Stromal Cell-derived Factor-1 Stimulation Research Articles

Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein-protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

Nitric oxide functions in stromal cell-derived factor-1-induced cytoskeleton changes and the migration of Jurkat cells.

Stromal cell-derived factor-1 (SDF-1) regulates multiple cell signal pathways in a variety of cellular functions, including cell migration, proliferation, survival and angiogenesis. SDF-1-induced chemotaxis is an important step of lymphocyte migration. However, the molecular mechanisms that modulate SDF-1-mediated lymphocyte migration are not well identified. Nitric oxide (NO) has been found to function as a signaling molecule in a number of signaling pathways, including migration. In the present study, the potential role of NO in SDF-1-induced migration and the association between NO and the cytoskeletal changes of Jurkat cells was investigated. The present study demonstrated that Jurkat cells induced the production of NO by SDF-1 stimulation, using Griess reaction method and western blot analysis, and that NO was involved in SDF-1-induced rearrangement and polymerization of the cytoskeleton, using NOS inhibitor L-NMMA. Furthermore, NO was required for the migration of Jurkat cells. The research suggested that NO signaling pathways exerted a critical role in SDF-1-induced cytoskeleton changes and the migration of Jurkat cells. This work provides insight into the migration mechanism of acute lymphoblastic leukemia and provides an effective theoretical basis for therapy strategies for acute lymphoblastic leukemia.

Effect of integrin α5β1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization.

To investigate the roles of integrins in choroidal neovascularization (CNV) and their associations with the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD) 3100 (CXCR4 inhibitor), and SDF-1+ATN161 (integrin α5β1 inhibitor) groups; their effects on CNV progression were observed using hematoxylin eosin (HE) staining, fundus fluorescein angiography (FFA) grading and optical coherence tomography (OCT), and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs) were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed. Integrin subunit α5 increased at 3rd and 7th day and decreased at 14th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 µm in HE images, significantly lower than the mean length in SDF-1 (345.70 µm) group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 µm2, significantly higher than control (1.25×104 µm2), which decreased to 1.78×104 µm2 after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation, which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment. ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1. SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.

Research on roles played by stromal cell-derived factor 1 and chemokine receptor 4 in diagnosis on proliferation, infiltration and metastasis of cervical cancer

Purpose: To understand the influence of Stromal Cell-Derived Factor 1 (SDF-1) and corresponding receptor CXCR4 on proliferation, infiltration and metastasis of cervical cancer HeLa cells. Method: The flow cytometry dyeing and the Western blot method are adopted to detect the expression conditions of CXCR4 in HeLa cells; the MTT method is adopted to detect the influence of SDF-1 and corresponding receptor CXCR4 on proliferation of HeLa cells; the Boyden chamber method is adopted to detect the influence of SDF-1 and corresponding receptor CXCR4 on infiltration and metastasis of HeLa cells. Results: There is high expression on receptor CXCR4, the receptor of SDF-1; after the SDF-1 stimulation, the proliferation ability of HeLa cells is significantly increased (P<0.05), and this effect is dose-dependent; after the knock-down of CXCR4 expression on surface of HeLa cells with RNAi, the proliferation of HeLa cells caused by SDF-1 stimulation is significantly interdicted (P<0.05). SDF-1 may give rise to infiltration and metastasis of HeLa cells. After the knock-down of CXCR4 expression on surface of HeLa cells with RNAi, the infiltration and metastasis of HeLa cells are significantly interdicted (P<0.05). Conclusion: The CXCR4 with high expression on surface of HeLa cells plays a significant role in inducing proliferation, infiltration and metastasis of HeLa cells.

The Function of SDF-1-CXCR4 Axis in SP Cells-Mediated Protective Role for Renal Ischemia/Reperfusion Injury by SHH/GLI1-ABCG2 Pathway.

Renal ischemia-reperfusion (I/R) injury ranks as the primary cause of acute renal injury with severe morbidity and mortality. Side population (SP) cells have recently drawn increasing attention due to their critical role in injury repair and regeneration. Unfortunately, the underlying mechanism involved in renal I/R remains poorly elucidated. Here, pronounced increases of stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) were substantiated in I/R kidneys from C57BL/6 mice subjected to clamp the bilateral renal pedicles to mimic renal ischemia. Similar up-regulation of them was also determined in SP cells upon simulated ischemia/reperfusion (SI/R). In contrast to non-SP cells, SP cells exhibited higher viability, apoptosis resistance, chemotaxis, and paracrine actions following SI/R treatment, and these were further enhanced after SDF-1 stimulation. Interestingly, blocking CXCR4 signaling with AMD3100 notably ameliorated the above effects. Mechanism analysis corroborated that SDF-1/CXCR4 further induced the expression of ATP-binding cassette transporter ABCG2, an essential element for SP-mediated kidney regeneration after renal I/R injury. Moreover, AMD3100 pretreatment strikingly attenuated ABCG2 elevation in SP cells. Additionally, sonic hedgehog (SHH)-Gli 1 signaling was involved in SDF-1/CXCR4-mediated ABCG2 expression. When SP cells pretreated with AMD3100 were intravenously injected into I/R mice, SP cell-mediated decreases in blood urea nitrogen, serum creatinine, and histological score of kidney were noticeably attenuated, indicating that blocking CXCR4 pathway mitigated the therapeutic function of SP cells in renal I/R injury. Together, this research suggests that SDF-1/CXCR4 axis might act, via Shh-Gli1-ABCG2 signaling, as a positive regulator of SP cell-based therapies for renal I/R by Shh-Gli 1-ABCG2 signaling.

SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3\u03b2/\u03b2-catenin pathways

SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

SDF-1/CXCR7 axis regulates the proliferation, invasion, adhesion, and angiogenesis of gastric cancer cells.

BackgroundMore recent studies have revealed that chemokine receptor CXCR7 plays an important role in cancer development. However, little is known about the effect of CXCR7 on the process of gastric cancer cell invasion and angiogenesis. The aim of this study is to investigate the expression of CXCR7 in gastric cancer cell lines and to evaluate the role of CXCR7 in the proliferation, invasion, adhesion, and angiogenesis of gastric cancer cells.MethodsReal-time PCR and Western blotting were used to examine the mRNA and protein levels of CXCR4 and CXCR7 in five gastric cancer cell lines (HGC-27, MGC-803, BGC-823, SGC-7901, and MKN-28). CXCR7-expressing shRNA was constructed and subsequently stably transfected into the human gastric cancer cells. In addition, the effect of CXCR7 inhibition on cell proliferation, invasion, adhesion, VEGF secretion, and tube formation was evaluated.ResultsThe mRNA and protein of CXCR7 were expressed in all five gastric cancer cell lines; in particular, the expression of CXCR7 was the highest in SGC-7901 cells. Stromal cell-derived factor-1 (SDF-1) was found to induce proliferation, invasion, adhesion, and tube formation. Moreover, the VEGF secretion in SGC-7901 cells was also enhanced by SDF-1 stimulation. These biological effects were inhibited by the silencing of CXCR7 in SGC-7901 cells.ConclusionsIncreased CXCR7 expression was found in gastric cancer cells. Knockdown of CXCR7 expression by transfection with CXCR7shRNA significantly inhibits SGC-7901 cells’ proliferation, invasion, adhesion, and angiogenesis. This study provides new insights into the significance of CXCR7 in the invasion and angiogenesis of gastric cancer.

RGS1 and RGS13 Regulate SDF-1-Mediated Responses and Homing in Human Cord Blood CD34+ Hematopoietic Stem/Progenitor Cells

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis is crucial to migration and homing of hematopoietic stem/progenitor cells (HSC). In a previous study, we reported the expression profile of human cord blood CD34+ cells in response to a short-term exposure of SDF-1 (Leung et al, Blood, 2011). We further identified that expression levels of several R4 Regulator of G-protein signaling (RGS) family proteins were upregulated upon SDF-1 treatment. RGS family proteins are known to negatively control G-protein-coupled receptor signal transduction through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes), responses to SDF-1 are subjected to regulation by RGS proteins. However, their expression patterns and functional roles in HSC are poorly characterized. We first assessed the basal expressions of 10 R4 RGS proteins by quantitative RT-PCR. Our results demonstrated that cord blood CD34+ cells expressed relatively high mRNA level of RGS1, RGS2 and RGS3, and moderate level of RGS5, RGS13, RGS16 and RGS18 (n = 3). Expression of RGS8 was barely detectable, while RGS4 and RGS21 expression was not detectable. A short-term exposure (1-24 hours) of CD34+ cells to SDF-1 (100 ng/mL, n = 4) significantly increased expressions of RGS1 (1.5 fold, P = .002), RGS13 (1.8 fold, P = .044), and to a lesser extent RGS2 and RGS3 (both 1.3 fold; P < .032). Expressions of RGS5, RGS16 and RGS18 were not affected by SDF-1 stimulation. Preincubation of CD34+ cells with the CXCR4 antagonist AMD3100 resulted in 82.2% and 56.2% inhibition of SDF-1-induced RGS1 and RGS13 expressions respectively. To investigate the functional roles of RGS1 and RGS13 in SDF-1-mediated responses, we introduced GFP-tagged cDNA clones into cord blood CD34+ cells by lentiviral transduction. Using the spleen focus-forming virus (SFFV) promoter, we achieved 55.2% transduction efficiency in control GFP vector-transduced cells, 39.4% in RGS1-transduced cells and 51.7% in RGS13-transduced cells. Quantitative RT-PCR further confirmed RGS1 (76 fold) and RGS13 (3,641 fold) overexpression in transduced CD34 cells. We showed that SDF-1-directed migration was inhibited by 18.5% in RGS1-transduced cells, but not in RGS13-transduced cells. In RGS1-transduced cells, significantly less GFP+ cells was found in the migrating fraction (51% in non-migrating vs 28% in migrating fraction). We further demonstrated that actin polymerization, an early response to SDF-1 stimulus, was reduced by 9.5% in RGS13-transduced cells but not in RGS1-transduced cells. In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing of CD34+ cells was impaired in RGS1-overexpressing (54.4% reduction) or RGS13-overexpressing (28.1% reduction) cells, when compared to control GFP-transduced cells. Splenic homing of CD34+ cells was not affected by either RGS1 or RGS13. Taken together, we provided the first expression profile of R4 RGS proteins in cord blood CD34+ cells, and their expressional changes in response to SDF-1 stimulation. We also provided evidence that RGS1 and RGS13 regulate specific SDF-1-mediated responses and, importantly, both negatively affect homing of CD34+ cells. Strategies to inhibit RGS1 or RGS13 signaling could be a potential method for enhancing HSC homing and their long-term engraftment for immunologic reconstitution. DisclosuresNo relevant conflicts of interest to declare.

The SDF‐1/CXCR4 axis utilizes mTOR signal transduction to promote angiogenesis (278.8)

Pathological angiogenesis, which occurs during chronic inflammation or tumor growth, results when positive and negative angiogenic regulators are disrupted. The chemokine stromal‐cell derived factor‐1 (SDF‐1) and its receptor CXCR4 promote endothelial cell (EC) migration and tube formation. PI3K is a key downstream regulator of CXCR4‐mediated chemotaxis and mammalian target of rapamycin (mTOR) is at the center of PI3K‐mediated signal transduction. We have hypothesized that the SDF‐1/CXCR4/mTOR signaling axis contributes to the molecular mechanism of angiogenesis. This study illustrates that SDF‐1 stimulation activates mTOR signal transduction in EC grown in monolayer cell culture. The pharmacological inhibition of CXCR4 or mTOR blocks sprouting in an in vitro 3D angiogenesis model, and also abolishes EC migration in a 3D angiogenic invasion assay. Specific inhibition of mTOR complex 1 or mTOR complex 2 (mTORC2) reveals that SDF‐1‐induced angiogenesis occurs via mTORC2 signal transduction. An analysis of targets downstream of mTOR indicates that 6‐phosphofructo‐2‐kinase (PFKFB3), which is a key regulator of glycolysis, is downregulated in EC in which mTORC2 is inhibited. Taken together these data indicate that the SDF‐1/CXCR4 axis activates angiogenesis by utilizing an mTORC2 signal transduction pathway. In addition, these data present a potential link between mTORC2, metabolism and angiogenesis, which deserves further exploration.Grant Funding Source: The project described was supported by Grant Number T32CA009054 from the National Cancer Institute.

The role of IL-8 in the SDF-1α/CXCR4-induced angiogenesis of laryngeal and hypopharyngeal squamous cell carcinoma

Stromal cell-derived factor-1 (SDF-1) (CXCL12) has been observed to promote laryngeal and hypopharyngeal squamous cell carcinomas (LHSCCs) invasion through cooperation with its receptor CXCR4. Here, we further explore the angiogenesis mechanism induced by SDF-1/CXCR4 interaction in LHSCCs. Immunohistochemistry (IHC) reveals the significant correlation between CXCR4 and angiogenesis in tumors. After blocking the function of CXCR4 by specific inhibitor AMD3100 and neutralized antibody 12G5 or inhibiting the expression by siRNA, we were able to disrupt the HUVECs tube formation, demonstrating that SDF-1/CXCR4 indeed regulated the angiogenesis mechanism. The angiogenesis profiling from angiogenesis array and reverse transcription polymerase chain reaction indicates that IL-8 can be significantly triggered by SDF-1/CXCR4 interaction in LHSCCs. We also demonstrate that IL-8 secretion mechanism is regulated by Akt phosphorylation after SDF-1 stimulation. These results point out the importance of SDF-1/CXCR4 interaction in LHSCCs angiogenesis. The angiogenic factor IL-8 would be triggered by the cooperation of SDF-1 and CXCR4 through an Akt-dependent pathway. This provides a new targeting therapy utility, disrupting SDF-1/CXCR4 interaction combined with downstream-induced angiogenic factors in LHSCCs would be beneficial to improve clinical outcome.

Stromal cell‐derived factor‐1/CXC receptor 4 and β1 integrin interaction regulates urokinase‐type plasminogen activator expression in human colorectal cancer cells

The stromal cell-derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) axis has been shown to play a role in colorectal cancer progression. In addition, the protease urokinase-type plasminogen activator (uPA) is an important factor in tumor cell invasion and metastasis. However, the mechanism by which SDF-1 mediates uPA expression in human colorectal cancer cells remains unknown. We investigated the molecular mechanism governing the interaction between SDF-1 stimulation and uPA expression in three human colon cancer cell lines (DLD-1, SW48, and COLO 205). We found that SDF-1 stimulation led to an increase in the expression and secretion of uPA in these cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways are critical for SDF-1-induced uPA expression. Analysis of transcription factor binding using ELISA and chromatin immunoprecipitation assays revealed that SDF-1 increased Sp1- and AP-1-DNA-binding activities in DLD-1 cells. Inhibition of Sp1 and AP-1 activation blocked the SDF-1-induced expression and activity of the uPA promoter. The effect of SDF-1 on DLD-1 signaling and uPA expression was mediated by the CXCR4/β1 integrin axis. In summary, our findings elucidate the mechanisms of SDF-1/CXCR4 downstream signaling and provide insights into the function of SDF-1 in colon cancer cells.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro

ObjectiveTo characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis....

Gα13 and Rho Mediate Endosomal Trafficking of CXCR4 into Rab11+ Vesicles upon Stromal Cell-Derived Factor-1 Stimulation

CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and, thereby, lead to gene upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11(+) vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11(+) compartment. Trafficking of CXCR4 into the Rab11(+), TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11(+) endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11(+), recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4-TCR heterodimers. To our knowledge, this is the first report of Gα13 as a mediator of receptor trafficking.

Activation of Signal Transducers and Activators of Transcription 3 and Focal Adhesion Kinase by Stromal Cell-Derived Factor 1 Is Required for Migration of Human Mesenchymal Stem Cells in Response to Tumor Cell-Conditioned Medium

Mesenchymal stem cells (MSCs) migrate to tumors both in vitro and in vivo. Gene expression profiling analysis reveals that stromal cell-derived factor 1 (SDF-1) is significantly upregulated in MSCs exposed to tumor cell-conditioned medium, when compared with cells treated with control medium, suggesting that SDF-1 signaling is important in mediating MSC migration. This study investigates downstream signaling during MSC migration in response to tumor cell-conditioned medium and recombinant SDF-1 protein treatments. We observed that both recombinant SDF-1 and tumor cell-conditioned medium were able to activate downstream signaling via signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) as revealed by increased phosphorylation of STAT3 and ERK1/2 in human MSCs (hMSCs). Significant impairment of in vitro migration was observed in the presence of MAPK/ERK kinase (MEK) inhibitor PD98059, whereas two Janus kinase 2 (Jak2) inhibitors completely abolished migration induced by tumor cell-conditioned medium. Impaired MSC migration correlated with decreased levels of phosphorylated STAT3 and ERK1/2, suggesting that SDF-1 stimulation activates Jak2/STAT3 as well as MEK/ERK1/2 signaling, which in turn promotes migration of MSCs toward tumor cells. Furthermore, stimulation of hMSCs with recombinant SDF-1 and tumor cell-conditioned medium also significantly activated the focal adhesion kinases (FAKs) and paxillin, which correlated with reorganization of F-actin filaments in hMSCs. Decreased phosphorylation of FAK and paxillin as well as disruption of cytoskeleton organization was observed following Jak2 and MEK inhibitor treatment. Taken together, our results provide insight into the molecular pathways responsible for MSC migration toward the tumor microenvironment and may provide the molecular basis for modifying MSCs for therapeutic purposes.

Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer

To investigate the effect of chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer. Human gastric cancer cells of the lie NUGC4 and mesothelial cells of the line HMrSV were cultured. RT-PCR was used to detect the expression of CXCR4 and SDF-1 mRNA in the NUGC4 and HMrSV cells. The proliferation of NUGC4 cells was detected by MTT method. In mesothelial cell adhesion teat NUGC4 cells were cultured with confluent HMrSV mesothelial cells in 24-well plate and then divided into 3 groups: chemokine group, added with SDF-1, antibody blocking group, in which the NUGC4 cells were pre-incubated with CXCR4 monoclonal antibody for 2 h and then SDF-1 was added, and control group added with only culture fluid. Microscopy was used to calculate the number of gastric cancer cells adhered with mesothelial cells. In the mesothelial cell migration test HMrSV cells were put in the upper chamber of a Transwell chamber so as to cover the infiltration membrane. This Transwell chamber was put into a culture plate, NUGC4 cells, divided into 3 groups as mentioned above were put into the upper chamber, 24 h later HE staining and microscopy were performed to calculate the number of the NUGC4 cells that penetrated the membrane. BALB/c nu/nu female nude mice underwent intraperitoneal injection of NUGC4 cells, and then with PBS or AMD3100, small molecular specific antagonist, one day after the cancer cell injection once a day for 2 weeks. Then the mice were killed to observe the intraperitoneal tumorigenesis. CXCR4 mRNA was highly expressed in the NUGC4 cells but only very weekly expressed in the HMrSV cells. SDF-1 mRNA expression was seen in the HMrSV cells but in the NUGC4 cells. Anti-CXCR4 monoclonal antibody (McAb) inhibited the proliferation of NUGC4 cells significantly (P < 0.05). In mesothelial cell adhesion test, the number of the NUGC4 cells adhered with HMrSV cells after SDF-1 stimulation was 84.4 +/- 21.2, significantly higher than that of the control group (43.6 +/- 12.4, P < 0.05). The number of migrating NUGC4 cells in the chemokine group was 170.8 +/- 24.2, significantly higher than hat of the control group (102.8 +/- 18.2, P < 0.05); and the number of migrating NUGC4 cells in the antibody blocking group was 114.7 +/- 20.3, significantly lower than that of the chemokine group (P < 0.05). The survival time of the mice injected with both NUGC4 cells and AMD3100 was (43.8 +/- 2.8) days, significantly longer than that of the control group [(28.2 +/- 2.5) days, P < 0.01]. The tumor number of the AMD3100 group was (64.6 +/- 8.2), significantly lower than that of the control group [(103 +/- 12.4), P < 0.01]. SDF-1 and its receptor CXCR4 play an important role in the development of peritoneal carcinometastasis from gastric cancer. Interfering with the SDF-1/CXCR4 biological axis may become a potential strategy in the prevention and treatment of peritoneal carcinometastasis from gastric cancer.

Stromal-cell derived factor-1 regulates epithelial ovarian cancer cell invasion by activating matrix metalloproteinase-9 and matrix metalloproteinase-2

The aim of this study was to test the hypothesis that stromal-cell derived factor-1 (SDF-1)/chemokine receptor type 4 (CXCR4)-mediated metastasis of ovarian cancer cells to peritoneal cavity would be related with matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) activation. Invasion assay was used to evaluate the functional interaction between SDF-1 and CXCR4. Real-time polymerase chain reaction was performed to analyze the changes of CXCR4, MMP-2 and MMP-9 at the mRNA level upon SDF-1 stimulation. Western blot was used for observing the changes of CXCR4 protein expression and zymograph assay for comparing MMP-2 and MMP-9 activities after SDF-1 induction. In ovarian cancer cells, SDF-1 stimulation resulted in increased CXCR4 expression at both the transcription and protein levels. SDF-1 increased cell invasion in a CXCR4-dependent manner, and this increase was related with the activation of MMPs. Moreover, SDF-1-CXCR4 interaction activated MMP-9 at the both transcription and protein levels and MMP-2 only at the post-translation level. SDF-1-CXCR4 interaction confers on ovarian cancer cells a remarkable potential to activate MMPs for subsequently invading the peritoneal cavity.

Protein Phosphatase 2A Plays an Important Role in Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12-Mediated Migration and Adhesion of CD34+ Cells

Migration of hemopoietic stem and progenitor cells (HSPC) is required for homing to bone marrow following transplantation. Therefore, it is critical to understand signals underlying directional movement of HSPC. Stromal cell-derived factor-1 (SDF-1)/CXCL12 is a potent chemoattractant for HSPC. In this study, we demonstrate that the serine-threonine protein phosphatase (PP)2A plays an important role in regulation of optimal level and duration of Akt/protein kinase B activation (a molecule important for efficient chemotaxis), in response to SDF-1. Inhibition of PP2A, using various pharmacological inhibitors of PP2A including okadaic acid (OA) as well as using genetic approaches including dominant-negative PP2A-catalytic subunit (PP2A-C) or PP2A-C small interfering RNA, in primary CD34(+) cord blood (CB) cells led to reduced chemotaxis. This was associated with impairment in polarization and slower speed of movement in response to SDF-1. Concomitantly, SDF-1-induced Akt phosphorylation was robust and prolonged. Following SDF-1 stimulation, Akt and PP2A-C translocate to plasma membrane with enhanced association of PP2A-C with Akt observed at the plasma membrane. Inhibition of PI3K by low-dose LY294002 partially recovered chemotactic activity of cells pretreated with OA. In addition to chemotaxis, adhesion of CD34(+) cells to fibronectin was impaired by OA pretreatment. Our study demonstrates PP2A plays an important role in chemotaxis and adhesion of CD34(+) CB cells in response to SDF-1. CD34(+) CB cells pretreated with OA showed impaired ability to repopulate NOD-SCID mice in vivo, suggesting physiological relevance of these observations.

Protein Phosphatase2A Regulates Stromal Cell- Derived Factor-1 Mediated Responses of CD34+ Cord Blood Cells.

The chemokine stromal cell- derived factor-1(SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. During steady-state hematopoiesis, CXCR4/SDF-1 interaction restricts hematopoietic stem progenitor cells in marrow and disruption of the SDF-1/CXCR4 axis leads to their egress into circulation. However, biological responses to SDF-1 are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. It is known that phosphorylation of G-protein coupled receptors (GPCRs) by several protein kinases including serine-threonine kinase, protein kinase C (PKC), can result in receptor desensitization. This prompted us to investigate whether the role of serine- threonine phosphatase in the regulation of SDF-1 induced responses of CD34+ cord blood (CB) cells. To investigate the role of serine- threonine phosphatase we evaluated the effect of okadaic acid, a specific serine-threonine phosphatase inhibitor, on SDF-1 induced chemotaxis and adhesion of CD34+ cells from cord blood. Pre-incubation of CD34+ CB cells with okadaic acid (100–1000nM) significantly reduced SDF-1 directed chemotaxis and adhesion of CD34+ CB cells. This correlated with an increase in PKC phosphorylation. Although primary SDF-1 induced calcium flux in CD34+CB cells was unaffected by okadaic acid, if the cells were pretreated with SDF-1 and then stimulated with SDF-1, calcium flux was significantly less in CD34+CB cells pretreated with okadaic acid compared to untreated cells. Further, confocal microscopy was used to demonstrate the co-localization of serine threonine phosphatase and PKC. Using antibodies specific to PP2A catalytic subunit (PP2Ac) and various classes of PKC super family, it was observed in unstimulated CD34+ CB cells PP2Ac was mainly localized in nucleus and some phosphor-PKC-delta was detected in the cytoplasm. However, upon SDF-1 stimulation, co-localization of PP2Ac and phospho-PKC-delta was observed on the membrane of CD34+CB cells. These results indicate that serine-threonine phosphatase regulate SDF-1 mediated responses in CD34+CB cells and may play an important role in the ability of hematopoietic stem progenitor cells to continually respond to SDF-1.

Long-term functional impairment of hemopoietic progenitor cells engineered to express the S1 catalytic subunit of pertussis toxin

A large body of data suggests that pertussis toxin (PTX)-sensitive G protein signals in mature and immature hemopoietic cells control their migration patterns in vitro and in vivo. These effects were derived after treatment of cells or animals with PTX. To circumvent several inherent problems of PTX holotoxin treatment, we expressed the S1 catalytic activity of PTX, thus blocking Gi protein signaling, in 32D murine myeloid progenitor cells and in primary human CD34+ cells, and studied its functional consequences. S1 was expressed using viral vectors. Effects of Gi protein blockade on proliferation, migration, adhesion, and gene expression were tested in vitro. S1 expression was nontoxic for the cells; expression and function were stable long-term and not overridden by compensatory mechanisms. S1-transduced 32D cells and primary CD34+ cells migrated poorly and did not contract their cytoskeleton upon treatment with the chemoattractant stromal cell-derived factor -1 (SDF-1), similar to the phenotype induced by PTX treatment. Gene expression studies comparing S1-transduced and control 32D cells uncovered four genes, expression of which was regulated by Gi protein blockade. Of interest, although SDF-1 signaling was inhibited, comparison between SDF-1-treated and untreated cells suggests that SDF-1 stimulation does not depend on de novo gene expression in these cells. Furthermore, when injected into nonobese diabetic/severe combined immunodeficient mice, seeding of S1-expressing 32D cells to bone marrow was largely blocked. Expression of S1 is an effective approach for studying long-term functional consequences of Gi protein blockade in hemopoietic cells in vitro and in vivo.

Unique SDF-1–induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling

AbstractThe mechanisms governing migration and extramedullary dissemination of leukemic cells remain obscure. In this study the migration and in vivo homing to the bone marrow of nonobese diabetic severe combined immunodeficient (NOD/SCID) mice injected with human precursor-B acute lymphoblastic leukemia (ALL) cells in comparison to normal CD34+progenitors (both cord blood and mobilized peripheral blood) was investigated. Although migration and homing of both cell populations was dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions, major differences in receptor expression as well as the migratory capacity toward various concentrations of SDF-1 were found. Furthermore, unlike normal CD34+progenitors, in vivo homing of the leukemic cells was superior when recipient NOD/SCID mice were not irradiated prior to transplantation. In addition, we report differences in the adhesion molecules activated following SDF-1 stimulation, documenting a major role for very late antigen 4 (VLA-4), but not VLA-5 and lymphocyte function-associated antigen-1 (LFA-1), in homing of precursor-B ALL cells. Interestingly, Toxin-B and pertussis toxin inhibited the homing of the leukemic cells but not that of normal CD34+progenitors or normal CD10+/CD19+precursor-B cells, revealing differences in CXCR4 signaling pathways that are based on changes that acquired by the leukemic cells. Altogether, our data provide new insights into different SDF-1–induced signaling, activation, and consequent motility between normal CD34+and precursor-B ALL progenitors, which may lead to improved clinical protocols. (Blood. 2004;103: 2900-2907)