Project description:The chemokine CXCL12 and its receptor CXCR4 play important roles in signaling and migration of T-cells, but little is known about the transcriptional events involved in CXCL12-mediated T-cell migration. In this study we performed microarray analysis on CXCL12- treated T-cells, and found that the Wnt family of proteins was significantly upregulated during CXCL12 treatment. Confirmation of these results by real-time PCR and Western analysis indicated that the non-canonical Wnt pathway was specifically upregulated during CXCL12 treatment. In vitro and in vivo knockdown studies confirm that b-catenin (the key mediator of canonical Wnt signaling) is not involved in the CXCL12-mediated migration of T-cells. However, Wnt5A, a non-canonical Wnt protein, increases signaling through the CXCL12/ CXCR4 axis via Protein Kinase C (PKC). Our results demonstrated that CXCL12 required Wnt5A to mediate T-cell migration, and the treatment of T-cells with recombinant Wnt5A sensitized T-cells to CXCL12 induced migration. Additionally, Wnt5A expression was required for the sustained expression of CXCR4, both transcriptionally and translationally. These results could be translated in vivo, using EL4 thymoma metastasis as a model of T-cell migration. Taken together our data indicate, for the first time, that Wnt5A is a critical mediator of the CXCL12/ CXCR4 signaling axis. Keywords: Wnt5A, CXCL12, CXCL12, CXCR4, T-cell Migration
Project description:The chemokine CXCL12 and its receptor CXCR4 play important roles in signaling and migration of T-cells, but little is known about the transcriptional events involved in CXCL12-mediated T-cell migration. In this study we performed microarray analysis on CXCL12- treated T-cells, and found that the Wnt family of proteins was significantly upregulated during CXCL12 treatment. Confirmation of these results by real-time PCR and Western analysis indicated that the non-canonical Wnt pathway was specifically upregulated during CXCL12 treatment. In vitro and in vivo knockdown studies confirm that b-catenin (the key mediator of canonical Wnt signaling) is not involved in the CXCL12-mediated migration of T-cells. However, Wnt5A, a non-canonical Wnt protein, increases signaling through the CXCL12/ CXCR4 axis via Protein Kinase C (PKC). Our results demonstrated that CXCL12 required Wnt5A to mediate T-cell migration, and the treatment of T-cells with recombinant Wnt5A sensitized T-cells to CXCL12 induced migration. Additionally, Wnt5A expression was required for the sustained expression of CXCR4, both transcriptionally and translationally. These results could be translated in vivo, using EL4 thymoma metastasis as a model of T-cell migration. Taken together our data indicate, for the first time, that Wnt5A is a critical mediator of the CXCL12/ CXCR4 signaling axis. Experiment Overall Design: Primary T cells were treated with human CXCL12 (Peprotech, Rocky Hill, NJ) at 100 ng/ml per 10 million cells overnight in a humidified incubator at 37ºC with 5% CO2. Control cells were incubated in media only. Cells were harvested and washed with ice cold PBS for 2 times followed by the addition of ice cold TRIzol (Invitrogen, Carlsbad, CA) and frozen at -80ºC overnight. Total RNA was isolated using the RNA isolation kit manufactured by Qiagen (Valencia, CA). The cDNA was prepared from equal amount of RNA using a cDNA preparation kit (Bio-Rad, Hercules, CA) followed by preparation of cRNA according to manufacturerâs instructions (Agilent, Santa Clara, CA). The cRNA was amplified and labeled with either Cy-3 or Cy-5, using the Agilent low-input linear amplification kit, according to manufacturerâs protocols. Labeled cRNA were applied to the Human 44K whole genome oligo array slides (Agilent, Santa Clara, CA). Slides were hybridized in a rotating chamber overnight at 60ºC in 6X SSC. Next day, slides were washed with 0.005% Triton X-102 for 10 minutes, and then in 0.1X SSC, 0.005% Triton X-102 for 5 minutes on ice. Slides were dried using a nitrogen-filled air gun, and scanned using an Agilent scanner. Images were analyzed using the Agilent Feature Extractor Software, Version A.7.5.1 and ratios for each spot were calculated.
Project description:Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and non-peptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key-factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine-triphosphate (ATP) and uridine-triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin. In vivo, pre-incubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Gαi) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5â-triphosphatases (GTPase) Rac2 and downstream effectors Rho GTPaseâactivated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the migration of HSCs and their homing to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases. Experiment Overall Design: Highly purified CD34+ cells from 6 healthy donors were seeded at 1000000 cells/ml in serum free medium (EX vivo 15) w/o cytokines and treated with 10 mM UTP, 150ng/ml CXCL12, or 10 mM UTP plus 150ng/ml CXCL12 respectively for 24 hours. As a control, CD34+ untreated cells were maintained in the same culture conditions at the same time.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.