Chemokines Induce Wnt-Frizzled Gene Expression in Human T Cells

趋化因子诱导人类 T 细胞中 Wnt 卷曲基因表达

• 批准号: 7592056
• 负责人: DENNIS D. TAUB
• 金额: $ 23.06万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592056
• 关键词: Actins; Adhesions; Adrenal Glands; Binding; Biology; Bone Marrow; CCL19 gene; CXCL12 gene; CXCR4 gene; Cell Line; Cells; Cellular biology; Chemokine, Other; Chemotactic Factors; Chemotaxis; Condition; Development; Electrophoretic Mobility Shift Assay; Family member; GTP-Binding Proteins; Gene Activation; Gene Expression; Gene Family; Genes; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV-1; Human; Immigration; Immune; In Vitro; Inflammation; Inflammatory; Leukocytes; Ligands; Ligation; Liver; Lung; Lymphocyte; Matrix Metalloproteinases; Mediating; Melanoma Cell; Membrane Microdomains; Microarray Analysis; Molecular; Numbers; Organogenesis; Pathway interactions; Play; Polymerase Chain Reaction; Protein Family; Receptor Activation; Receptor Signaling; Reporting; Rest; Rodent; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Stream; Structure; Surface; System; T-Cell Activation; T-Lymphocyte; Thymus Gland; Time; Tissues; Transcriptional Activation; Up-Regulation; Virus; Western Blotting; Wnt proteins; Work; blastomere structure; cell motility; cell type; chemokine; chemokine receptor; in vivo; insight; lymph nodes; member; migration; polymerization; promoter; receptor; receptor expression; receptor function; response; seven-transmembrane G-protein-coupled receptor; small hairpin RNA; trafficking

Chemokines have been shown to induce and direct adhesion, chemotaxis, activation, and degranulation of human and rodent leukocytes both in vitro and in vivo. CXCL12 and CCL19 are two important chemokines that regulate T cell motility and activation under normal and inflammatory conditions. Despite numerous reports examining the function of chemokines, little is known about the molecular changes induced by these ligands. We have identified by microarray analysis differentially expressed genes in human T cells following treatment with CXCL12 or CCL19. In primary T cells, it was observed that the genes associated with non canonical Wnt signaling pathway are significantly induced by chemokines compared to vehicle control-treated cells. The non-canonical Wnts, Wnt5a and Wnt10a, along with their receptors Fzd2 and Fzd3 were significantly induced upon CXCL12 and CCL19 treatment, respectively, in a time-dependent fashion. The up regulation of these genes was confirmed by real time PCR and western blot analysis. In addition, the transcriptional activation of both Wnt5a and Wnt10a promoter by CXCL12 and CCL19 was further confirmed by electrophoretic mobility shift assay (EMSA). siRNA and shRNA knockdown of Wnt5a in primary T cells and transformed T cell lines, respectively, significantly inhibited CXCR4 and CCR7 signaling and function suggesting an important intermediary role for the Wnt-Fzd pathways in chemokine-chemokine receptor function. Moreover, these chemokine-induced Wnt and Fzd molecules appear to play a critical role in PKC and Rac activation, MMP expression and actin polymerization in response to chemokine stimulation. Similar effects were observed using melanoma cell lines. In vivo studies are currently underway to support the function role of this pathway in lymphocyte migration and trafficking. Together, these results provide insight into chemokine-induced gene activation and implicate the possible involvement of the non-canonical Wnt signaling pathway in chemokine activation and T cell trafficking. Moreover, we are also currently verifying and characterizing several additional gene families that are highly expressed in T cells after migration in response to or simply stimulation with CXCL12, CCL19, gp120 and HIV-1 virus. Moreover, the role of lipid rafts in chemokine biology and HIV infectivity are also under examination using microarray analysis. A greater understanding of the transcriptional signals differentially induced by the ligation of various chemokine receptors may provide a means to dissect the pathways by which these chemoattractants induce cell migration and activation as well as any host transcriptional signals important in HIV entry and replication.

趋化因子已被证明在体外和体内诱导和指导人和啮齿动物白细胞的粘附、趋化、活化和脱粒。CXCL 12和CCL 19是两种重要的趋化因子，在正常和炎症条件下调节T细胞运动和活化。尽管有许多研究趋化因子功能的报道，但对这些配体诱导的分子变化知之甚少。我们已经通过微阵列分析鉴定了用CXCL 12或CCL 19处理后人类T细胞中差异表达的基因。在原代T细胞中，观察到与媒介物对照处理的细胞相比，与非经典Wnt信号传导途径相关的基因被趋化因子显著诱导。在CXCL 12和CCL 19处理后，非典型Wnt，Wnt 5a和Wnt 10a，沿着其受体Fzd 2和Fzd 3分别以时间依赖性方式显著诱导。 通过真实的时间PCR和蛋白质印迹分析证实这些基因的上调。此外，通过电泳迁移率变动分析（EMSA）进一步证实了CXCL 12和CCL 19对Wnt 5a和Wnt 10a启动子的转录激活。在原代T细胞和转化T细胞系中Wnt 5a的siRNA和shRNA敲低分别显著抑制CXCR 4和CCR 7信号传导和功能，表明Wnt-Fzd途径在趋化因子-趋化因子受体功能中的重要中介作用。此外，这些趋化因子诱导的Wnt和Fzd分子似乎在响应于趋化因子刺激的PKC和Rac活化、MMP表达和肌动蛋白聚合中起关键作用。 使用黑色素瘤细胞系观察到类似的效果。 目前正在进行体内研究，以支持该途径在淋巴细胞迁移和运输中的功能作用。总之，这些结果提供了深入了解趋化因子诱导的基因激活，并暗示可能参与的非经典Wnt信号通路在趋化因子激活和T细胞运输。 此外，我们目前还在验证和表征几个额外的基因家族，这些基因家族在迁移后在T细胞中高度表达，以响应或简单地刺激CXCL 12，CCL 19，gp 120和HIV-1病毒。此外，脂筏在趋化因子生物学和HIV感染性中的作用也正在使用微阵列分析进行检查。 更好地了解不同的转录信号诱导的各种趋化因子受体的连接可能提供一种手段来解剖这些化学引诱剂诱导细胞迁移和激活的途径，以及任何主机的转录信号在HIV的进入和复制的重要。