Mechanism of PMT-Induced Anchorage-Independent Growth and mTOR Signaling

PMT 诱导锚定非依赖性生长和 mTOR 信号转导的机制

• 批准号: 8746644
• 负责人: P. BOON Chock
• 金额: $ 86.51万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/8746644
• 关键词: 1,2-diacylglycerol; 3T3 Cells; ATP Synthesis Pathway; Anchorage-Independent Growth; Apoptosis; Area; Bacterial Infections; Bacterial Proteins; Biological; Biological Assay; Cancer Biology; Cell Line; Cell Proliferation; Cell division; Cell membrane; Cells; Chronic; Citric Acid Cycle; Conditioned Culture Media; Culture Media; Development; Diglycerides; Disease; Epidemiology; Epidermal Growth Factor Receptor; Extracellular Matrix Proteins; Fibrosis; GTP-Binding Proteins; Genes; Glutamine; Guanosine Triphosphate; In Vitro; Infection; Inositol; Knock-out; Link; MAP Kinase Gene; MEKs; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Messenger RNA; Metabolism; Microarray Analysis; Mitogens; Molecular; Neoplasm Metastasis; Normal Cell; Paracrine Communication; Pasteurella multocida toxin; Pathway interactions; Phenotype; Phosphatidic Acid; Phosphorylation; Play; Production; Property; Protein Biosynthesis; Protein Tyrosine Kinase; Proteins; Receptor Activation; Reporting; Ribosomal Protein S6; Ribosomal Protein S6 Kinase; Ribosomes; Role; Serum; Signal Pathway; Signal Transduction; Sirolimus; Suspension substance; Suspensions; Swiss 3T3 Cells; Time; Tumor Promoters; Up-Regulation; Virulence Factors; Wound Healing; alpha ketoglutarate; aurora B kinase; autocrine; cell motility; cell type; connective tissue growth factor; deamidation; glucose receptor; in vivo; inhibitor/antagonist; intercellular communication; mTOR protein; migration; overexpression; phospholipase C beta; survivin; tumor; uptake

It is believed that bacterial virulence factors that interfere with cell signaling and result in disruption of normal cell division could promote anchorage-independent growth. Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections. The detailed mechanism behind mitogenic properties of PMT is unknown. Recent reports show that PMT exerts its biological effects, in part, via the deamidation of a conserved glutamine residue in the alpha subunit of heterotrimeic G proteins, including Gaq, Gai, Ga12, and Ga13, and leads to a constitutively active phenotype of the G proteins. Our mechanistic study showed that rPMT caused a significant increase in protein synthesis in adherent serum-starved Swiss 3T3 cells, a cell line has been extensively used for studying cellular effect of PMT, as well as in serum-starved cells kept in suspension. Protein synthesis is energetically costly, requiring not only ATP and GTP but also the production of ribosomes. Indeed, rPMT treatment of serum-starved cells for 24h induced a 30% increase in their cellular ATP content in comparison to the control non-treated cells. Furthermore, rPMT treatment also induced migration and proliferation in quiescent 3T3 cells as demonstrated by an in vitro wound healing assay. Addition of rPMT to serum-starved 3T3 cells resulted in an increase of cells migration toward the denuded area compared with control non-treated cells. Concomitantly rPMT induces a sustained phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (S6). This phosphorylation is inhibited by rapamycin and Torin1, two specific inhibitors of mammalian target of rapamycin (mTOR). The PMT-mediated mTOR activation was observed in MEF WT but not in MEF Gaq/11 knockout cells, consistent with our results indicating that PMT-induced mTOR activation proceeds via the deamidation of Gaq/11 and leads to the activation of PLC-beta; to generate diacylglycerol (DAG) and inositol trisphosphate (IP3), two known activators of PKC pathway. Exogenously added DAG or PMA, activators of PKC, leads to S6 phosphorylation in a manner dependent on rapamycin. Furthermore, PMT-induced S6 phosphorylation is inhibited by PKC inhibitor, Go6976. These findings reveal for the first time that PMT activates mTORC1 through the Gaq/11/PLCβ/PKC pathway to, in part, mediate cellular protein synthesis. In addition, we did not observe any increase in phosphatidic acid in rPMT treated cells. However, rPMT did induce EGF receptor activation and glucose receptor I (Glut1) upregulation, yet they exert no effect on rpS6 phosphorylation. Immunohistochemical analysis revealed that Clut1 was not translocated to the plasma membrane, and thus, the rPMT-induced cellular ATP synthesis may involve glutamine uptake and metabolism to generate alpha-ketoglutarate to drive the TCA cycle to generate ATP. It is worth mentioning, that cell treatment with rPMT in the presence of rapamycin did not completely inhibit rPMT-induced protein synthesis and cell proliferation. This result is consistent with the observation that rPMT is capable of inducing some degree of protein synthesis in Gq/11-deficient cells, independent of mTORC1 activation. Taken together, our findings indicate that activation of mTORC1 pathway and the stimulation of additional signaling cascades are responsible for the rPMT-mediated protein synthesis and cell proliferation. In addition, an increasing body of evidence supports the idea that extracellular matrix (ECM) proteins are major players in the global control of intercellular communication and integration of environmental signals. We hypothesized that the mitogenic action of rPMT may involve the expression and secretion into the culture medium substance(s) capable of activating autocrine and/or paracrine signaling pathways. To this end, we found that the conditioned medium from rPMT-treated cells activates mTORC1 and MAPK signaling, but not membrane-associated tyrosine kinase signaling. Surprisingly, this diffusible factor(s) is (are) capable of activating mTORC1 and MAPK pathways even in MEF Gq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene, with a 140 folds enhancement, in 3T3 cells, along with other genes, e.g. those encode survivin and aurora kinase B, known to involve in cell proliferation and cancer biology. In accord with the elevation of mRNA, CTGF protein was also elevated. CTGF, an ECM protein, is known to be upregulated in certain cancers and in fibrosis. In accord with rPMT-induced mTOR activation, upregulation of CTGF was mediated by deamidation of Gq/11, and was independent of TGF, a well-known inducer of CTGF, which is also involved in fibrotic disease. Furthermore, MEK/ERK but not mTOR regulates rPMT-induced upregulation of CTGF at the translational level. Importantly, overexpression of CTGF in mammalian cells leads to rpS6 phosphorylation, a readout of mTOR activation. However, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells, indicating that while CTGF plays an important role, there are additional factors involved in the mitogenic action of PMT.

人们认为，干扰细胞信号传导并导致正常细胞分裂的细菌毒力因子可以促进非锚定生长。多杀性巴氏杆菌毒素（PMT）是一种细胞内作用的细菌蛋白，以其在体外和体内的强有丝分裂特性以及诱导某些类型细胞的强非锚定生长的能力而闻名。这些特性表明PMT可能具有作为肿瘤促进剂的潜力，特别是在慢性感染的情况下。PMT有丝分裂特性背后的详细机制尚不清楚。最近的报道表明，PMT发挥其生物学作用，部分是通过在异三聚体G蛋白（包括Gaq， Gai， Ga12和Ga13）的α亚基中保守的谷氨酰胺残基的脱酰胺作用，并导致G蛋白的组成活性表型。我们的机制研究表明，rPMT引起贴壁血清饥饿的Swiss 3T3细胞的蛋白质合成显著增加，该细胞系被广泛用于研究PMT的细胞效应，以及悬浮状态下的血清饥饿细胞。蛋白质合成消耗大量能量，不仅需要ATP和GTP，还需要核糖体的产生。事实上，与未处理的对照细胞相比，rPMT处理血清饥饿细胞24小时诱导其细胞ATP含量增加30%。此外，体外伤口愈合实验表明，rPMT治疗还能诱导静止3T3细胞的迁移和增殖。与未处理的对照细胞相比，在血清饥饿的3T3细胞中添加rPMT导致细胞向剥落区迁移的增加。同时，rPMT诱导核糖体S6激酶（S6K1）及其底物核糖体S6蛋白（S6）的持续磷酸化。这种磷酸化被雷帕霉素和Torin1这两种哺乳动物雷帕霉素靶蛋白（mTOR）的特异性抑制剂所抑制。在MEF WT中观察到pmt介导的mTOR激活，但在MEF Gaq/11敲除细胞中没有观察到，这与我们的结果一致，表明pmt诱导的mTOR激活通过Gaq/11的脱酰胺进行，并导致plc - β的激活；生成二酰基甘油（DAG）和肌醇三磷酸（IP3），这两种已知的PKC通路激活剂。外源添加的DAG或PMA， PKC的激活剂，以依赖雷帕霉素的方式导致S6磷酸化。此外，pmt诱导的S6磷酸化被PKC抑制剂Go6976抑制。这些发现首次揭示PMT通过Gaq/11/PLC&#946；/PKC途径激活mTORC1，部分介导细胞蛋白合成。此外，我们没有观察到rPMT处理细胞中磷脂酸的任何增加。然而，rPMT确实诱导EGF受体激活和葡萄糖受体1 （Glut1）上调，但它们对rpS6磷酸化没有影响。免疫组织化学分析显示，Clut1没有转移到质膜上，因此，rpmt诱导的细胞ATP合成可能涉及谷氨酰胺摄取和代谢生成α -酮戊二酸，从而驱动TCA循环生成ATP。值得一提的是，在雷帕霉素存在的情况下，用rPMT处理细胞并没有完全抑制rPMT诱导的蛋白质合成和细胞增殖。这一结果与rPMT能够在Gq/11缺陷细胞中诱导一定程度的蛋白质合成而独立于mTORC1激活的观察结果一致。综上所述，我们的研究结果表明mTORC1通路的激活和其他信号级联的刺激是rpmt介导的蛋白质合成和细胞增殖的原因。