Studies on the mechanism of action of the potent mitogen Pasteurella multocida toxin

The mechanisms of action of bacterial toxins have provided novel approaches to elucidating cellular and molecular regulatory mechanisms. Recently Pasteurella muHocida toxin (PMT) was shown to be an extremely effective mitogen for Swiss 3T3 cells and stimulates a phospholipase C-mediated increase in inositol phosphates in these cells. Hence, rPMT provided a novel tool for studying this signalling pathway. Here we analysed the effect of recombinant PMT (rPMT) on the G protein and tyrosine kinase-mediated pathways for the activation of phospholipase 0. The results in this thesis demonstrate that rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. Treatment of Swiss 3T3 cells with a subsaturating concentration of rPMT markedly potentiated the production inositol phosphates induced by bombesin, vasopressin and endothelin in both a time- and dose-dependent manner. Under similar conditions rPMT had no potentiating effect on the production of inositol phosphates induced by PDGF (AA and BB homodimers). rPMT treatment also markedly enhanced bombesin-induced enhancement of inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In addition, rPMT pretreatment greatly reduces the Ca2+-mobilising action of bombesin, consistent with Ca2+ mobilisation from a common intracellular pool. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase Cy. Depletion of protein kinase C markedly increased the accumulation of inositol phosphates induced by rPMT in a manner similar to that observed for bombesin but not PDGF. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis also persisted in permeabilized cells. The addition of guanosine 5'-O-(β-thiodiphosphate) to permeabilized cells markedly reduced rPMT induced accumulation of inositol phosphates in a time and dose dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. These results strongly suggest that the action of rPMT is to facilitate the coupling of G protein to phospholipase C. Recently, the pertussis toxin-insensitive Gq subfamily of G proteins have been found to couple neuropeptide receptors (including vasopressin) to the β isoform of phospholipase C. Overexpression of the alpha subunits of this G-protein family (αq and α11) by transfection in COS-1 cells resulted in an increased production of inositol phosphates induced by rPMT. These results demonstrate that Gq may be involved in the production of inositol phosphates stimulated by PMT. Serum and other growth factors are known to transiently induce the expression of the early protooncogenes c-fos and c-myc. The induction of these genes is subject to strong feedback inhibition. At least part of this autoregulatory mechanism could be the result of receptor desensitization. At the beginning of this work nothing was known about the ability of rPMT to stimulate gene expression. Since PMT by-passes receptor-mediated signal generation we have analysed the effect of rPMT on the induction of these early protooncogenes. The results in this thesis demonstrate that rPMT also stimulates the induction of c-fos and c-myc in Swiss 3T3 cells. The induction of these genes by rPMT occurs after a lag period of 3-4 h and levels of c-fos and in particular c-myc message can be detected for prolonged time periods. Maximal levels of c-fos induced by rPMT were approximately 50% of the maximum levels induced by bombesin. In contrast, maximum levels of c-myc induced by rPMT were slightly higher than maximum levels induced by bombesin. In PKC-down-regulated cells the levels of c-fos and c-myc mRNA induced by rPMT were severely attenuated but not abolished. Similar results are observed for bombesin-stimulation of c-fos and c-myc in PKC-depleted cells. These results demonstrate that rPMT stimulates early gene expression through both PKC-dependent and independent pathways. At least in the case of c-myc, the striking and prolonged expression of this protooncogene may be attributed to the persistent activation of early signalling pathways by rPMT