Upregulation Of CXCR4 Research Articles

Morphine Increases Brain Levels of Ferritin Heavy Chain Leading to Inhibition of CXCR4-Mediated Survival Signaling in Neurons

This study focuses on the effect of mu-opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of mu-opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced G alpha(i)/G betagamma activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective mu-opioid agonist DAMGO (D-Ala2-N-Me-Phe(4)-glycol(5)-enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

SDF-1α/OXCR4 is implicated in THP-1 cells chemotaxis and it is enhanced by OX-LD

Objective To explore the effect of SDF-1 α /CXCR4 on THP-1 cells chemotaxis and the influence of OX- LDL on THP-1 cells migration esplsed to SDP-1 α,the effect of OX-LDL in the expression of CXCR4 in THP-1 cells.THP-1 cells chemotaxis was examined with transwell permeable supports.THP-1 cells with or withort pretreatde with OX-LDL were input in upper chambers,SDF-1αwas added in low chambers.Number of migrated cells in the low chamber was counted after incubation for 10h.CXCR4 mRNA and protein was revealed by RT-PCR and Western blkt respectively in THP-1 cells incubated with different concentrations/time of OX-LDL.Results SDF-1αinduced a pronounced migration of input cells in a concentration-related manner,but this effect was obviously inhibited by the antibody to CXXR4 was constitutionally expressde in THP-1 cells.Comparing with control group,a foru-to fivefold induction of CXCR4 occurred in THP-1 treated with 50 ug/ml OX-LDL.Upregulation of CXCR4 was within 6h,peaked at 12h then followed with a decline,mirroring the expression pattern in macrophage.Conclusions SDF-1 α/CXCR4 is implicated in THP-1 cells chemotaxis and chemotactic response of monocyte to SDF-1 α is enhanced by Ox-LDL; OX-LDL up-regulates CXCR4 expression. Key words: SDF-1α/CXCR; ox-LDL; Chemotaxis; Monocyte

Dexamethasone and hypoxia upregulate CXCR4 expression in myeloma cells

We investigated the modulation of CXCR4 expression by cytokines, dexamethasone, and hypoxia in myeloma cells in vitro. Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta1 (TGF-β1), and vascular endothelial growth factor (VEGF) enhanced CXCR4 expression in RPMI8226 cells. In the myeloma cell lines examined and primary bone marrow (BM) CD138+ cells, dexamethasone enhanced CXCR4 expression both in the cytoplasm and on the cell surface, while downregulating SDF-1 expression and secretion in BM stromal cells. Incubation of cells under hypoxic conditions (1% O2) also induced upregulation of CXCR4 in the cytoplasm and on the cell surface and enhanced chemotaxis in response to stromal cell-derived factor-1 (SDF-1). Cell surface CXCR4 expression was more prominent in annexin V-positive apoptotic cells. Given the roles of the SDF-1/CXCR4 axis in the development and progression of myeloma, CXCR4-downregulating agents may enhance the antitumor effects of dexamethasone.

Neural Stem Cell Tropism to Glioma: Critical Role of Tumor Hypoxia

Hypoxia is a critical aspect of the microenvironment in glioma and generally signifies unfavorable clinical outcome. Effective targeting of hypoxic areas in gliomas remains a significant therapeutic challenge. New therapeutic platforms using neural stem cells (NSC) for tumor-targeted drug delivery show promise in treatment of cancers that are refractory to traditional therapies. However, the molecular mechanisms of NSC targeting to hypoxic tumor areas are not well understood. Therefore, we investigated the role of hypoxia in directed migration of NSCs to glioma and identified the specific signaling molecules involved. Our data showed that hypoxia caused increased migration of human HB1.F3 NSCs to U251 human glioma-conditioned medium in vitro. In HB1.F3 NSCs, hypoxia led to up-regulation of CXCR4, urokinase-type plasminogen activator receptor (uPAR), vascular endothelial growth factor receptor 2 (VEGFR2), and c-Met receptors. Function-inhibiting antibodies to these receptors inhibited the migration of HB1.F3 cells to glioma-conditioned medium. Small interfering RNA knockdown of hypoxia-inducible factor-1alpha in glioma cells blocked the hypoxia-induced migration of NSCs, which was due to decreased expression of stromal cell-derived factor-1 (SDF-1), uPA, and VEGF in glioma cells. Our in vivo data provided direct evidence that NSCs preferentially distributed to hypoxic areas inside intracranial glioma xenografts, as detected by pimonidazole hypoxia probe, as well as to the tumor edge, and that both areas displayed high SDF-1 expression. These observations indicate that hypoxia is a key factor in determining NSC tropism to glioma and that SDF-1/CXCR4, uPA/uPAR, VEGF/VEGFR2, and hepatocyte growth factor/c-Met signaling pathways mediate increased NSC-to-glioma tropism under hypoxia. These results have significant implications for development of stem cell-mediated tumor-selective gene therapies.

Correction of the Abnormal Trafficking of Primary Myelofibrosis CD34+ Cells by Treatment with Chromatin Modifying Agents

Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by abnormal trafficking of CD34+ cells, resulting in their constitutive mobilization. This abnormal trafficking of PMF CD34+ cells has been related to the reduced expression of the chemokine receptor CXCR4 (Shi et al, Cancer Research 67: 6417, 2007). We further tested this hypothesis by studying the homing of PMF CD34+ cells to the marrow and the spleens of sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and determining if this defect could be corrected by treatment with chromatin modifying agents. Following the infusion of PMF (n=10) or G-CSF mobilized peripheral blood (mPB) (n=4) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and assayable human CFU-GM were detected in the marrow of these mice (139±47 PMF CD34+ cells/106 BMCs vs. 1493±946 mPB CD34+ cells/106 BMCs, 2.0±0.8% of input PMF CFU-GM vs. 12.6±6.6% of input mPB CFU-GM, P<0.05). The CFU-GM were shown to be of human origin by plucking individual colonies and analyzing by RT-PCR for the human 17-alpha satellite gene and the murine GAPDH gene. Of the total CD34+ cells homing to the spleen and marrow of the NOD/SCID mice, 25% of PMF CD34+ cells and 2.4% of mPB CD34+ cells homed to the spleen. The abnormal trafficking of PMF CD34+ cells was associated with reduced expression of CXCR4 (15.3% vs.4.8%, P<0.05). PMF CD34+ cells from patients with (n=6) or without (n=4) the JAK2V617F mutation exhibited similar degrees of defective bone marrow(BM) homing, suggesting that the impairement of PMF CD34+ cell trafficking was not related to the JAK2 mutation. We have previously shown that the sequential treatment of PMF CD34+ cell with the DNA methyltransferase inhibitor, decitabine [5-aza-2′-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), resulted in the upregulation of CXCR4 (Shi et al, Cancer Research 67: 6417, 2007). We next determined if this upregulation of CXCR4 was associated with correction of the impaired trafficking of PMF CD34+ cell to the marrow of NOD/SCID mice. The percentage of CD34+ cells expressing CXCR4 was significantly enhanced by exposure of PMF CD34+ cells to 5azaD/TSA (primary CD34+ cells: 2.55±0.87%, cytokines alone: 6.51±1.80%, cytokines plus 5azaD/TSA: 12.47±3.65%). Treatment of PMF CD34+ cells in vitro with cytokines plus 5azaD/TSA for 9 days resulted in a 3-fold increase in the absolute numbers of CD34+CXCR4+ cells when compared to CD34+ cells exposed to cytokines alone and 61-fold increase of CD34+CXCR4+ cells when compared to primary PMF CD34+ cells. Treatment of PMF CD34+ cells with cytokines alone resulted in 3- to 9-fold greater homing to NOD/SCID marrow than that of primary PMF CD34+ cells (P=0.12), while equal number of PMF CD34+ cells treated with chromatin modifying agents had a 7- to 20-fold greater ability to home to mouse marrow than that of primary PMF CD34+ cells (P<0.05) and a 2- to 3-fold greater marrow homing ability than PMF CD34+ cells treated with cytokines alone (P<0.05). Treatment with 5azaD/TSA reduced by 36% the homing of PMF CD34+ cells to the spleen as compared to the treatment with cytokines alone. We conclude that PMF CD34+ cells preferentially home to the spleen rather than the marrow of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin modifying agents. Chromatin modifying agents represent a novel means of correcting the abnormal cellular trafficking characteristic of PMF, a therapeutic strategy which is about to be evaluated in the clinic.

CD300f Triggering Modifies Myeloid Cell Function

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. Blood 2006; 108:225B-225B and Zhao X et al Blood 2007; 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.

CXCL12 is essential for migration of activated Langerhans cells from epidermis to dermis

The initial step in Langerhans cell (LC) migration from the epidermis to the lymph node involves migration of maturing LC into the dermis. Here, we investigated the migration of LC out of the epidermis after exposure of the skin to contact allergens. Ex vivo intact human skin, epidermal sheets, and LC derived from the MUTZ-3 cell line (MUTZ-LC) were used to determine whether dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of epidermal sheets or MUTZ-LC to allergens (nickel sulphate, 2,4-dinitrochlorobenzene, and cinnamaldehyde) or a cytokine maturation cocktail resulted in LC migration towards dermal fibroblasts. This was due to upregulation of CXCR4 on maturing LC and secretion of CXCL12/stromal derived factor-1 chemokine by fibroblasts. Neutralizing antibodies to either CXCL12 or CXCR4 completely blocked migration. Injection of CXCL12 neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. In contrast, neutralizing antibodies to CCL19/CCL21 did not inhibit migration into the dermis. We describe a novel and essential role of dermis-derived CXCL12 in initiating migration of maturing human LC to the dermis thus permitting their further journey to the draining lymph nodes.

CD19 regulates the development of bleomycin‐induced pulmonary fibrosis in a mouse model

The contribution of CD19 and B lymphocytes to pulmonary fibrosis is controversial. The aim of this study was to address the role of CD19 during the development of pulmonary fibrosis. Mice lacking or overexpressing the B cell surface molecule CD19, which is known as a positive regulator of B cell activation, were used in a model of bleomycin-induced pulmonary fibrosis. Ten or sixteen days after intratracheal injection of bleomycin, lung sections from mice were evaluated by histologic analysis. Seven days after instillation, the total leukocyte count and the number of B cells in bronchoalveolar lavage fluid (BALF) were determined, using a hemocytometer and flow cytometry. Bleomycin was also administered into selectin-deficient or intercellular adhesion molecule 1-deficient mouse strains. The level of CXCR3 expression on B cells was determined by flow cytometry. CD19 deficiency significantly reduced susceptibility to intratracheal bleomycin challenge on day 16, while CD19 overexpression augmented fibrosis even on day 10. Furthermore, the survival rate and number of B cells in BALF also correlated with CD19 expression levels. The accumulation of B cells in BALF was dependent on CD19 levels, whereas there was no association with the levels of selectins or intercellular adhesion molecule 1. Additionally, CXCR3 was up-regulated in BALF B cells, while it was rarely expressed on circulating B cells. Furthermore, CD19 signaling facilitated B cell CXCR3 up-regulation in response to stimulation in vitro. These results suggest that CD19 signaling is associated with the development of pulmonary fibrosis by controlling B cell infiltration into lung tissue, which may be associated with CXCR3 up-regulation.

Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genes

A subgroup of medulloblastomas shows constitutive activation of the Sonic hedgehog pathway with expression of GLI1. We identified the subset of GLI1 transforming target genes specifically expressed in medulloblastomas by comparing GLI1 targets in RK3E cells transformed by GLI1 with the gene expression profile of Sonic hedgehog signature medulloblastomas. We identified 1,823 genes whose expression was altered more than 2-fold in 2 independent RK3E + GLI1 cell lines. We identified 25 whose expression was altered similarly in medulloblastomas expressing GLI1. We identified potential GLI binding elements in the regulatory regions of 10 of these genes, confirmed that GLI1 binds the regulatory regions and activates transcription of select genes, and showed that GLI1 directly represses transcription of Krox-20. We identified upregulation of CXCR4, a chemokine receptor that plays roles in the proliferation and migration of granule cell neuron precursors during development, supporting the concept that reinitiation of developmental programs may contribute to medulloblastoma tumorigenesis. In addition, the targets suggest a pathway through which GLI1 may ultimately affect medulloblastoma cell proliferation, survival and genomic stability by converging on p53, SGK1, MGMT and NTRK2. We identify a p53 mutation in RK3E + GLI1 cells, suggesting that p53 mutations may sometimes shift the balance toward dysregulated tumor cell survival.

Post-transcriptional regulation of chemokine receptor CXCR4 by estrogen in HER2 overexpressing, estrogen receptor-positive breast cancer cells

Expression of the chemokine receptor CXCR4, a G protein-coupled receptor, and HER2, a receptor tyrosine kinase, strongly correlates with the aggressive and metastatic potential of breast cancer cells. We studied estrogen regulation of CXCR4 in estrogen receptor (ER)-positive MCF-7 breast cancer cells overexpressing HER2 (MCF7-HER2). Although estrogen evoked no change in CXCR4 mRNA levels, CXCR4 protein was significantly up-regulated after estrogen treatment of these cells, whereas estrogen had no effect on CXCR4 protein level in parental MCF7 cells that are low in HER2. Use of the CXCR4 specific inhibitor, AMD 3100, indicated that this increase in CXCR4 protein was partially responsible for the increase in estrogen-induced migration of these cells. The estrogen-induced increase in CXCR4 protein in MCF-7-HER2 cells was abrogated by the antiestrogen ICI 182780 and by gefitinib (Iressa; a phospho-tyrosine kinase inhibitor), indicating an ER-mediated effect and confirming involvement of receptor tyrosine kinases, respectively. Using specific pathway inhibitors, we show that the estrogen-induced increase in CXCR4 involves PI3K/AKT, MAPK and mTOR pathways. PI3K/AKT and MAPK pathways are known to result in the phosphorylation and functional inactivation of tuberin (TSC2) of tuberous sclerosis complex thereby negating its inhibitory effects on mTOR, which in turn stimulates the translational machinery. Small interfering RNA (siRNA) mediated knockdown of tuberin elevated the level of CXCR4 protein in MCF7-HER2 cells and also nullified further estrogen up-regulation of CXCR4. This study suggests a pivotal role of PI3 K, MAPK and mTOR pathways, via tuberin, in post-transcriptional control of CXCR4, initiated through estrogen-stimulated crosstalk between ER and HER2. Thus, post-transcriptional regulation of CXCR4 by estrogens acting through ER via kinase pathways may play a critical role in determining the metastatic potential of breast cancer cells.

T Cells from Leishmania major-Susceptible BALB/c Mice Have a Defect in Efficiently Up-Regulating CXCR3 upon Activation

Genetic background influences the outcome of Leishmania major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-gamma-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes (LN) early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-gamma-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-gamma-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the LN and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared the ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3(+) T cells in regional LN and lesions, whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naive BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-gamma and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major susceptibility.

Down-regulation of CXCL12 mRNA expression by promoter hypermethylation and its association with metastatic progression in human breast carcinomas

Not only is the expression of CXCR4 on breast cancers a key determinant of tumor metastasis, CXCL12 exhibiting peak levels of constitutive expression in organs representing the first destinations of cancer metastasis, but is proposed to be also essential for the organ-specific metastatic process. In this study, the expressions of CXCR4 and CXCL12 were investigated using quantitative RT-PCR and immunohistochemistry in samples of 63 primary breast carcinomas and 20 normal breast tissues. Using methylation-specific PCR, we also analyzed the methylation status of CXCL12. Both up-regulation of CXCR4 and down-regulation of CXCL12 were observed in primary breast carcinomas. Over-expression of CXCR4 mRNA was significantly related to lymph node metastasis status and strong Her-2 expression, while decreased expression of CXCL12 mRNA was significantly associated with positive lymph node metastasis and estrogen receptor negativity. Methylation-specific PCR showed that 52.4% of breast tumors were hypermethylated in the CXCL12 promoter region. The expression levels of DNA methyltransferase (DNMT) 1 and DNMT3B were significantly higher in the CXCL12-methylated breast carcinomas than in the CXCL12-unmethylated ones. In summary, DNA hypermethylation of CXCL12 plays an important role in the down-regulation of CXCL12 expression in breast carcinomas. Cancer cells lacking expression of CXCL12, but maintaining over-expression of CXCR4, can selectively spread to target organs in which the ligand is highly secreted.

Interferon-α2b Restores the Impaired Chemotactic Activity of Peripheral Blood Mononuclear Cells from Head and Neck Squamous Cell Carcinoma Patients by Modulating CXC Receptor Ligand Interaction

We have studied the immunomodulatory effects of interferon-alpha2b (IFN-alpha2b) in rectification of the dysregulated IFN-gamma-dependent chemokines and their receptor CXCR3 splice variants in head and neck squamous cell carcinoma-peripheral blood mononuclear cells (HNSCC-PBMC). CXCR3 expression was upregulated in HNSCC-PBMC, with the demonstration of poor chemotactic function. CXCR3 upregulation possibly represents the increased synthesis of CXCR3B splice variant, without significant change in CXCR3A. Upregulated expression of CXCR3B was downregulated following in vitro IFN-alpha2b treatment of HNSCC-PBMC. Upregulation of CXCR3A+B by IFN-alpha2b with downregulation of the CXCR3B indirectly suggests that the upregulation of the CXCR3A splice variant induces cellular migration. On the other hand, the stimulation of PBMC with IFN-alpha2b maintains physiological homeostasis of CXCR3 ligands, CXCL10 and CXCL9, and increases the secretion of IFN-gamma. The suppressed chemotactic ability of HNSCC-PBMC could be restored either by in vitro treatment of PBMC with IFN-alpha2b or during the use of IFN-alpha2b stimulated PBMC supernatant as a chemoattractant. Chemoattraction process is guided at the level of both receptor and its ligands, as confirmed by neutralization studies. IFN-alpha2b possibly controls chemotaxis by regulating the interaction between CXCL10 and CXCR3A. Neutralization of IFN-gamma downregulates the IFN-alpha2b mediated CXCL10 release, suggesting the active role of IFN-gamma in the transduction of chemotactic signal for the migration of cytotoxic T/NK cells at the tumor site.

Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke

Murine olfactory ensheathing cells (OECs) promote central nervous system axonal regeneration in models of spinal cord injury. We investigated whether OECs could induce a neuroplastic effect to improve the neurological dysfunction caused by hypoxic/ischemic stress. In this study, human OECs/olfactory nerve fibroblasts (hOECs/ONFs) specifically secreted trophic factors including stromal cell-derived factor-1alpha (SDF-1alpha). Rats with intracerebral hOEC/ONF implantation showed more improvement on behavioral measures of neurological deficit following stroke than control rats. [18F]fluoro-2-deoxyglucose PET (FDG-PET) showed increased glucose metabolic activity in the hOEC/ONF-treated group compared with controls. In mice, transplanted hOECs/ONFs and endogenous homing stem cells including intrinsic neural progenitor cells and bone marrow stem cells colocalized with specific neural and vascular markers, indicating stem cell fusion. Both hOECs/ONFs and endogenous homing stem cells enhanced neuroplasticity in the rat and mouse ischemic brain. Upregulation of SDF-1alpha and CXCR4 in hOECs/ONFs promoted neurite outgrowth of cocultured primary cortical neurons under oxygen glucose deprivation conditions and in stroke animals through upregulation of cellular prion protein (PrP C) expression. Therefore, the upregulation of SDF-1alpha and the enhancement of CXCR4 and PrP C interaction induced by hOEC/ONF implantation mediated neuroplastic signals in response to hypoxia and ischemia.

Chemokine receptor expression in non‐melanoma skin cancer

Previous studies suggest that chemokines and chemokine receptors have a role in the metastatic process. A correlation exists between the specific expression of these chemoattractive, pro-inflammatory cytokines and the ability of cancer to disseminate. Prior studies have shown that in metastatic melanoma and squamous cell carcinoma of the head and neck upregulation of CXC (alpha) chemokine receptor (CXCR)4 and CC (beta) chemokine receptor (CCR)7 expression is accompanied by downregulation of the chemokine receptor CCR6. However, the expression patterns of CCR6, CCR7 and CXCR4 in non-melanoma skin cancer have yet to be elucidated. The expression patterns of CCR6, CCR7 and CXCR4 were determined using an immunohistochemical approach on formalin-fixed, paraffin-embedded normal, pre-cancerous actinic (solar) keratosis, squamous cell carcinoma and basal cell carcinoma tissues. Analysis of chemokine receptor expression showed downregulation of CCR6 and upregulation of CCR7 and CXCR4 in potentially metastatic non-melanoma skin cancer, invasive squamous cell carcinoma, but this pattern did not exist in non-melanoma skin cancer with no metastatic potential, basal cell carcinoma; or actinic keratosis, when compared with normal skin. Chemokine receptor expression may influence the biological behavior of non-melanoma skin cancer. The exact mechanism by which this occurs requires further study.

Oligomerization of Vibrio Cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.

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.

Glycogen Synthase Kinase-3β Inhibition Preserves Hematopoietic Stem Cell Activity and Inhibits Leukemic Cell Growth

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

CXCR7 transcription in human non-small cell lung cancer and tumor-free lung tissues; possible regulation upon chemotherapy

Various chemokine receptors are expressed by malignant tumors and have been associated with tumor progression and metastasis [4]. A recent study demonstrated the promotion of tumor growth in breast and lung cancer by CXCR7, formerly known as orphan receptor CMKOR1/RDC1 utilizing cell lines and animal models. CXCR7 expression in human breast and lung cancer specimens, as well as in the tumor vasculature was shown on the protein level by immunohistochemistry. Here, we employed human lung tissues treated with the novel hepes–glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique [5] to analyze the overall expression of CRCX7 on the mRNA level in non-small cell lung cancer (NSCLC) and tumor-free lung tissues. Additionally, the effect of chemotherapy upon the transcription of CRXR7 was addressed, employing a currently established human model system [1, 3]. Applying RT-PCR with a specific primer set spanning an amplicon of 156 bp (5′–TGCTGGACATCTTCTCCATC-3′ forward, 5′-CTTCATCAGCTCCTACCT-3′ reverse), we found transcription of CXCR7 in all of the 30 NSCLC samples analyzed. A set of ten examples is shown in Fig. 1a. Specificity of the reverse-transcription polymerase chain reaction (RTPCR) was verified by sequencing of the PCR products. The results of RT-PCR were then validated by in situ hybridization (N=21) using a double stranded DNA probe labeled with digoxigenin, which revealed transcripts of CXCR7 within the tumor cells of 17 samples (Fig. 1b) with only some occasional signals in the tumor-associated vasculature [2]. Moreover, 20 specimens of tumor-free human lungs were also subjected to in situ hybridization and all revealed transcripts localized mainly in alveolar macrophages (Fig. 1c) and in cells, which reflect the morphology of alveolar epithelial cells type II (Fig. 1d). Stimulation of tumor tissues with physiological concentrations of carboplat and gemcitabine (N=30) was performed within the human model system designated short-term stimulation of tissues in comparison to corresponding tissues, which received cultivation without stimulation [3]. Here, we found an upregulation of CXCR7 transcription upon chemotherapy in 13.4% of the cases, downregulation in 33.3%, and no regulation in 53.3%, as determined by semiquantitative RT-PCR in relation to glyceraldehyde-3-phosphate dehydrogenase (exemplified in the lower panel of Fig. 1a; note upregulation of CXCR7 in sample 4 upon chemotherapy). Taken together, our study verifies the recently reported results on the mRNA level in a comparably large amount of specimens [4]. However, although some signals were occasionally observable, we did not detect strong expression within the tumor-associated vasculature on the RNA level. The effects of chemotherapy onto CXCR7 are heterogeneous. Nevertheless, the same holds true for the overall characteristics of the tumors themselves and their Virchows Arch (2008) 452:347–348 DOI 10.1007/s00428-008-0579-8

CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells

Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML. We proposed that tyrosine kinase inhibitors, imatinib or INNO-406, may restore CXCR4 expression and cause the migration of CML cells to bone marrow microenvironment niches, which in turn results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib, INNO-406, or IFN-alpha increased CXCR4 expression and migration. This increase in CXCR4 levels on CML progenitor cells was likewise found in samples from CML patients treated with imatinib or IFN-alpha. Imatinib induced G0-G1 cell cycle block in CML cells, which was further enhanced in a mesenchymal stem cell (MSC) coculture system. MSC coculture protected KBM-5 cells from imatinib-induced cell death. These antiapoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by inhibitor of integrin-linked kinase QLT0267. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to bone marrow stroma, causing the G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells.