yao Zhang. "Exosomes Derived from RM-1 Cells Promote the Recruitment of MDSCs into Tumor Microenvironment by Upregulating CXCR4 via TLR2/NF- κ B Pathway." Journal of Oncology 2021 (October 8, 2021): 1–9. http://dx.doi.org/10.1155/2021/5584406.
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Myeloid-derived suppressor cells (MDSCs) play a critical role in tumor immune escape because of its remarkable immunosuppressive effect. However, the mechanism of MDSCs migrated into tumor microenvironment remains unclear. In this study, we demonstrated the recruitment of MDSCs can be promoted by exosomes derived from prostate cancer cells, which could upregulate chemokine (CXC motif) receptor 4 (CXCR4) via the TLR2/NF- κ B signalling pathway. Flow cytometry detected that the percentage of MDSCs in the mice spleen and tumor tissue was significantly increased after injection with exosomes via mouse tail vein. Transwell chemotaxis assay showed the recruitment of MDSCs toward the lower chamber was enhanced after stimulation with exosomes, and the migration ability could be inhibited by AMD3100 (a CXCR4 specific inhibitor) both in vivo and in vitro. Additionally, Western blot and flow cytometry verified a remarkably increase of CXCR4 in MDSCs after incubation with exosomes; meanwhile, the protein level of TLR2 and activation of NF- κ B were also strengthened obviously. Nevertheless, after blocking TLR2 by C29 (a TLR2-specific inhibitor), the expression of p-p65 and CXCR4, which were hypothesized as the downstream target of TLR2, was prominently reduced. In conclusion, prostate cancer-derived exosomes could reinforce CXCR4 expression in MDSCs through the TLR2/NF- κ B signalling pathway, eventually promoting migration of MDSCs into tumor microenvironment in a CXCR4-CXCL12 axis-dependent manner.
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Ervin, J. M., S. Z. McIntosh, C. L. Runyan, and R. L. Ashley. "63 Inhibition of CXCR4 at the fetal-maternal interface during placentation results in altered production of vascular endothelial growth factor receptors in the placenta on Day 90 of pregnancy." Reproduction, Fertility and Development 32, no. 2 (2020): 157. http://dx.doi.org/10.1071/rdv32n2ab63.
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Placental development is characterised by extensive angiogenesis and vascularization; if these processes are compromised, placental dysfunction occurs, which is the underlying cause of complications such as preeclampsia and intrauterine growth restriction. The signalling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulate angiogenesis critical to placental vascularization. Our laboratory and others demonstrated stimulation of vascular endothelial growth factor (VEGF) synthesis by CXCL12/CXCR4 signalling, and recently, we reported less production of the VEGF receptor, FLT-1, on Day 20 in pregnant sheep following interference of intrauterine CXCL12-dependent signalling. While no animal model fully recapitulates human placentation, the sheep is arguably the most applicable animal model to study fetal-maternal interactions and placentation. Based on our studies, we hypothesised that inhibiting CXCR4 at the fetal-maternal interface during initial placentation alters placental production of VEGF receptors, FLT-1 and KDR, at mid-gestation. To test this hypothesis, AMD3100, a CXCR4 antagonist, was used to elucidate the role of CXCL12/CXCR4 signalling at the ovine fetal-maternal interface. On Day 12 post-breeding, osmotic pumps were surgically installed and delivered either AMD3100 or phosphate-buffered saline (PBS) into the uterine lumen ipsilateral to the corpus luteum for either 7 days (n=7 PBS and n=8 AMD3100) or 14 days (n=7 PBS and n=8 AMD3100). The objectives were to determine whether disruption of the CXCL12/CXCR4 axis during placentation affects fetal survival and alters VEGF receptor synthesis and whether duration of CXCR4 inhibition affects placental vascular remodelling. On Day 90 of pregnancy, ewes were anaesthetised; reproductive tracts were removed; and maternal caruncle (CAR) and fetal cotyledon (COT) components were separated, snap frozen in liquid nitrogen, and stored at −80°C until protein isolation. Pregnancy success was not affected by treatment or duration of treatment (71% PBS vs. 62% AMD3100 for 7 days; 85% PBS vs. 62% AMD3100 for 14 days). In addition, fetal weight on Day 90 (530.8±28.2 g PBS vs. 540.5±20.3g AMD3100 for 7 days; 494.3±23.9g PBS vs. 532.7±11.8g AMD3100 for 14 days) was not affected by treatment. Immunoblotting was used to detect protein abundance, and an unpaired two-tailed Student's t-test was used to determine significant changes. Greater FLT-1 (P<0.05) was evident in CAR and COT tissue on Day 90 for both the 7-day treatment (0.92±0.16 CAR PBS vs. 1.48±0.18 CAR AMD3100; 0.12±0.16 COT PBS vs. 0.62±0.16 COT AMD3100) and the 14-day treatment (0.18±0.05 CAR PBS vs. 0.43±0.001 CAR AMD3100; 0.04±0.005 COT PBS vs. 0.11±0.02 COT AMD3100) of CXCR4 inhibition compared with controls, whereas KDR levels did not change (P>0.05). Interestingly, elevated FLT-1, but not KDR, is a marker of preeclampsia in women, and because of its role as a VEGF scavenger, overexpression of FLT-1 often leads to an anti-angiogenic state. We suggest that CXCL12/CXCR4 signalling during initial placental development serves as an upstream regulator of placental vascularization, thereby ensuring appropriate placental development.
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Terheyden-Keighley, Daniel, Xiaoqing Zhang, Beate Brand-Saberi, and Carsten Theiss. "CXCR4/SDF1 signalling promotes sensory neuron clustering in vitro." Biology Open 7, no. 9 (August 22, 2018): bio035568. http://dx.doi.org/10.1242/bio.035568.
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Zieker, D., I. Koenigsrainer, S. Beckert, J. Glatzle, M. Loeffler, R. S. Taichman, and A. Koenigsrainer. "Effect of changes in the glycolytic metabolism on tumor progression and dissemination in gastric cancer." Journal of Clinical Oncology 29, no. 4_suppl (February 1, 2011): 47. http://dx.doi.org/10.1200/jco.2011.29.4_suppl.47.
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47 Background: Metastases are a frequent finding in gastric cancer patients and are associated with a poor prognosis. Recent data indicate that there might be a link between metabolic changes in the glycolytic system and dissemination in gastric cancer. The enhanced expression of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and its signalling targets CXCR4 and CXCL12 seem to play a crucial role in enabling gastric tumours to develop dissemination. Methods: Microarray analysis was conducted investigating human specimens from consecutive gastric cancer patients with dissemination versus gastric cancer patients without dissemination. Subsequently siRNA-knock- down concerning PGK1 and CXCR4 and transfection (overexpression) of PGK1 was performed. Further, our thesis was investigated in a metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumour implantation. Tumour growth was detected by PET-MR-imaging and section of the animals. Results: Microarray analysis revealed a significant overexpression of PGK1, CXCR4 and CXCL12 in gastric cancer specimens with dissemination. Further siRNA-knock-down of PGK1 and CXCR4 showed a significant co-regulation on expression and protein level in vitro. The transfection (overexpression) of PGK1 also revealed a significant upregulation of its signalling targets CXCR4 and CXCL12 on expression and protein level. In addition the transfected cells showed a 50-fold distinctive property in the invasion assay compared to cancer cells without PGK1 overexpression. The in-vivo results obtained in the mouse model showed that elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumours significantly and PET-MR-imaging and section correlated very well comparing tumour growth and metastasis. Conclusions: The overexpression of the glycolytic enzyme PGK1 and its signalling targets in gastric cancer might be a promising regulation-pathway promoting dissemination. This data may provide new prognostic markers and/or potential therapeutic targets to prevent migration of gastric carcinoma cells and has intriguing connection to an old hypothesis advocated by Otto Warburg for tumour metabolism. No significant financial relationships to disclose.
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Sun, Zejia, Xin Li, Xiang Zheng, Peng Cao, Baozhong Yu, and Wei Wang. "Stromal cell-derived factor-1/CXC chemokine receptor 4 axis in injury repair and renal transplantation." Journal of International Medical Research 47, no. 11 (October 3, 2019): 5426–40. http://dx.doi.org/10.1177/0300060519876138.
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Stem cell therapy has shown promise in treating a variety of pathologies, such as myocardial infarction, ischaemic stroke and organ transplantation. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) axis plays a key role in stem cell mobilization. This review describes the important role of SDF-1 in tissue injury and how it works in tissue revascularization and regeneration via CXCR4. Furthermore, factors influencing the SDF-1/CXCR4 axis and its clinical potential in ischaemia reperfusion injury, such as renal transplantation, are discussed. Exploring signalling pathways of the SDF-1/CXCR4 axis will contribute to the development of stem cell therapy so that more clinical problems can be solved. Controlling directional homing of stem cells through the SDF-1/CXCR4 axis is key to improving the efficacy of stem cell therapy for tissue injury. CXCR4 antagonists may also be effective in increasing circulating levels of adult stem cells, thereby exerting beneficial effects on damaged or inflamed tissues in diseases that are currently not treated by standard approaches.
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Poltavets, Valentina, Jessica W. Faulkner, Deepak Dhatrak, Robert J. Whitfield, Shaun R. McColl, and Marina Kochetkova. "CXCR4-CCR7 Heterodimerization Is a Driver of Breast Cancer Progression." Life 11, no. 10 (October 7, 2021): 1049. http://dx.doi.org/10.3390/life11101049.
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Metastatic breast cancer has one of the highest mortality rates among women in western society. Chemokine receptors CXCR4 and CCR7 have been shown to be linked to the metastatic spread of breast cancer, however, their precise function and underlying molecular pathways leading to the acquisition of the pro-metastatic properties remain poorly understood. We demonstrate here that the CXCR4 and CCR7 receptor ligands, CXCL12 and CCL19, cooperatively bind and selectively elicit synergistic signalling responses in invasive breast cancer cell lines as well as primary mammary human tumour cells. Furthermore, for the first time, we have documented the presence of CXCR4-CCR7 heterodimers in advanced primary mammary mouse and human tumours where number of CXCR4-CCR7 complexes directly correlate with the severity of the disease. The functional significance of the CXCR4-CCR7 association was also demonstrated when their forced heterodimerization led to the acquisition of invasive phenotype in non-metastatic breast cancer cells. Taken together, our data establish the CXCR4-CCR7 receptor complex as a new functional unit, which is responsible for the acquisition of breast cancer cell metastatic phenotype and which may serve as a novel biomarker for invasive mammary tumours.
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Borna, Simon, Ales Drobek, Jarmila Kralova, Daniela Glatzova, Iva Splichalova, Matej Fabisik, Jana Pokorna, et al. "Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis." Journal of Cellular and Molecular Medicine 24, no. 2 (December 17, 2019): 1980–92. http://dx.doi.org/10.1111/jcmm.14895.
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Pritchett, J., C. Wright, L. Zeef, and B. Nadarajah. "[P140]: Sdf‐1/Cxcr4 signalling regulates proliferation during cortical development." International Journal of Developmental Neuroscience 24, no. 8 (November 16, 2006): 556. http://dx.doi.org/10.1016/j.ijdevneu.2006.09.202.
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Ma, M., and G. C. F. Chan. "Mesenchymal Stem Cells Enhanced Metastasis of Neuroblastoma Via SDF-1/CXCR4 and SDF-1/CXCR7 Signalling." Biology of Blood and Marrow Transplantation 18, no. 2 (February 2012): S374—S375. http://dx.doi.org/10.1016/j.bbmt.2011.12.459.
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Wang, Jing, Tailang Yin, Yanqi Wen, Fuju Tian, Xiaojun He, Danni Zhou, Yi Lin, and Jing Yang. "Potential effects of interferon regulatory factor 4 in a murine model of polyinosinic-polycytidylic acid-induced embryo resorption." Reproduction, Fertility and Development 28, no. 10 (2016): 1631. http://dx.doi.org/10.1071/rd14499.
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Interferon regulatory factor (IRF) 4 has been reported to modulate Toll-like receptor (TLR) signalling. Polyinosinic-polycytidylic acid (poly(I:C)) can be specifically recognised by TLR3, triggering the innate immune response and subsequently resulting in pregnancy loss. In the present study, poly(I:C) was administered to mice with or without TLR3 blockade. Chemokine (C-X-C motif) receptor 4 (CXCR4) expression was measured with or without chemokine (C-X-C motif) ligand 12 (CXCL12) inhibition. In cultured murine splenic mononuclear cells, IRF4 was knocked down by a specific short interference (si) RNA. IRF4 mRNA and protein levels and T helper (Th) 17 cell frequencies in the poly(I:C)-treated group were significantly higher than in the phosphate-buffered saline (PBS)-treated control group, and were correlated with a significantly higher embryo resorption rate. Interleukin (IL)-17A and IL-21 levels were markedly lower in the IRF4 siRNA-treated group than in the non-specific siRNA- or vehicle control-treated groups. The CXCR4+ cell frequency was significantly higher among IRF4+ uterine mononuclear and granular cells (UMGCs) compared with IRF4– cells. Inhibition of CXCL12 significantly abrogated poly(I:C)-induced increases in the frequency of IRF4+CXCR4+ cells in UMGCs. IRF4 might play a critical role in TLR3 signalling, which mediates Th17 cell activation and upregulates the expression of IL-17A and IL-21, which results in pregnancy loss. CXCL12 may modulate IRF4+CXCR4+ cell migration at the fetomaternal interface. TLR3 and IRF4 blockade could potentially prevent spontaneous abortion under certain conditions.
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Zhao, Yaqian, Guanglan Pu, Yanan Li, Hong Jiang, Qiang Zhang, Ping Chen, Qing Lu, Mingjun Wang, and Rui Yang. "Serum Levels of CXCR4, SDF-1, MCP-1, NF-κB and ERK1/2 in Patients with Skeletal Fluorosis." International Journal of Environmental Research and Public Health 19, no. 24 (December 9, 2022): 16555. http://dx.doi.org/10.3390/ijerph192416555.
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C-X-C motif chemokine receptor 4 (CXCR4), stromal cell-derived factor-1 (SDF-1), monocyte chemoattractant protein-1 (MCP-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) affect bone cells and play an important role in bone and joint diseases, but the data on CXCR4, SDF-1, MCP-1, ERK1/2 and NF-κB in the serum of skeletal fluorosis (SF) patients are inconclusive. Thus, according to the “Diagnostic Criteria for Endemic Skeletal Fluorosis” (WS 192 - 2008), we enrolled patients with SF (n = 60) as the SF group and those without SF as the controls (n = 60). Serum levels of CXCR4, SDF-1, MCP-1, ERK1/2 and NF-κB were detected by enzyme-linked immunosorbent assays (ELISAs). Serum SDF-1, CXCR4, MCP-1 and NF-κB levels were significantly higher in the SF group than in the control group. Within the serum of SF patients, CXCR4 and SDF-1 levels were positively correlated with NF-κB levels. There was no correlation between MCP-1 levels and those of ERK1/2 or NF-κB. SDF-1 and CXCR4 may activate the NF-κB pathway, and MCP-1 affects the occurrence and development of SF by regulating osteocytes through other pathways. The SDF-1/CXCR4 axis and MCP-1 signalling pathway provide a new theoretical basis for the occurrence and development of SF.
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Bouamar, Hakim, Yanyan Zhang, Dima Jouni, Monika Wittner, Morad Bensidhoum, Peggy Jarrier, Hervé Petite, William Vainchenker, Olivier Albagli, and Fawzia Louache. "CXCR7 Expression Restricts CXCR4/SDF-1 Mediated Hematopoietic-Supporting Activity of Stromal Cells by Decreasing Extracellular SDF-1 Availability." Blood 114, no. 22 (November 20, 2009): 1452. http://dx.doi.org/10.1182/blood.v114.22.1452.1452.
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Abstract Abstract 1452 Poster Board I-475 Stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays a major role in regulating the interactions between hematopoietic stem and progenitor cells (HPSC) and their stromal microenvironment within the bone marrow. A second SDF-1/CXCL12 receptor, CXCR7 binds SDF-1 with high affinity but little is known about its function in hematopoiesis. In the present study, we demonstrate that the activity of CXCR7 is crucial for proper maintenance of hematopoietic activity on stromal layers. Using quantitative reverse transcription-PCR analysis, we demonstrate that CXCR7 is highly expressed in stromal cells in contrast to hematopoietic cells showing that it functions primarily in the stromal microenvironment compartment. CXCR7 stable expression in UT7 hematopoietic cells fails to support SDF-1 induced migration and signalling but inhibits migration of CXCR4 expressing cells in paracrine manner. Overexpression of CXCR7 in MS-5 stromal cells lead to a reduction of SDF-1 concentration in the supernatants. In addition, supernatants from these cells had substantially lower efficiency in promoting integrin alpha-4 beta-1–mediated adhesion and migration of Mo7e cells to vascular cell adhesion molecule-1 (VCAM-1) and CS-1/fibronectin than their control GFP counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1–dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to VCAM-1 when supernatants from CXCR7 expressing MS-5 cells were used compared with supernatants from GFP expressing cells. Finally, phenotypical primitive murine cells displayed reduced hematopoietic activity when cultured on CXCR7 expressing MS-5 cells. This reduced hematopoietic activity is partly reverted when recombinant SDF-1 was added to CXCR7 overexpressing MS-5 cells. Taken together, our results indicate that CXCR7-controlled disponibility of SDF-1 expression influences BM cell migration, adhesion and hematopoietic activity and thus behaves as a decoy receptor regulating the SDF-1 level. Disclosures Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.
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Simon, Anna, Dagmar Wider, Marie Follo, Johannes Waldschmidt, Martina Kleber, Ralph Waesch, and Monika Engelhardt. "Targeting The Protective Microenvironment In Multiple Myeloma (MM): An Analysis Of The CXCL12/CXCR4-Axis and Its Inhibitors AMD3100 and Nox-A12 Combined With Antimyeloma Substances, Such As Pomalidomide and Carfilzomib." Blood 122, no. 21 (November 15, 2013): 3851. http://dx.doi.org/10.1182/blood.v122.21.3851.3851.
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Abstract Introduction The interaction of malignant plasma cells (PC) with the bone marrow (BM) microenvironment is crucial for MM pathogenesis. The CXCL12/CXCR4-axis plays a key role in this cross-talk.CXCL12 induces homing of normal hematopoietic progenitor cells to the BM and also drives MM cells to their protective niche. The binding of CXCL12 to CXCR4 directly promotes PC survival and facilitates environment-mediated drug resistance, which ultimately renders antimyeloma substances ineffective. Therefore, targeting the CXCL12/CXCR4-axis in combination with the use of new antimyeloma therapeutics represents a promising approach in developing new treatment strategies. Methods Comparative analysis of three different CXCR4 antibody clones (12G5, 44717, 4G10) and their interaction with the CXCR4 inhibitor AMD3100 was performed by flow cytometry. The effect of CXCL12 on CXCR4 expression using the human MM cell lines (MMCLs) U266 and L363, with and without AMD3100, was assessed by flow- and image cytometry. CXCR4 downstream pathways were analysed by western blot. Pomalidomide and the specific proteasome inhibitor carfilzomib were tested in various concentrations, with and without AMD3100, using MMCLs (U266, L363) and BM samples from MM patients (n=4, BM infiltration >50%), with and without M210B4 stroma support. Cell viability was evaluated by trypan blue- and PI/annexin staining. Treatment effects on the expression of CD138, CD38 and CXCR4 were detected by flow- and image cytometry. The combination of carfilzomib with AMD3100 or NOX-A12 is currently being analysed regarding synergistic cytotoxicity and drug resistance. NOX-A12 is a PEGylated mirror-image oligonucleotide (Spiegelmer®) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity, thereby inhibiting CXCL12 signalling on both its receptors, CXCR4 and CXCR7. The effect of NOX-A12 is of eminent interest when compared to AMD3100 and with use of various antimyeloma agents. Results AMD3100 treatment of U266 cells reduced CXCR4 expression with the commonly used antibody (ab)-clone 12G5 by 44-fold and with clone 44717 by 5-fold. The binding of the ab-clone 4G10 (FITC- and PE-coupled) was not influenced by AMD3100 at the concentrations tested (20-200 µl/ml), making 4G10 the most reliable for CXCR4 analysis. We also established a protocol for image cytometry that allows imaging of high cell throughputs, assessing viability and the expression of intra- and extracellular CXCR4 with no need for transfection. Human U266 cells showed high extra- and intracellular CXCR4-expression, whereas the lower expression of CXCR4 in L363 cells was confirmed. CXCL12 induced a notable decrease in extracellular CXCR4 and increased intracellular CXCR4 expression, confirming the reliability of our image cytometry protocol. AMD3100 alone inhibited cell proliferation (p<0.01) in U266 monoculture at different time points (t1=24h, t2=3d), although it was not cytotoxic. In terms of CXCR4 expression, CXCL12-induced CXCR4 internalization was inhibited by AMD3100, even after long incubation periods (t=3d). The use of carfilzomib on MMCLs confirmed prior cytotoxic concentrations (20-100nM), whereas L363 cells were even more sensitive than U266 cells. M210B4 co-culture induced CXCR4 expression and tumor cell protection, however was not able to completely induce resistance to carfilzomib: use of 100nM carfilzomib remained substantially cytotoxic, decreasing overall cell numbers, CD138 positive cells and CXCR4-expression in U266 cells. Co-cultivation of MM patient-derived BM cells with M210B4 stroma substantially reduced cell apoptosis as described (Zlei,Engelhardt, Ex Hematol 2007, Udi, Engelhardt, BJH 2013). When primary MM cells were grown without stroma support, AMD3100 did not enhance pomalidomide-induced cytotoxic effects; however, when primary MM cells were co-cultured with M210B4, pomalidomide (100nM) and AMD3100 (50µM) inhibited cell proliferation, and the combination was significantly more effective than AMD3100 alone (p=0.018). Conclusion Our findings stress the importance of CXCL12 and its receptor CXCR4 in MM progression and environment-mediated drug resistance. Analysis of the CXCL12/CXCR4-axis, of its inhibitors AMD3100 and NOX-A12 as well as their combined effects with antimyeloma substances may pave the way to novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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De Toni, L., A. Di Nisio, S. Magagna, A. Michielan, M. Martinato, G. C. Sturniolo, R. D’Incà, C. Foresta, and A. Garolla. "Altered Chemokine Signalling in Endothelial Progenitor Cells from Acute Ulcerative Colitis Patients." Gastroenterology Research and Practice 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/843980.
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Ulcerative colitis (UC) is a chronic, idiopathic, inflammatory bowel disease, characterized by alternating stages of clinically active and inactive disease. UC exhibits several inflammatory characteristics, including immune activation, leukocyte infiltration, and altered vascular density. In UC, many of the upregulated inflammatory cytokines are proangiogenic and are released by diverse cell populations, such as infiltrating immune cells and endothelial cells (EC). Increasing evidences suggest that neovascularisation may involve also endothelial progenitor cells (EPCs). In this study we evaluated EPCs recruitment and homing, assessed by CXCR4 expression, in both acute and remitting phase of UC. We report an overall decrease of EPCs in UC patients (controls = 97,94 ± 37,34 cells/mL; acute = 31,10 ± 25,38 cells/mL; remitting = 30,33 ± 19,02 cells/mL;P<0.001for both UC groups versus controls). Moreover CXCR4+-EPCs, committed to home in inflammatory conditions, were found to be reduced in acute UC patients compared to both remitting patients and controls (acute = 3,13 ± 4,61 cells/mL; controls = 20,12 ± 14,0; remitting = 19,47 ± 12,83;P<0,001). Interestingly, we found that administration of anti-inflammatory drugs in acute UC is associated with an increase in circulating EPCs, suggesting that this therapy may exert a strong influence on the progenitor cells response to inflammatory processes.
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Foresta, Carlo, Luca De Toni, Sabina Magagna, Alessandro Galan, and Andrea Garolla. "Phosphodiesterase-5 Inhibitor Tadalafil Acts on Endothelial Progenitor Cells by CXCR4 Signalling." Current Drug Delivery 7, no. 4 (October 1, 2010): 274–82. http://dx.doi.org/10.2174/156720110793360595.
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Verma, Anant, Ganapathy Ayappa, and Deepak K. Saini. "Understanding the loss of CXCR4-mediated signalling in the presence of oxysterols." Biophysical Journal 121, no. 3 (February 2022): 194a. http://dx.doi.org/10.1016/j.bpj.2021.11.1762.
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Kovtonyuk, Larisa V., Markus G. Manz, and Hitoshi Takizawa. "Thrombopoietin-Receptor Signalling Induces Proliferation of Dormant HSC." Blood 120, no. 21 (November 16, 2012): 2343. http://dx.doi.org/10.1182/blood.v120.21.2343.2343.
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Abstract Abstract 2343 Lifelong blood production is maintained by a very rare population of self-renewing hematopoietic stem cells (HSCs) in bone marrow (BM). Proliferation, differentiation and survival of HSC toward stepwise hematopoietic cell development needs to be tightly controlled by cell intrinsic and extrinsic factors, as excess or insufficient production of mature blood cells potentially leads to neoplasia or aplasia. HSCs and progenitors (HSPCs) are equipped with cell surface receptors for different cytokines or chemokines (Kaushansky, NEJM 2006), and thus can integrate external signals, leading finally to proliferation and subsequent increase of hematopoietic cells in demand. Some of these regulatory pathways are already exploited in clinical settings: CXCR4 antagonists for HSPC mobilization, human granulocyte colony-stimulating factor (hG-CSF) for HSC mobilization and myeloid regeneration, and thrombopoietin agonists (THPO) for improving thrombocytopenia. However, despite their clinical use, little is known about the influence of these molecules on HSC. We established in vivo HSC divisional tracking with CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester). This allows to track single HSC division with high resolution, and subsequently to test biological activity of HSC-containing fractions (LKS) with different divisional history (Takizawa et al., JEM 2011). Using this system we evaluated the effects of systemic administration of human fms-related tyrosine kinase 3 ligand (hFlt3L), hG-CSF (Filgrastim), CXCR4 antagonist (AMD3100), and the THPO receptor (cMpl) agonist (Romiplostim) on HSC division. CFSE-labeled LKS were transferred into non-irradiated steady-state recipients. The non-dividing cell fraction was defined by the CFSE profile of CD4+CD62L+ T cells. One week after transplantation mice were injected with PBS or respective reagents daily or every other day for over one week. Three weeks after transfer, phenotypic BM analysis demonstrated that most of donor LKS had undergone several divisions while a small fraction of LKS remained undivided in PBS treated control mice (Figure 1a), containing long term self-renewing HSCs with at least 20–30% frequency (Takizawa et al., JEM 2011). Administration of hFlt3L increased the percentage of intermediate (1–5x divided) and fast cycling (>5x divided) LKS, which mainly contains CD150- Flt3+ multipotent progenitor cells (Figure 1b). Upon injections of cMpl agonist all donor LKS divided more than once, leaving no cells in quiescent fraction, with substantial expansion of CD150+ cells in the divided fraction. CXCR4 antagonist and hG-CSF administration had little effect on LKS proliferation. These data suggest that cMpl agonist drives dormant cells into proliferation, whereas hG-CSF has little effect on LKS division. To determine whether cMpl agonist increases the turnover of functionally defined, bona fide HSCs, we performed secondary transplantation of 0–1, 2–4, and ≥5x divided LKS. Twenty fast- (≥5x divided cells at 3 weeks), slow-cycling (2–4x divided) or relatively dormant LKS Flt3- cMpl+ cells (0–1x divided) were sorted from mice treated with PBS or cMpl agonist, and transplanted into lethally irradiated mice. Early results demonstrate increased percentage of secondary recipient engrafted with 2–4x divided cells from primary animals treated with cMpl agonist compared to those cells from PBS treated control Our results thus suggest that cMpl agonists have mitogenic activity not only for megakaryocyte progenitors but also for HSCs. How far this holds true in the human species needs to be determined. However, it should be taken in consideration given clinical data on evolution of pre-existing clonal myeloid diseases under cMpl agonist treatment (Dantoni, ASH abstract 2011), and also when treatment is applied long-term to patients with primary non-clonal hematopoietic diseases as immune thrombocytopenia or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.
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Pastore, Domenico, Anna Mestice, Margherita Giannoccaro, Arcangelo Liso, Maria Paola Martelli, Paola Carluccio, Francesco Albano, et al. "CXCR4 as a Predictor of Response in Acute Myeloid Leukemia." Blood 112, no. 11 (November 16, 2008): 2941. http://dx.doi.org/10.1182/blood.v112.11.2941.2941.
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Abstract The expression of CXCR4 (CD184) has been associated with poor prognosis in Acute Myeloid Leukemia (AML) and it has also been suggested that the CXCL12(SDF-1a)/CXCR4 interaction contributes to the resistance of leukemia cells to chemotherapy-induced apoptosis. Inhibition of CXCR4 was found to enhance chemotherapy-induced apoptosis in a subset of leukemic myeloblasts that carry Flt3 mutations and to overcome chemoresistance associated with stromal activity. NPM variants with a cytoplasmic localization represent the most common mutation detected in myeloid malignancies and are associated with a favourable clinical outcome. A recent study provides biological evidence for a novel role for NPM as a negative regulator of CXCR4 signalling induced by CXCL12: suppression of NPM expression enhanced chemotactic responses to CXCL12, and conversely, over-expression of a cytosolic NPM mutant reduced chemotaxis induced by CXCL12. We investigated whether CD184 expression is a negative predictor factor for response to chemotherapy and if there is clinical evidence that NPM mutations could overcome chemoresistance to induction therapy in this subset of patients. The expression of CD184 was analyzed by flow cytometric methods in a group of 70 cases of adult AML at onset of disease, diagnosed at our Institution since January 2006. The diagnosis was performed according to FAB/WHO criteria; all patients received intensive chemotherapy according to institutional protocols. There were 34 males and 36 females and median age was 46 years (range 18–65). AML cells were gated based upon their CD45 expression and samples were considered positive if CD184 was expressed by more than 20% of blasts. CD184 was positive in 45 and negative in 25 cases. There was no significant difference between the two groups in terms of sex, age, Hb level, WBC and Plt counts, percentage of blasts, and occurrence of the NPM mutation. The CR rate was 45% in CD184+ and 82% in CD184- (p=0.03); among CD184+ cases, the CR rate was significantly higher in NPMc+ cases, (p=0.03). Our results show that CD184 expression is associated with a lower rate of CR after induction therapy and this association is stronger in NPM unmutated cases, suggesting that CD184 expression is a negative predictive factor for response to chemotherapy. Further data are needed to verify if the biological role of the cytosolic NPM mutant as a negative regulator of CXCR4 signalling induced by CXCL12 could have a clinical role contributing to overcome the resistance of leukemic cells to induction chemotherapy.
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Vitale, Candida, Valentina Griggio, Chiara Riganti, Maria Todaro, Joanna Kopecka, Rebecca Jones, Chiara Salvetti, et al. "Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL." Cancers 13, no. 12 (June 9, 2021): 2883. http://dx.doi.org/10.3390/cancers13122883.
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The hypoxia-inducible factor 1 (HIF-1) and the CXCL12/CXCR4 axis regulate the interaction of chronic lymphocytic leukemia cells and the tumor microenvironment. However, the interconnections occurring between HIF-1 and the CXCL12/CXCR4 axis are not fully elucidated. Here, we demonstrate that the CXCL12/CXCR4 axis plays a pivotal role in the positive regulation of the α subunit of HIF-1 (HIF-1α) that occurs in CLL cells co-cultured with stromal cells (SC). Inhibitors acting at different levels on CXCR4 downstream signalling counteract the SC-induced HIF-1α upregulation in CLL cells, also hindering the SC-mediated pro-survival effect. HIF-1α inhibition also exerts off-tumor effects on the SC component, inducing the downregulation of target genes, including CXCL12. Consistently, our data show that pretreatment of leukemic cells and/or SC with idelalisib effectively abrogates the SC-mediated survival support. A combined on-tumor and off-tumor inhibition of HIF-1α was also observed in idelalisib-treated patients, who showed, along with a downregulation of HIF-1α target genes in leukemic cells, a significant decrease in CXCL12 serum concentration and changes in the bone marrow microenvironment. Our data demonstrate that the targeting of HIF-1α or its regulatory pathways acts at the tumor- and SC-level, and may be an appealing strategy to overcome the microenvironment-mediated protection of CLL cells.
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Bendall, Linda, Rana Baraz, Julius Juarez, Sylvie Shen, and Ken Bradstock. "Defective P38 MAPK Signalling Impairs Chemotactic but Not Proliferative Responses to SDF-1 in Acute Lymphoblastic Leukemia." Blood 104, no. 11 (November 16, 2004): 997. http://dx.doi.org/10.1182/blood.v104.11.997.997.
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Abstract The chemokine SDF-1 regulates leukemic cell motility and proliferation but the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1, a subset of cases (18%) did not undergo chemotaxis in response to SDF-1 (unresponsive cases). These unresponsive cases also failed to increase b1 integrin-mediated adhesion to fibronectin or adhesion to bone marrow fibroblast layers. However the unresponsive cases could still elicit a calcium flux in response to SDF-1 and three of the four cases internalised the receptor, CXCR4, following exposure to SDF-1. In contrast, the CXCR4 antagonist, TC14012, inhibited proliferation of both responsive and unresponsive cases in stromal-dependent cultures, demonstrating that the unresponsive cases were still able to undergo proliferative responses to SDF-1. Examination of the signalling pathways activated by SDF-1 in responsive cells revealed increased phosphorylation of AKT, ERK and p38 MAPK 2 to 5 minutes following stimulation. However, cells from the unresponsive cases failed to phosphorylate p38 MAPK kinase when stimulated with SDF-1, while phosphorylation of AKT and ERK were comparable with that observed in responsive ALL cases. Inhibition of p38 MAPK by SB203500 completely inhibited the chemotaxis of responsive ALL cells to SDF-1 gradients suggesting that signalling through p38 MAPK is essential for ALL cell chemotaxis. Therefore it is likely that the absence of p38 MAPK phosphorylation in unresponsive cases underlies their lack of chemotaxis to this chemokine. The ability of the unresponsive cases to undergo SDF-1 driven proliferation in the absence of p38 MAPK phosphorylation suggests that, despite being absolutely required for chemotactic responses, induction of phosphorylation of p38 MAPK is not required for proliferative responses. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in NOD/SCID mice. This study suggests that signalling through p38 MAPK is required for ALL cell chemotaxis, but not for proliferation, and that chemotactic responses to SDF-1 are not essential for leukemic cell engraftment.
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Khatun, Hazera, Amna Anwar, Majid A. Kazmi, Stephen Schey, and Yolanda Calle. "The Wiskott Aldrich Syndrome Protein (WASP) Is Involved in Dexamethasone-Signalling Pathways Leading to Apoptosis of Multiple Myeloma Cells and in Cell Adhesion Mediated Drug Resistance Against Dexamethasone." Blood 118, no. 21 (November 18, 2011): 1809. http://dx.doi.org/10.1182/blood.v118.21.1809.1809.
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Abstract Abstract 1809 Direct contact of multiple myeloma (MM) cells with the bone marrow (BM) stroma promotes cell survival leading to cell adhesion mediated drug resistance (CAM-DR). Dexamethasone is a conventional anti-MM drug that effectively induces MM cell death at presentation and in relapsed patients but the signalling pathways involved in its mechanisms of action are not completely understood. Resistance of MM to Dexamethasone may result from genetic changes in MM cells or through the contact of MM cells with the BM microenvironment. It has been shown that CAM-DR blocks the effect of Dexamethasone by the binding of MM cells to the BM stroma. Our data indicate a key role of the binding of MM CXCR4 receptor on stromal cell derived SDF1-a in CAM-DR against Dexamethasone. Dexamethasone induced upregulation of CXCR4 in the MM1.S cell line (sensitive to Dexamethasone) whilst, as expected, having no effect on the MM1.R cell line (resistant to Dexamethasone by expression of a mutated form of the glucocorticoid receptor). Bortezomib induced down regulation of CXCR4 in both MM1.S and MM1.R cell lines in a dose dependent manner that correlated with decreased adhesion on BM stromal cells and increased sensitisation of MM cells in the presence of the BM stroma. The Wiskott Aldrich Syndrome Protein (WASP) is an adaptor protein that regulates actin polymerization and organization of cell adhesion molecules of the integrin family in haematopoietic cells. WASP is involved in the signalling pathway downstream of CXCR4 in various leukocytes and we hypothesised that blocking this pathway would prevent MM cell adhesion on stromal cells and sensitise them to treatment with Dexamethasone. We found that downregulation of WASP in the Dexamethasone-sensitive cell line MM1.S using shRNA reduced the adhesion to the BM stroma. However, it also rendered MM1.S cells resistant to treatment with Dexamethasone but not with Bortezomib, Doxorubicin or Melphalan independently of the presence of BM stromal cells. Similarly, downregulation of WASP in MM1.R cells did not affect their sensitivity to Bortezomib, Doxorubicin or Melphalan. Dexamethasone has been shown to induce changes in actin dynamics or in levels or activity of actin and/or adhesion related proteins. Western blot analysis showed both Dexamethasone and Bortezomib to cause modulation of WASP expression, inducing partial downregulation and upregulation, respectively. Downregulation of WASP expression in MM1.S cells using shRNA or treatment with Dexamethasone or Bortezomib did not affect the expression of other proteins involved in WASP-mediated F-actin remodelling or cell adhesion such as WIP, Arp 2/3 or vinculin. We conclude that WASP is involved in the signalling pathways that promote cell-adhesion of MM cells on BM stroma but it is also a vital component of the signalling pathway of Dexamethasone's mechanism of action. These data indicate that while blocking WASP signalling could theoretically have potential therapeutic benefits to prevent CAM-DR to Dexamethasone, it would inhibit the action of Dexamethasone rendering cells resistant to treatment with this drug. Our study suggest a possible paradox of a dual pro-apoptotic and pro-survival effect of certain therapies targeting pathways involved in the interaction of MM cells with the microenvironment. Detailed studies of the intricate connexion between the intracellular pathways involved in the process of adhesion and drug induced cell death through modulation of actin dynamics may lead to improved therapeutic strategies to overcome drug resistance against Dexamethasone and perhaps other drugs. Disclosures: No relevant conflicts of interest to declare.
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Dong, Yonghui, Hui Liu, Xuejun Zhang, Fei Xu, Liang Qin, Peng Cheng, Hui Huang, Fengjing Guo, Qing Yang, and Anmin Chen. "Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis." International Journal of Molecular Sciences 17, no. 6 (June 16, 2016): 943. http://dx.doi.org/10.3390/ijms17060943.
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Selvaraj, P., D. He, C. Boi-Doku, J. Kearney, R. Yellon, S. Davidson, and D. Yellon. "253 REMOTE ISCHAEMIC PRECONDITIONING IS MEDIATED VIA THE SDF 1Α/CXCR4 SIGNALLING AXIS." Heart 99, suppl 2 (May 2013): A134.1—A134. http://dx.doi.org/10.1136/heartjnl-2013-304019.253.
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Fiegl, Michael, Ismael J. Samudio, Karen Clise Dwyer, Jared Burks, Herbert Fritsche, Zakar H. Mnjoyan, and Michael Andreeff. "CXCR4 Expression and Biological Activity Is Dependent on Oxygen Partial Pressure in Acute Myeloid Leukemia." Blood 112, no. 11 (November 16, 2008): 938. http://dx.doi.org/10.1182/blood.v112.11.938.938.
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Abstract CXCR4, the receptor for bone marrow stroma derived SDF-1, has recently been studied in normal hematopoiesis and hematologic malignancies. Increased expression of CXCR4 by leukemic blasts has been reported by us and others (Konoplev S. et al, Cancer 2007) to be associated with poor prognosis in acute myeloid leukemia (AML). However, all in-vitro studies are usually carried out under unphysiological, i.e. normoxic (21% O2) conditions. We hypothesized that the pO2 in vitro has major impact on the expression of CXCR4, a key receptor for cell migration and intracellular signalling. Thus, pO2 of bone marrow aspirates was measured using the i-STAT Portable Clinical Analyzer and a hypoxic workstation was used providing constant low oxygen content. Surface and total CXCR4 expression was examined in leukemic cell lines and patient samples by flow cytometry, confocal microscopy and Western blotting (WB). In 19 patients, the median pO2 of the bone marrow was determined as 46.1±12.8 mmHg (6.1±1.7%) with no significant difference between patients with AML (n=7, pO2 41.3±11.2 mmHg) and patients in CR (n=12, pO2 48.3±15.9 mmHg). This level of hypoxia significantly increases surface and total expression of CXCR4 in the leukemic cell lines U937 and OCI-AML3 as well as in samples from patients with AML, as compared to normoxic conditions (~2.8fold increase). This increase happened mainly within the first 2–8 hours of hypoxia and was unrelated to increased CXCR4 transcription, as shown by PCR. Re-oxygenation of leukemic cells resulted in a statistical significant degradation of CXCR4 (~3fold decrease) in all examined cell lines and patient samples (n=10). This loss of CXCR4 is very rapid (within 5 minutes of re-oxygenation) and was detected by flow cytometry, confocal microscopy and WB. This phenomenon was independent of proteasome activity and ATP. Detailed analysis of membraneous lipid rafts by sucrose density separation, cholesterol depletion and flow cytometry analysis for GM1 gangliosides showed structural (distinct re-distribution of Lck in lipid rafts) and quantitative changes (loss of cholesterol and CXCR4) during re-oxygenation. Moreover, part of the loss of CXCR4 can be attributed to sequestration of microparticles into the extracellular environment as shown by WB of supernatant of re-oxygenated cells and by a significant increase (~1.5fold) in the amount of microparticles released by cells (cell lines U937 and OCI-AML3 and additional patient samples) during the process of re-oxygenation, as measured by flow cytometry. In summary, this study determined the oxygen content of CR and leukemic bone marrow samples as 6.1±1.7%. This pO2 is associated with an increase in CXCR4 expression on AML cells, while re-oxygenation leads to a rapid decrease of CXCR4, perhaps in part by shedding of CXCR4- containing microparticles. These studies point to the importance of studying leukemic blasts under physiologic, i.e. hypoxic conditions.
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Vitale, Candida, Valentina Griggio, Chiara Riganti, Ivana Campia, Marta Robino, Micol Rigoni, Patrizia Sciancalepore, et al. "The Mevalonate Metabolic Pathway and the CXCL12/CXCR4 Axis Reciprocally Interact and Are Implicated in Fludarabine Resistance of Chronic Lymphocytic Leukemia Cells." Blood 124, no. 21 (December 6, 2014): 833. http://dx.doi.org/10.1182/blood.v124.21.833.833.
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Abstract BACKGROUND: Treatment of fludarabine-resistant chronic lymphocytic leukemia (CLL) patients is an unmet clinical need. Fludarabine resistance in CLL depends on intrinsic molecular features of the tumor cells, and on bidirectional interactions occurring between CLL cells and stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis. CXCR4 is a G protein-coupled receptor constitutively expressed on CLL cells. The binding of CXCR4 with CXCL12 activates the Ras/ERK1-2/Akt and the RhoA-dependent signalling pathways. To be active transducers Ras and RhoA need to undergo a post-translational modification (i.e. isoprenylation) by means of small molecules produced by the mevalonate (Mev) pathway. We have recently demonstrated that the Mev pathway is more active in IGHV unmutated than in mutated CLL cells, and is amenable to pharmacological manipulation by statins (i.e. simvastatin [Sim]). It is currently unknown whether the Mev pathway and its pharmacological targeting are implicated in the modulation of the CXCL12/CXCR4 axis and in the SC-induced fludarabine resistance of CLL cells. AIM: The aim of this study was to investigate the reciprocal interactions between the Mev pathway and the CXCL12/CXCR4 axis, in order to identify potential targets to counteract the constitutive and SC-induced fludarabine resistance of CLL cells. METHODS: Immuno-magnetically purified patient-derived CLL cells were cultured alone or with murine SC (M2-10B4 cell line). In selected experiments, cell cultures were exposed to human recombinant CXCL12 (100 μg/ml), CXCR4 inhibitor AMD3100 (5 μg/ml), fludarabine (F-ara-A, 10 μM), Sim (1 μM), ERK1-2 kinase inhibitor PD98059 (10 μM), RhoA kinase inhibitor Y276 (10 μM), HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by the quantification of metabolites [i.e. cholesterol and farnesyl pyrophosphate (FPP)] produced by CLL cells after 24 h incubation with 1 μCi of [3H]acetate. Ras and RhoA activities were evaluated measuring their GTP-bound fraction, taken as an index of the G-protein activation, respectively by pull-down assay and by an ELISA based assay. ERK1-2 and HIF-1α phosphorylation were evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay kit. The amount of CXCL12 in culture supernatants was assessed by ELISA assay. Cell viability was determined by Annexin-V/propidium Iodide immunostaining and flow cytometry analysis. RESULTS: Co-culture with SC upregulated the Mev pathway activity of CLL cells, as shown by the increased production of cholesterol and FPP. This SC-induced increase in the Mev pathway activity was followed by the activation of the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, the upregulation of the pro-survival factor Akt, and an increase in the transcriptional activity of HIF-1α. These biological and molecular effects were identically observed when CLL cells were exposed to recombinant CXCL12, and were completely abrogated by the CXCR4 antagonist AMD3100, thus showing the key role of the CXCL12/CXCR4 axis in the SC-induced modulation of the Mev pathway and the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling. On the other hand, blocking the Mev pathway by Sim and targeting ERK1-2 kinases, RhoA kinase and HIF-1α by specific small-molecule inhibitors significantly reduced the constitutive activity and the SC-induced upregulation of the Ras/ERK1-2 and RhoA/RhoA kinase signal transduction. A regulatory role of the Mev pathway on the CXCL12/CXCR4 axis was observed not only in CLL cells but also in SC, as shown by the significant reduction in CXCL12 secretion by SC exposed to Sim. In the last set of experiments, we found that the inhibition of the Mev pathway by Sim potentiated the direct cytotoxic effect of fludarabine against CLL cells. Even more importantly, both Sim and the downstream HIF-1α inhibitor YC-1 were capable of counteracting the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. CONCLUSIONS: Our data demonstrate that the Mev pathway has a regulatory role on the CXCL12/CXCR4 axis and on the SC-mediated protective effects toward spontaneous and fludarabine-induced CLL cell death. The upstream inhibition of the Mev pathway and the downstream targeting of HIF-1α are promising strategies to circumvent fludarabine resistance in CLL. Disclosures Boccadoro: Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Verdelli, C., L. Avagliano, P. Creo, V. Guarnieri, A. Scillitani, L. Vicentini, G. B. Steffano, et al. "Tumour-associated fibroblasts contribute to neoangiogenesis in human parathyroid neoplasia." Endocrine-Related Cancer 22, no. 1 (December 16, 2014): 87–98. http://dx.doi.org/10.1530/erc-14-0161.
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Components of the tumour microenvironment initiate and promote cancer development. In this study, we investigated the stromal component of parathyroid neoplasia. Immunohistochemistry for alpha-smooth muscle actin (α-SMA) showed an abundant periacinar distribution of α-SMA+ cells in normal parathyroid glands (n=3). This pattern was progressively lost in parathyroid adenomas (PAds; n=6) where α-SMA+cells were found to surround new microvessels, as observed in foetal parathyroid glands (n=2). Moreover, in atypical adenomas (n=5) and carcinomas (n=4), α-SMA+ cells disappeared from the parenchyma and accumulated in the capsula and fibrous bands. At variance with normal glands, parathyroid tumours (n=37) expressed high levels of fibroblast-activation protein (FAP) transcripts, a marker of tumour-associated fibroblasts. We analysed the ability of PAd-derived cells to activate fibroblasts using human bone-marrow mesenchymal stem cells (hBM-MSCs). PAd-derived cells induced a significant increase in FAP and vascular endothelial growth factor A (VEGFA) mRNA levels in co-cultured hBM-MSCs. Furthermore, the role of the calcium-sensing receptor (CASR) and of the CXCL12/CXCR4 pathway in the PAd-induced activation of hBM-MSCs was investigated. Treatment of co-cultures of hBM-MSCs and PAd-derived cells with the CXCR4 inhibitor AMD3100 reduced the stimulated VEGFA levels, while CASR activation by the R568 agonist was ineffective. PAd-derived cells co-expressing parathyroid hormone (PTH)/CXCR4 and PTH/CXCL12 were identified by FACS, suggesting a paracrine/autocrine signalling. Finally, CXCR4 blockade by AMD3100 reduced PTH gene expression levels in PAd-derived cells. In conclusion, i) PAd-derived cells activated cells of mesenchymal origin; ii) PAd-associated fibroblasts were involved in tumuor neoangiogenesis and iii) CXCL12/CXCR4 pathway was expressed and active in PAd cells, likely contributing to parathyroid tumour neoangiogenesis and PTH synthesis modulation.
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Hart, Derek, Courtney Modra, and Georgina Clark. "CD300f Triggering Modifies Myeloid Cell Function." Blood 112, no. 11 (November 16, 2008): 1255. http://dx.doi.org/10.1182/blood.v112.11.1255.1255.
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Abstract CD300f, a member of the CD300 family of immunoregulatory molecules, is capable of signalling through association with both SHP-1 phosphatase and the p85α subunit of phosphoinositide 3-kinase. On normal cells, CD300f is expressed on monocytes and dendritic cells in the blood and bone marrow. CD300f is expressed on myeloid derived cell lines and Acute Myeloid Leukaemias (AMLs) and is an acknowledged candidate for antibody targeting of AML (Modra CJ et al. Blood2006;108:225B-225B and Zhao X et al Blood2007;110:531a–532a). We have generated a monoclonal antibody, MMRI- 23, specific for the extracellular domain of CD300f. MMRI-23 immunoprecipitates a 57kd protein from the myeloid derived cell lines HEL, THP-1 and U937 and this protein binds to a polyclonal antibody to CD300f (LMIR3) in Western blot analysis. Binding of MMRI-23 to myeloid cells was blocked by the CD300f recombinant proteins or the polyclonal CD300f antibody. The MMRI-23 mAb was used as a surrogate ligand to study the functional consequences of crosslinking CD300f on normal monocytes and myeloid derived cell lines. Purified peripheral blood monocytes were cultured for 18 hours in the presence of immobilised MMRI-23 or control mAb. MMRI-23 binding was not altered by activation of peripheral blood monocytes but crosslinking monocytes with MMRI- 23 induced downregulation of CD14 and CD33. There was significant inhibition of IL-6 but not IL-1β, IL-8 or TNFα secretion. Crosslinking monocytes or the U937 cell line with MMRI-23 increased specific chemotaxis towards CXCL12 (SDF-1). This increased migration index following MMRI-23 crosslinking was reversed in the presence of wortmannin indicating that MMRI-23 induces signalling through phosphoinositide 3-kinase. The effect of crosslinking did not enhance CXCR4 upregulation but did induce localization of CD300f and CXCR4 to the lipid rafts in myeloid cell line, U937. Thus CD300f plays a significant role in the regulation of monocyte migration to CXCR4. As CD300f is upregulated on around 70% of AMLs, this regulation of homing has major implications for the treatment of AML.
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Luo, Tingting, Hongrui Liu, Wei Feng, Di Liu, Juan Du, Jing Sun, Wei Wang, et al. "Adipocytes enhance expression of osteoclast adhesion-related molecules through the CXCL12/CXCR4 signalling pathway." Cell Proliferation 50, no. 3 (November 21, 2016): e12317. http://dx.doi.org/10.1111/cpr.12317.
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Zuccarello, D., A. Ferlin, A. Garolla, M. Menegazzo, L. Perilli, G. Ambrosini, and C. Foresta. "How the human spermatozoa sense the oocyte: a new role of SDF1-CXCR4 signalling." International Journal of Andrology 34, no. 6pt2 (May 30, 2011): e554-e565. http://dx.doi.org/10.1111/j.1365-2605.2011.01158.x.
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Zheng, F., V. Flamini, R. Bradbury, Z. Zhang, W. G. Jiang, and Y. Cui. "CXCR4 promotes adhesion capacity and activates the AKT signalling pathway in colorectal cancer cells." European Journal of Cancer 72 (February 2017): S68. http://dx.doi.org/10.1016/s0959-8049(17)30302-7.
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Dimova, Ivanka, Swapna Karthik, Andrew Makanya, Ruslan Hlushchuk, David Semela, Vladislav Volarevic, and Valentin Djonov. "SDF‐1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception." Journal of Cellular and Molecular Medicine 23, no. 6 (April 4, 2019): 3916–26. http://dx.doi.org/10.1111/jcmm.14269.
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Cuthill, Kirsty, Yan Zhang, Andrea Buggins, Eve Coulter, Piers E. Patten, Derek Macallan, and Stephen Devereux. "In-Vivo Labelling Studies in Patients with Chronic Lymphocytic Leukemia Studies Demonstrate the Existence of Apparently Distinct Subpopulations That Differ in Phenotype and Proliferative Capacity." Blood 126, no. 23 (December 3, 2015): 615. http://dx.doi.org/10.1182/blood.v126.23.615.615.
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Abstract It is now generally believed that proliferation of the neoplastic clone in chronic lymphocytic leukemia (CLL) takes place in lymphoid tissues where interactions involving the B-cell receptor (BCR) and other microenvironmental elements take place. Previous studies using in-vivo labelling with deuterated water have shown that recently proliferated emigrants from lymphoid tissues express low levels of the chemokine receptor CXCR4 and high levels of CD5 (CXCR4loCD5hi). It has been proposed that, following entry into the peripheral blood (PB), these cells become quiescent, re-express CXCR4 and downregulate CD5 allowing re-entry into tissues and further rounds of proliferation. In the present study we used in-vivo labelling with deuterated glucose (6,6-2H2-glucose, D2G) to investigate the proliferation and release of CLL cells into PB. In contrast to deuterated water, this technique allows pulse labelling of a distinct cohort of cells, which can then be tracked over time in-vivo. Labelling studies were performed in 10 patients with previously untreated, non-progressive CLL. Patients underwent 10 hours of labelling with oral 2DG, after which peripheral blood and lymph node compartments were serially sampled. DNA Deuterium enrichment was measured in the entire CLL population and in flow-sorted subsets defined by CXCR4/CD5 and surface IgM (sIgM) expression. Maximum release of labelled cells into PB occurred after a median of 14 days (4-56 days). The disappearance rate was very slow, with labelled cells detectable after 56 days in half of the subjects. In one case we were able to track labelled cells in both lymph node (LN) and PB and demonstrated an increase in the fraction of labelled cells in the LN between days 7 and 28, consistent with re-entry into this compartment. Subpopulations of PB CLL cells, defined by CXCR4 and CD5 expression were studied over time to investigate the dynamic nature of these molecules in circulating cells. As previously reported, maximum rapid incorporation of deuterium was observed in the CXCR4loCD5hi fraction, equivalent to 1.15 ± 0.04 %/d fractional synthesis at 7 days. Conversely, CXCR4hiCD5lo cells remained largely unlabelled throughout the 8-week study, reaching a maximum of only 0.01 ± 0.003 %/d, suggesting that they represent a non-proliferating population, not derived from the CXCR4loCD5hi subset. In contrast, CXCR4/CD5 intermediate cells exhibited delayed and intermediate labelling, peaking at 0.1 ± 0.02 %/d at 28 days. This sequential labelling pattern suggests that they derive from the CXCR4loCD5hi subset. Since both CXCR4 and CD5 expression are modulated by BCR signalling, we went on to sort cells according to sIgM expression and found maximum labelling in the sIgM high subset with little or no deuterium incorporation in the sIgM low fraction at any time. Again, the sIgM intermediate subset showed delayed labelling, suggesting that they are derived from sIgM-high cells. These observations provide further evidence for clonal heterogeneity in CLL and suggest the existence of distinct but interdependent subpopulations. Recently-divided cells are CXCR4loCD5hi, but appear to transition to an intermediate phenotype by down-regulation of CD5 and sIgM and upregulation of CXCR4 over the ensuing weeks, but do not appear to transition to CXCR4hiCD5lo cells over the time-course of the experiment. Our findings have clinical relevance, since these functionally distinct subsets might also differ in their responsiveness to therapeutic agents, such as drugs that block BCR signalling. Figure 1. Deuterium enrichment in CLL subsets over eight week study Figure 1. Deuterium enrichment in CLL subsets over eight week study Disclosures No relevant conflicts of interest to declare.
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Vlad, Amalia, Pierre-Antoine Deglesne, Remi Letestu, Nathalie Chevallier, Fanny Baran-Marszak, Nadine Varin-Blank, Florence Cymbalista, and Dominique Ledoux. "CXCR4 and CD62L Down-Regulation Following B-Cell Receptor Ligation Is Restricted to Progressive Chronic Lymphocytic Leukemia (CLL) Cases." Blood 110, no. 11 (November 16, 2007): 1122. http://dx.doi.org/10.1182/blood.v110.11.1122.1122.
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Abstract B cell receptor (BCR) mediated survival plays a central role in disease progression of CLL. We have previously shown that BCR engagement allowed the identification of two groups of patients with a strong correlation between in vitro cell survival response capacity and clinical stage or prognostic factors. The aim of this study was to determine the implication of BCR stimulation in the accumulation of CLL cells in the enlarged lymph nodes of progressive cases. Therefore, we investigated the in vitro migratory capacity of CLL cells and the level of expression of microenvironment interacting molecules upon BCR stimulation. Surface expression of two membrane proteins: CXCR4 and CD62L were dramatically reduced in 40% of CLL cases after BCR engagement. CXCR4/CXCL12 axis and the L-selectin (CD62L) are critical for trafficking of B cells into lymph node, germinal center organization as well as lymphocyte exit from the lymph node. Peripheral blood mononuclear cells obtained from 27 untreated patients were purified and stimulated with immobilized anti-IgM for 48h. Presence of CXCR4 and CD62L at the cell surface was then measured by flow cytometry in CD19-positive cells. The CXCL12-dependant migratory capacity of B-CLL cells was analysed using a Transwell chemotaxis assay before and after BCR stimulation. BCR stimulation induced over 90% decrease of both CXCR4 and CD62L membrane expression in 11/27 cases. Importantly, this strong down-regulation of CXCR4 and CD62L was restricted to progressive cases with lymphadenopathy and unfavourable prognostic markers (unmutated IgVH, expression of ZAP70, high level of proliferation markers). These cases also showed marked increase of in vitro cell survival upon BCR engagement. Conversely, in the 6/27 cases corresponding to stable stage A patients with favourable prognostic markers, and absence of BCR mediated in vitro survival enhancement, no decrease of CXCR4/CD62L expression upon BCR stimulation was observed. We demonstrated that the down-regulation of CXCR4 from cell surface was associated with the internalization of the receptor mainly through clathrin-mediated endocytosis. CXCR4 down-regulation was associated with a reduced capacity of the cells to migrate in response to CXCL12 gradient. Indeed, migration was not affected in the 6 stable cases. Finally, the 10/27 remaining cases exhibited an intermediate down-regulation of CXCR4 and CD62L. The remaining level of expression of CXCR4 was strikingly correlated to CD62L level in all cases(y= 0.99x + 4.44; R2=0.893). This matching variation of both surface molecules reflects the cellular heterogeneity of response to BCR engagement in a given patient. In conclusion, our results show that BCR engagement induces a strong down-regulation of CXCR4 and CD62L, and the subsequent decrease of migratory capacity of the cells in progressive CLL cases only. These experiments strongly suggest that BCR signalling capacity is linked to the down-regulation of cell surface markers that favour a reduced lymphocyte trafficking and the maintenance of a proliferative cellular pool within the lymph nodes.
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Wang, Rongrong, Jiaming Qian, Da Ji, Xingyu Liu, and Ranran Dong. "Transcriptome Analysis Reveals Effect of Dietary Probiotics on Immune Response Mechanism in Southern Catfish (Silurus meridionalis) in Response to Plesiomonas shigelloides." Animals 13, no. 3 (January 28, 2023): 449. http://dx.doi.org/10.3390/ani13030449.
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To explore whether a probiotic complex composed of Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei can prevent or inhibit the inflammatory response caused by the invasion of Plesiomonas shigelloides in the southern catfish, we screened differentially expressed genes and enriched inflammation-related pathways among a control and three experimental groups and conducted analysis by transcriptome sequencing after a 21-day breeding experiment. Compared with those in the PS (Plesiomonas shigelloides) group, southern catfish in the L-PS (Lactobacillus-Plesiomonas shigelloides) group had no obvious haemorrhages or ulcerations. The results also showed that inflammation-related genes, such as mmp9, cxcr4, nfkbia, socs3, il-8, pigr, tlr5, and tnfr1, were significantly upregulated in the PS group compared with those in the L-PS groups. In addition, we verified six DEGs (mmp9, cxcr4, nfkbia, socs3, rbp2, and calr) and three proteins (CXCR4, NFKBIA, and CALR) by qRT-PCR and ELISA, respectively. Our results were consistent with the transcriptome data. Moreover, significantly downregulated genes (p < 0.05) were enriched in inflammation-related GO terms (lymphocyte chemotaxis and positive regulation of inflammatory response) and immune-related pathways (intestinal immune network for IgA production and IL-17 signalling pathway) in the L-PS vs. the PS group. Our results indicate that the infection of P. shigelloides can produce an inflammatory response, and probiotics could inhibit the inflammatory response caused by P. shigelloides to some extent.
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Triantafilou, Martha, Kensuke Miyake, Douglas T. Golenbock, and Kathy Triantafilou. "Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation." Journal of Cell Science 115, no. 12 (June 15, 2002): 2603–11. http://dx.doi.org/10.1242/jcs.115.12.2603.
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The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.