Expression Of Functional CXCR4 Research Articles

The independent, unfavorable prognostic factors endothelin A receptor and chemokine receptor 4 have a close relationship in promoting the motility of nasopharyngeal carcinoma cells via the activation of AKT and MAPK pathways

BackgroundRecent studies have indicated that the expression of endothelin A receptor (ETAR) and chemokine receptor 4 (CXCR4) could be used as an indicator of the metastatic potential of nasopharyngeal carcinoma (NPC). The aim of this study was to determine the prognostic value of ETAR and CXCR4 in NPC patients and to reveal the interplay of the endothelin-1 (ET-1)/ETAR and stromal-derived factor-1(SDF-1)/CXCR4 pathways in promoting NPC cell motility.MethodsSurvival analysis was used to analyze the prognostic value of ETAR and CXCR4 expression in 153 cases of NPC. Chemotaxis assays were used to evaluate alterations in the migration ability of non-metastatic 6-10B and metastatic 5-8F NPC cells. Real-time PCR, immunoblotting, and flow cytometric analyses were used to evaluate changes in the expression levels of CXCR4 mRNA and protein induced by ET-1.ResultsThe expression levels of ETAR and CXCR4 were closely related to each other and both correlated with a poor prognosis. A multivariate analysis showed that the expression levels of both ETAR and CXCR4 were independent prognostic factors for overall survival (OS), progression-free survival (PFS), and distant metastasis-free survival (DMFS). The migration of 6-10B and 5-8F cells was elevated by ET-1 in combination with SDF-1α. The knockdown of ETAR protein expression by siRNA reduced CXCR4 protein expression in addition to ETAR protein expression, leading to a decrease in the metastatic potential of the 5-8F cells. ET-1 induced CXCR4 mRNA and protein expression in the 6-10B NPC cells in a time- and concentration-dependent fashion and was inhibited by an ETAR antagonist and PI3K/AKT/mTOR and MAPK/ERK1/2 pathway inhibitors.ConclusionsETAR and CXCR4 expression levels are potential prognostic biomarkers in NPC patients. ETAR activation partially promoted NPC cell migration via a mechanism that enhanced functional CXCR4 expression.

Abstract 2701: Expression and function of CXCR4 in human salivary gland cancers.

Abstract Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. On the contrary to the CXCR4 expression, CXCR7, a recently identified second receptor for SDF-1, was detected in all SGC cell lines, expression levels were considerably lower on average compared with that of CXCR4 expression. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Yasusei Kudo, Makoto Kinouchi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. Expression and function of CXCR4 in human salivary gland cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2701. doi:10.1158/1538-7445.AM2013-2701

Expression and function of CXCR4 in human salivary gland cancers

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.

HGF-induced PKCζ activation increases functional CXCR4 expression in human breast cancer cells.

The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to mediate the metastasis of many malignant tumors including breast carcinoma. Interaction between hepatocyte growth factor (HGF) and the Met receptor tyrosine kinase mediates development and progression of cancers. HGF is able to induce CXCR4 expression and contributes to tumor cell invasiveness in breast carcinoma. However, the mechanism of the CXCR4 expression modulated by c-Met-HGF axis to enhance the metastatic behavior of breast cancer cells is still unclear. In this study, we found that HGF induced functional CXCR4 receptor expression in breast cancer cells. The effect of HGF was specifically mediated by PKCζ activity. After transfection with PKCζ-siRNA, the phosphorylation of PKCζ and CXCR4 was abrogated in breast cancer cells. Interference with the activation of Rac1, a downstream target of HGF, prevented the HGF-induced increase in PKCζ activity and CXCR4 levels. The HGF-induced, LY294002-sensitive translocation of PKCζ from cytosol to plasma membrane indicated that HGF was capable of activating PKCζ, probably via phosphoinositide (PI) 3-kinases. HGF treatment also increased MT1-MMP secretion. Inhibition of PKCζ, Rac-1 and phosphatidylinositol 3-kinase may attenuate MT1-MMP expression in cells exposed to HGF. Functional manifestation of the effects of HGF revealed an increased ability for migration, chemotaxis and metastasis in MDA-MB-436 cells in vitro and in vivo. Our findings thus provided evidence that the process of HGF-induced functional CXCR4 expression may involve PI 3-kinase and atypical PKCζ. Moreover, HGF may promote the invasiveness and metastasis of breast tumor xenografts in BALB/c-nu mice via the PKCζ-mediated pathway, while suppression of PKCζ by RNA interference may abrogate cancer cell spreading.

CXCL12 receptor preference, signal transduction, biological response and the expression of 5T4 oncofoetal glycoprotein

CXCL12 is a pleiotropic chemokine capable of eliciting multiple signal transduction cascades and functions, via interaction with either CXCR4 or CXCR7. Factors that determine CXCL12 receptor preference, intracellular signalling route and biological response are poorly understood but are of central importance in the context of therapeutic intervention of the CXCL12 axis in multiple disease states. We have recently demonstrated that 5T4 oncofoetal glycoprotein facilitates functional CXCR4 expression leading to CXCL12 mediated chemotaxis in mouse embryonic cells. Using wild type (WT) and 5T4 knockout (5T4KO) murine embryonic fibroblasts (MEFs), we now show that CXCL12 binding to CXCR4 activates both the ERK and AKT pathways within minutes, but while these pathways are intact, they are non-functional in 5T4KO cells treated with CXCL12. Importantly, in the absence of 5T4 expression, CXCR7 is upregulated and becomes the predominant receptor for CXCL12, activating a distinct signal transduction pathway with slower kinetics involving transactivation of the epidermal growth factor receptor (EGFR), eliciting proliferation rather than chemotaxis. Thus the surface expression of 5T4 marks the use of the CXCR4 rather than the CXCR7 receptor, with distinct consequences for CXCL12 exposure, relevant to the spread and growth of a tumour. Consistent with this hypothesis, we have identified human small cell lung carcinoma cells with similar 5T4/CXCR7 reciprocity that is predictive of biological response to CXCL12 and determined that 5T4 expression is required for functional chemotaxis in these cells.

PGE2-Induced CXCL12 Production and CXCR4 Expression Controls the Accumulation of Human MDSCs in Ovarian Cancer Environment

Signals mediated by CXCL12 (SDF1) and its receptor CXCR4 are centrally involved in cancer progression, both directly by activating cancer cells and indirectly by inducing angiogenesis plus recruiting T regulatory and plasmacytoid dendritic immune cells. Here, we show that in ascites isolated from ovarian cancer patients, both CXCL12 and CXCR4 are controlled by the tumor-associated inflammatory mediator prostaglandin E(2) (PGE(2)), which attracts myeloid-derived suppressor cells (MDSC) into the ascites microenvironment. In this setting, PGE(2) was essential both for expression of functional CXCR4 in cancer-associated MDSCs and for production of its ligand CXCL12. Frequencies of CD11b(+)CD14(+)CD33(+)CXCR4(+) MDSCs closely correlated with CXCL12 and PGE(2) levels in patient ascites. MDSCs migrated toward ovarian cancer ascites in a CXCR4-dependent manner that required COX2 activity and autocrine PGE(2) production. Inhibition of COX2 or the PGE(2) receptors EP2/EP4 in MDSCs suppressed expression of CXCR4 and MDSC responsiveness to CXCL12 or ovarian cancer ascites. Similarly, COX2 inhibition also blocked CXCL12 production in the ovarian cancer environment and its ability to attract MDSCs. Together, our findings elucidate a central role for PGE(2) in MDSC accumulation triggered by the CXCL12-CXCR4 pathway, providing a powerful rationale to target PGE(2) signaling in ovarian cancer therapy.

17β-Estradiol enhances the recruitment of bone marrow-derived endothelial progenitor cells into infarcted myocardium by inducing CXCR4 expression

Background17β-Estradiol (E2) has been thought to produce cardioprotective effects by mediating bone marrow-derived endothelial progenitor cells (EPC) for cardiac repair in the setting of acute myocardial infarction (AMI). However, the underlying mechanism of action of E2 on EPC remains unclear. CXCR4 is a critical modulator in homing of EPC. Accordingly, we hypothesized that E2 exerts beneficial effects through enhancing EPC homing to infarcted myocardium via mediating CXCR4 pathway. Methods and resultsMigratory capacity and CXCR4 expression of EPC from ovariectomized BALB/C mice were detected after being incubated with various E2 concentrations for various incubation times. For in vivo studies, EPC were labeled with superparamagnetic ion oxide (SPIO) for tracing, and ovariectomized mice were grouped (n=11) after inducing AMI to receive saline without cells or with 3×106 non-preconditioned EPC, 100nmol/L E2 preconditioned EPC, CXCR4 inhibitor AMD3100 (5μg/mL) preconditioned EPC, or EPC pretreated with E2 plus AMD3100. The number of homing EPC in infarcted myocardium and left ventricular (LV) function, dimensions and fibrosis were measured. In vitro data showed that E2 increased migratory activity and functional CXCR4 expression of EPC. However, these effects were completely blocked by AMD3100. In vivo data in E2 group displayed a greater number of homing EPC, decreased fibrosis of LV, and significant improvement in cardiac function. Nevertheless, effects of E2 preconditioning were abrogated by AMD3100. ConclusionsWe conclude that E2 enhances the recruitment of EPC into infarcted myocardium by up-regulating functional CXCR4 expression, resulting in improving recovery after myocardial infarction.

The EGFR Ligands Amphiregulin and Heparin-Binding EGF-like Growth Factor Promote Peritoneal Carcinomatosis in CXCR4-Expressing Gastric Cancer

Peritoneal carcinomatosis, often associated with malignant ascites, is the most frequent cause of death in patients with advanced gastric cancer. We previously showed that the CXCR4/CXCL12 axis is involved in the development of peritoneal carcinomatosis from gastric cancer. Here, we investigated whether epidermal growth factor receptor (EGFR) ligands are also involved in the development of peritoneal carcinomatosis from gastric cancer. The functional involvement of expression of the ErbB family of receptors and/or EGFR ligands was examined in CXCR4-expressing human gastric cancer cells and fibroblasts, clinical samples (primary tumors and ascites), and an animal model. High concentration of the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF), as well as of CXCL12, were present in malignant ascites. Human gastric cancer cell lines and primary gastric tumors, with high potential to generate peritoneal carcinomatosis, expressed high levels of EGFR and CXCR4 mRNA and protein. Both amphiregulin and HB-EGF enhanced the proliferation, migration, and functional CXCR4 expression in highly CXCR4-expressing gastric cancer NUGC4 cells. Amphiregulin strongly enhanced the proliferation of NUGC4 cells, whereas HB-EGF markedly induced the migration of fibroblasts. Moreover, HB-EGF and CXCL12 together enhanced TNFα-converting enzyme (TACE)-dependent amphiregulin shedding from NUGC4 cells. In an experimental peritoneal carcinomatosis model in mice, cetuximab effectively reduced tumor growth and ascites formation. Our results strongly suggest that the EGFR ligands amphiregulin and HB-EGF play an important role, interacting with the CXCL12/CXCR4 axis, in the development of peritoneal carcinomatosis from gastric cancer, indicating that these two axes may be potential therapeutic targets for peritoneal carcinomatosis of gastric carcinoma.

CXCR4 signaling mediates morphine-induced tactile hyperalgesia

Morphine and related compounds are the first line of therapy in the treatment of moderate to severe pain. Over time, individuals taking opioids can develop an increasing sensitivity to noxious stimuli, even evolving into a painful response to previously non-noxious stimuli (opioid-induced hyperalgesia; OIH). The mechanism underlying OIH is not well understood although complex intracellular neural mechanisms, including opioid receptor desensitization and down-regulation, are believed to be major mechanisms underlying OIH. However, OIH may also be associated with changes in gene expression. A growing body of evidence suggests that cellular exposure to mu agonists upregulate chemokines/receptors and recent work from our laboratory implicates chemokine upregulation in a variety of neuropathic pain behaviors. Here we characterized the degree to which chemokines/receptors signaling is increased in primary afferent neurons of the dorsal root ganglion (DRG) following chronic morphine sulfate treatment and correlated these changes with tactile hyperalgesic behavior in rodents. We demonstrate that mRNA expression of the chemokine, stromal-derived factor-1 (SDF1/CXCL12) is upregulated following morphine treatment in sensory neurons of the rat. The release of SDF1 was found to be constitutive when compared with the activity dependent release of the C–C chemokine, monocyte chemoattractant protein-1 (MCP1/CCL2) in a line of F11 neuroblastoma-sensory neuron hybrid cells. We further determined that there is pronounced CXCR4 expression in satellite glial cells and following morphine treatment, increased functional CXCR4 expression in sensory neurons of the DRG. Moreover, intraperitoneal administration of the specific CXCR4 antagonist, AMD3100, completely reversed OIH in the rat. Taken together; the data suggest that opioid-induced SDF1/CXCR4 signaling is central to the development of long lasting OIH and that receptor antagonists represent a promising novel approach to the management of the side effects associated with the use of opioids for chronic pain management.

1307 CXCL12/SDF1 expression by breast cancers is an independent prognostic marker of progression

Morphine and related compounds are the first line of therapy in the treatment of moderate to severe pain. Over time, individuals taking opioids can develop an increasing sensitivity to noxious stimuli, even evolving into a painful response to previously non-noxious stimuli (opioid-induced hyperalgesia; OIH). The mechanism underlying OIH is not well understood although complex intracellular neural mechanisms, including opioid receptor desensitization and down-regulation, are believed to be major mechanisms underlying OIH. However, OIH may also be associated with changes in gene expression. A growing body of evidence suggests that cellular exposure to mu agonists upregulate chemokines/receptors and recent work from our laboratory implicates chemokine upregulation in a variety of neuropathic pain behaviors. Here we characterized the degree to which chemokines/receptors signaling is increased in primary afferent neurons of the dorsal root ganglion (DRG) following chronic morphine sulfate treatment and correlated these changes with tactile hyperalgesic behavior in rodents. We demonstrate that mRNA expression of the chemokine, stromal-derived factor-1 (SDF1/CXCL12) is upregulated following morphine treatment in sensory neurons of the rat. The release of SDF1 was found to be constitutive when compared with the activity dependent release of the C–C chemokine, monocyte chemoattractant protein-1 (MCP1/CCL2) in a line of F11 neuroblastoma-sensory neuron hybrid cells. We further determined that there is pronounced CXCR4 expression in satellite glial cells and following morphine treatment, increased functional CXCR4 expression in sensory neurons of the DRG. Moreover, intraperitoneal administration of the specific CXCR4 antagonist, AMD3100, completely reversed OIH in the rat. Taken together; the data suggest that opioid-induced SDF1/CXCR4 signaling is central to the development of long lasting OIH and that receptor antagonists represent a promising novel approach to the management of the side effects associated with the use of opioids for chronic pain management.

Suppression of CXCR4 expression in mast cells upon IgE-mediated antigen stimulation

Recent studies have demonstrated that a variety of chemokine receptors are expressed in mast cells. We investigated the changes in mRNA expression of CXCRs in murine IL-3-dependent bone marrow-derived mast cells (BMMCs) to clarify how the CXCR expression is regulated in mast cells. Expression of CXCR mRNA was measured by RNase protection assay. Functional expression of CXCRs was confirmed by monitoring intracellular Ca(2+) mobilization. CXCR4 mRNA expression was transiently induced in BMMCs in serum-dependent fashion and was completely suppressed upon IgE-mediated antigen stimulation. In contrast, CXCR5 mRNA expression was induced upon IgE-mediated antigen stimulation. Changes in the intracellular Ca(2+) mobilization induced by CXCL12 strongly indicated the functional expression of CXCR4. The decrease in CXCR4 and the increase in CXCR5 mRNA expression was also observed in BMMCs stimulated with thapsigargin, a phorbol ester, and stem cell factor. The mRNA expression of CXCR4 is differentially regulated in BMMCs upon various stimuli including IgE-mediated antigen stimulation.

Chemokine receptor CXCR4 expression and its regulation in gastric cancer cells

e22173 Background: The chemokine receptor CXCR4 is associated with the biological behavior in several kinds of cancer, but few studies have addressed the expression and regulation of CXCR4 in gastric cancer. Methods: Five gastric cancer cell lines were studied. The expression of CXCR4 was investigated using RT-PCR, Westerning blotting, flow cytometry, and immunofluorescence staining. The regulation of CXCR4 expression by hypoxia, dexamethasone, and the proinflammatory cytokines was evaluated. Results: CXCR4 mRNA and proteins were detectable by RT-PCR and Western blot analysis, respectively, in all five cell lines. However, MKN-28, MKN-45, MKN-74, and SNU 16 cells did not express membrane CXCR4, but had abundant CXCR4 in their cytoplasm, as determined by flow cytometry and immunofluorescence staining. In contrast, a small population of KATO III cells expressed membrane CXCR4. Hypoxia up-regulated CXCR4 proteins and enhanced membrane expression of CXCR4 in human gastric cancer KATO III cells, which constitutively expressed membrane CXCR4 in a steady state, as revealed by Western blotting and flow cytometry, respectively. This hypoxia-induced expression of CXCR4 was mediated via hypoxia-inducible factor (HIF)-1α. In addition, KATO III cells exposed to hypoxia demonstrated enhanced migration in response to stromal cell-derived factor (SDF)-1α, a specific ligand for CXCR4. However, MKN-28, MKN-45, MKN-74, and SNU-16 cells, which lack membrane CXCR4 in a steady state, showed no change in CXCR4 expression in hypoxic condition. Treatment with IFN-γ, TGF-β, TNF-α, and dexamethasone did not induce any change in CXCR4 expression in all five gastric cancer cells. Conclusions: This study suggests that hypoxia up-regulates the membrane expression of functional CXCR4 via HIF-1α in human gastric cancer cells that basally express membrane CXCR4 in a steady state in vitro. No significant financial relationships to disclose.

Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML

The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.

CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis

BackgroundMetastasis to regional lymph nodes is a common step in the progression of cancer. Recent evidence suggests that tumor production of CXCR4 promotes lymph node metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers.MethodsNitrite/nitrate levels and functional CXCR4 expression were assessed in K1 and B-CPAP papillary thyroid carcinoma (PTC) cells after induction and/or inhibition of NO synthesis. CXCR4 expression was also analyzed in primary human PTC. The relationship between nitrotyrosine levels, which are a biomarker for peroxynitrate formation from NO in vivo, CXCR4 expression, and lymph node status was also analyzed.ResultsProduction of nitrite/nitrate and functional CXCR4 expression in both cell lines was increased by treatment with the NO donor DETA NONOate. The NOS inhibitor L-NAME eliminated this increase. Positive CXCR4 immunostaining was observed in 60.7% (34/56) of PTCs. CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis in human PTC.ConclusionOur data indicate that NO stimulates CXCR4 expression in vitro. Formation of the NO biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in human PTC. NO may induce lymph node metastasis via CXCR4 induction in papillary thyroid carcinoma.

Functional expression of CXCR4 in somatotrophs: CXCL12 activates GH gene, GH production and secretion, and cellular proliferation

The interaction of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR4 may play an important role in the regulation of anterior pituitary function. In this study, we investigated the expression of CXCL12 and CXCR4 and their role in normal rat pituitary and GH-producing GH3 tumor cell line. RT-PCR analysis and immunohistochemistry revealed that CXCR4 was expressed in normal rat anterior pituitary and GH3 tumor cells. Double immunofluorescent staining showed the complete colocalization of CXCR4 with GH in rat pituitary, indicating that CXCR4 is specifically expressed in rat somatotrophs. Using rat primary pituitary cell cultures and GH releasing hormone receptor expressing stable GH3 cells (GH3-GHRHR), we evaluated the function of CXCL12 compared with GHRH. CXCL12 stimulated GH gene activation in both primary rat anterior pituitary cells and GH3-GHRHR cells. CXCL12 also stimulated GH secretion from primary rat pituitary cells in a dose-dependant manner. BrdU incorporation was increased in response to CXCL12 addition in GH3 cell culture, indicating CXCL12-induced cell proliferation. CXCL12-dependent phosphorylation of ERK1/2 was also confirmed by western blot analysis, supporting the evidence that MAPK is an intracellular mediator of CXCL12/CXCR4 interaction in GH3 cell proliferation. In conclusion, these results indicate that CXCL12/CXCR4 interaction plays an important role in GH production, secretion, and the proliferation of somatotrophs.

Culturing of Human Mesenchymal Stem Cells as Three-dimensional Aggregates Induces Functional Expression of CXCR4 That Regulates Adhesion to Endothelial Cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.

Abstract 859: Hypoxia-activated CXCR4 Expression In Cardiac Progenitor Cells For Replenishing Stem Cell Pool In Acute Ischemic Myocardium

The resident cardiac progenitor cells (CPCs) have been identified in the adult myocardium, however, the CPC pool is rapidly depleted after acute myocardial infarction (AMI). The strategy to home ex vivo cultured CPCs to ischemic myocardium might be an important strategy to replenish resident CPC pool, and improve cardiac function. The CXC chemokine SDF-1α and its receptor CXCR4 have been identified as critical mediator for the ischemia-specific recruitment of progenitor cells, yet the level of CXCR4 expression in CPCs is quite low in normal conditions. Our previous studies showed that hypoxia can optimize CPCs via inducing functional CXCR4 expression in vitro, therefore, in this study, we want to investigate whether the transplantation of hypoxia treated CPCs (Hypo:CPCs) have the benefit of improving cardiac function resulted from increased cell homing in ischemic myocardium through mechanisms of hypoxia induced CXCR4 expression. To compare the homing capability between CPCs and Hypo:CPCs in response to endogenous SDF-1 signal in acute ischemic myocardium, we used retrovirus (pCL-MFG-β-gal) to label c-kit + Lin − CPCs with β-gal, and then injected labeled CPCs and Hypo:CPCs (1×10 6 ) intravenously via intra-jugular vein to mice 10 min after induction of MI in the C56BL/6 mice. We harvested the hearts 1d after cell transplantation to quantify the cell retention by chemiluminescent β-galactosidase assay. We observed that hypoxia treatment resulted in about 2.5 fold increase in β-gal cell recruitment in ischemic hearts (p<0.001, n=7– 8/group), however, AMD3100 can partially reduce Hypo:CPCs homing, therefore, the beneficial effects of Hypo:CPCs transplantation were mediated primarily through increasing cell homing via SDF-1:CXCR4 binding within the ischemic hearts. In addtion, the infusion of Hypo:CPCs also led to improved left ventricular performance, as assessed by LV development pressure by Millar catheter, by 23.67% compared with CPCs at 1 month after cell therapy (p<0.001, n=6/group). These results indicate that replenish CPC pool after AMI using ex vivo cultured CPC transplantation has significant beneficial effects on injured heart function dependent of hypoxia induced CXCR4 upregulation.

Integrin-mediated laminin-1 adhesion upregulates CXCR4 and IL-8 expression in pancreatic cancer cells

We have shown recently that alpha(2)beta(1) integrin-mediated type I collagen adhesion promotes a more malignant phenotype in pancreatic cancer cell lines than other extracellular matrix (ECM) proteins. MiaPaCa-2 cells, by contrast, do not express collagen-binding integrins, but are metastatic in our orthotopic mouse model and migrate maximally on laminin-1 (Ln-1). It has also been shown that CXCR4 and IL-8 expression correlates directly with metastasis in pancreatic cancer in vivo. We therefore examined the potential of the ECM to regulate CXCR4 and IL-8 expression in pancreatic cancer cells. We cultured 8 pancreatic cancer cell lines on fibronectin (Fn), types I and IV collagen, Ln-1 and vitronectin (Vn), and examined cell lysates for CXCR4 by immunoblotting and media for IL-8 by ELISA. We also conducted cell migration assays with stromal-derived factor-1 (SDF-1) as the chemoattractant to examine integrin-binding specificity and CXCR4 function. All cell lines expressed CXCR4 protein. MiaPaCa-2 cell growth on Ln-1 increased significantly CXCR4 and IL-8 expression relative to other ECM proteins. Migration inhibition studies showed that both the alpha(6)beta(1) and alpha(3)beta(1) integrins mediate MiaPaCa-2 migration on Ln-1. Growth studies showed further that CXCR4 expression on Ln-1 was mediated by the alpha(6)beta(1) integrin whereas IL-8 expression was mediated by both the alpha(6)beta(1) and alpha(3)beta(1) integrins. The expression of functional CXCR4 was also shown in migration assays, where SDF-1 significantly increased pancreatic cancer cell chemotaxis on Ln-1. These data indicate that integrin-mediated Ln-1 adhesion upregulates CXCR4 and IL-8 expression and may play a mechanistic role in pancreatic cancer metastases.

The anti-cancer compound Nordy inhibits CXCR4-mediated production of IL-8 and VEGF by malignant human glioma cells

The chemokine receptor CXCR4 plays an important role in tumor growth, angiogenesis and metastasis. Our previous studies showed that Nordy, a synthetic chiral compound of nordihydroguaiaretic acid, inhibited the growth and angiogenesis of various malignant tumors. In this study we examined the capacity of Nordy to regulate CXCR4-mediated production of angiogenic factors by human glioblastoma cells. We found that Nordy potently inhibited CXCR4 ligand SDF-1-induced production of IL-8 and vascular endothelial cell growth factor, two important angiogenic factors implicated in the progression of malignant tumors. Further study revealed that the effect of Nordy was attributable to its down-regulation of the expression of functional CXCR4 in glioblastoma cells. These results suggest that the anti-cancer activity of Nordy is due, at least in part, to its suppression of the chemokine receptor CXCR4 thus reducing the production of angiogenic factors by tumor cells.

Up-regulation of functional CXCR4 expression on human lymphocytes in sepsis

Lymphocyte dysfunction has been documented in sepsis, and evidence suggests that lymphocyte infiltration contributes to tissue injury. The purpose of this study was to examine chemokine receptor expression and function in lymphocytes from septic patients and healthy donors. Observational study of septic patients and laboratory investigation of normal controls. Tertiary care intensive care unit. Nine critically ill patients fulfilling criteria for the systemic inflammatory response syndrome and with a Sepsis Score of >/=3 were included in this study. Lymphocytes were also obtained from healthy volunteers. None. The total number of circulating leukocytes in septic patients was markedly increased; however, lymphocyte counts were decreased. Chemokine receptor expression on lymphocytes was examined by flow cytometry. CXCR4 expression on lymphocytes from septic patients was increased whereas CCR5 was decreased and CCR7 was unchanged. Lipopolysaccharide stimulation of normal lymphocytes increased CXCR4 expression but decreased CCR5 and did not change CCR7 expression. This lipopolysaccharide-stimulated CXCR4 expression required 20 hrs of stimulation and was accompanied by increased messenger RNA. Lymphocytes from septic patients or after lipopolysaccharide treatment demonstrated enhanced actin polymerization and migration in response to CXCL12. Taken together, sepsis and lipopolysaccharide up-regulated CXCR4 expression and enhanced lymphocyte activation and migration in response to CXCL12. Blocking CXCR4 and CXCL12 function may provide a novel therapeutic method for controlling systemic inflammation and tissue injury in sepsis.