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.
|Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells. J Biol Chem 288:2805-15|
|Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells. Cell Signal 25:1136-48|