Pasteurella multocida toxin: potent mitogen for cultured fibroblasts.

Abstract

Native Pasteurella multocida toxin (PMT) is shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37C, but not at 4°C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH

“…It is therefore likely that a conformational change occurs within the toxin at pH 5 allowing proteolytic cleavage of PMT. This supports the suggestion that PMT, whose action is blocked by methylamine [1], is taken up by RME and trafficked to a low pH compartment where it is proteolytically activated.…”

“…Pasteurella multocida toxin (PMT) is the most potent mitogen identified for the Swiss murine 3T3 fibroblast cell line [1], and is the first intracellularly acting toxin that activates phosphatidyl inositol-specific phospholipase C (PI-PLC) [2]. The toxin is also mitogenic for the following established cell lines, Rat-1 [3], BALB/c 3T3, NIH 3T3, 3T6 and human fibroblasts [1].…”

“…The toxin is also mitogenic for the following established cell lines, Rat-1 [3], BALB/c 3T3, NIH 3T3, 3T6 and human fibroblasts [1]. The toxin has been cloned, sequenced and expressed in Escherichia co# [4][5][6][7][8][9].…”

“…There is evidence that PMT may enter the cytosol from an acidic compartment (endosome or lysosome) after being internalised by RME, since weak bases such as methylamine, which increase intracellular pH, inhibited the mitogenic effects of PMT [1]. This indicates that low pH processing of PMT probably occurs to yield an active fragment.…”

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

“…The latter disease is characterized by osteoclastic bone resorption of nasal turbinates, an effect that is uniquely caused by PMT. PMT activates a number of mitogenic signaling pathways, including MAP kinases (e.g., ERK) and JAK/STAT pathways (6,7) and is one of the most potent known mitogens of fibroblasts (8,9). Therefore, a role of PMT in tumor development and cancer has been proposed (10).…”