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

多杀性巴氏杆菌毒素(PMT)是一种细胞内作用的细菌蛋白，在体内和体外都有很强的促有丝分裂能力，并能诱导某些类型细胞的强烈非贴壁生长。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的生物学效应部分是通过使异源三聚体G蛋白(包括Gaq、Gai、GA12和Ga13)的α亚基中保守的谷氨酰胺残基去酰胺化而实现的，并导致G蛋白的结构性活性表型。我们发现rPMT能诱导血清饥饿的瑞士3T3细胞蛋白质和ATP合成、细胞迁移和增殖。同时，PMT诱导核糖体S6蛋白(S6)及其底物S6K1的持续磷酸化。这种磷酸化被雷帕霉素和Torin1这两种哺乳动物靶向雷帕霉素(MTOR)的特异性抑制剂抑制。在MEF WT中观察到PMT介导的mTOR激活，而在MEF Gaq/11基因敲除细胞中未观察到，这与我们的结果一致，表明PMT诱导的mTOR激活是通过Gaq/11的脱酰胺化进行的，并导致已知的PKC途径的两个激活剂--二酰甘油(DAG)和三磷酸肌醇(IP3)的激活。外源加入PKC激活剂DAG或PMA，可导致依赖于雷帕霉素的S6磷酸化。此外，PKC抑制剂Go6976可抑制PMT诱导的S6磷酸化。综上所述，我们的发现首次揭示了PMT通过Gaq/11/PLC/PKC途径激活mTORC1。PMT还可诱导EGF受体激活和葡萄糖受体I(Glut1)表达上调。然而，它们对S6的磷酸化没有影响。PMT诱导的蛋白质合成和细胞迁移被雷帕霉素部分抑制的事实表明，PMT可能刺激额外的信号级联反应。 此外，越来越多的证据支持这样一种观点，即细胞外基质(ECM)蛋白是全球控制细胞间通信和环境信号整合的主要参与者。目前尚不清楚经rPMT处理的细胞是否能够表达和分泌一种底物(S)，该底物能够激活自分泌和/或旁分泌信号。我们发现，来自rPMT处理细胞的条件培养液激活了mTOR和MAPK信号，但不能激活膜相关酪氨酸激酶信号。令人惊讶的是，这种扩散因子(S)即使在MEF Gaq/11双基因敲除细胞中也能够激活mTOR和MAPK通路。微阵列分析发现，结缔组织生长因子(CTGF)mRNA是3T3细胞中表达最高的基因，与其他参与细胞增殖和癌症生物学的基因一样。CTGF是一种ECM蛋白，在某些癌症和纤维化中上调，RT-PCR和Western印迹分析证实了CTGF的上调。与rPMT诱导的mTOR激活相一致，CTGF的上调是由Gaq/11的脱酰胺介导的，并且不依赖于众所周知的CTGF的诱导剂--转化生长因子。此外，MEK/ERK而不是mTOR在翻译水平上调节rPMT诱导的CTGF表达上调。重要的是，CTGF在哺乳动物细胞中的过度表达导致S6磷酸化，这是mTOR激活的读数。这些发现表明CTGF发挥了重要作用，但PMT的促有丝分裂作用还涉及其他因素。

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. We showed that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT 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 PLCβ 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. Furtheremore, PMT-induced S6 phosphorylation is inhibited by PKC inhibitor, Go6976. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gaq/11/PLCβ/PKC pathway. PMT also induces EGF receptor activation and glucose receptor I (Glut1) upregulation. However, they exert no effect on S6 phosphorylation. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that PMT could likely stimulate additional signaling cascades. 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. It was not known whether rPMT-treated cells are able to express and secrete into the medium a substrate(s) capable of activating autocrine and/or paracrine signaling. We found that the conditioned medium from rPMT-treated cells activates mTOR and MAPK signaling, but not membrane-associated tyrosine kinase signaling. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gaq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in 3T3 cells, along with other genes involved in cell proliferation and cancer biology. The elevation of CTGF, an ECM protein upregulated in certain cancers and in fibrosis, was confirmed by RT-PCR and Western blot analysis. In accord with rPMT-induced mTOR activation, upregulation of CTGF was mediated by deamidation of Gaq/11, and was independent of TGFβ, a well known inducer of CTGF. 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 S6 phosphorylation, a readout of mTOR activation. These findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.