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

细菌毒力因子干扰细胞信号转导，导致正常细胞分裂中断，被认为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，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。我们的机制研究表明，rPMT显著增加了粘附性血清饥饿的Swiss 3T3细胞的蛋白质合成，该细胞系已被广泛用于研究PMT的细胞效应，以及悬浮状态下的血清饥饿细胞。蛋白质合成在能量上是昂贵的，不仅需要ATP和GTP，而且还需要生产核糖体。事实上，rPMT处理血清饥饿的细胞24小时，与对照的未处理细胞相比，细胞内的ATP含量增加了30%。此外，体外伤口愈合实验表明，rPMT处理还能诱导静止的3T3细胞迁移和增殖。在血清饥饿的3T3细胞中加入rPMT，与未处理的对照细胞相比，细胞向裸露区域迁移的数量增加。同时，rPMT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PLC-β的激活，从而产生已知的PKC途径的两种激活剂--二酰甘油(DAG)和三磷酸肌醇(IP3)。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。这些发现首次揭示了PMT通过Gaq/11/PLC/PKC途径激活mTORC1，部分地调节细胞蛋白质的合成。此外，我们没有观察到rPMT处理的细胞中磷脂酸的任何增加。但rPMT可诱导EGF受体激活和葡萄糖受体I(Glut1)表达上调，但对rpS6的磷酸化无影响。免疫组织化学分析表明，Clut1没有移位到质膜上，因此，rPMT诱导的细胞ATP合成可能涉及谷氨酰胺的摄取和代谢，以产生α-酮戊二酸，从而驱动TCA循环产生ATP。值得一提的是，在雷帕霉素存在的情况下，rPMT处理细胞并不能完全抑制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的观察结果一致，而不依赖于mTORC1的激活。综上所述，我们的研究结果表明，mTORC1通路的激活和额外信号级联的刺激对rPMT介导的蛋白质合成和细胞增殖起作用。 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。我们推测，rPMT的促有丝分裂作用可能涉及到能够激活自分泌和/或旁分泌信号通路的培养液物质(S)的表达和分泌。为此，我们发现，来自rPMT处理细胞的条件培养液激活了mTORC1和MAPK信号，但不激活膜相关酪氨酸激酶信号。令人惊讶的是，即使在MEF GQ/11双基因敲除细胞中，这种扩散因子(S)也能够激活mTORC 1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，上调了140倍，此外还有其他基因，如编码Survivin和极光激酶B的基因，已知涉及细胞增殖和癌症生物学。CTGF蛋白的表达也随着mRNA水平的升高而升高。CTGF是一种ECM蛋白，已知在某些癌症和纤维化中上调。与rPMT诱导的mTOR激活一致，CTGF的上调是由GQ/11的脱酰胺介导的，并且不依赖于众所周知的CTGF的诱导剂，后者也参与了纤维化疾病。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，在哺乳动物细胞中过表达CTGF会导致rpS6的磷酸化，这是mTOR激活的读数。然而，单独上调CTGF并不能像在rPMT处理的细胞中观察到的那样引起形态变化，这表明尽管CTGF起着重要作用，但PMT的促有丝分裂作用还涉及其他因素。

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.