Differential Modulation and Subsequent Blockade of Mitogenic Signaling and Cell Cycle Progression byPasteurella multocidaToxin

Abstract

The intracellularly acting protein toxin of Pasteurella multocida (PMT) causes numerous effects in cells, including activation of inositol 1,4,5-trisphosphate (IP 3 ) signaling, Ca 2؉ mobilization, protein phosphorylation, morphological changes, and DNA synthesis. The direct intracellular target of PMT responsible for activation of the IP 3 pathway is the G q/11 ␣-protein, which stimulates phospholipase C (PLC) ␤1. The relationship between PMT-mediated activation of the G q/11 -PLC-IP 3 pathway and its ability to promote mitogenesis and cellular proliferation is not clear. PMT stimulation of p42/p44 mitogen-activated protein kinase occurs upstream via G q/11 -dependent transactivation of the epidermal growth factor receptor. We have further characterized the effects of PMT on the downstream mitogenic response and cell cycle progression in Swiss 3T3 and Vero cells. PMT treatment caused dramatic morphological changes in both cell lines. In Vero cells, limited multinucleation, nuclear fragmentation, and disruption of cytokinesis were also observed; however, a strong mitogenic response occurred only with Swiss 3T3 cells. Significantly, this mitogenic response was not sustained. Cell cycle analysis revealed that after the initial mitogenic response to PMT, both cell types subsequently arrested primarily in G 1 and became unresponsive to further PMT treatment. In Swiss 3T3 cells, PMT induced up-regulation of c-Myc; cyclins D1, D2, D3, and E; p21; PCNA; and the Rb proteins, p107 and p130. In Vero cells, PMT failed to up-regulate PCNA and cyclins D3 and E. We also found that the initial PMT-mediated up-regulation of several of these signaling proteins was not sustained, supporting the subsequent cell cycle arrest. The consequences of PMT entry thus depend on the differential regulation of signaling pathways within different cell types.The protein toxin from Pasteurella multocida (PMT) is the primary etiological agent of progressive atrophic rhinitis (11), and purified PMT experimentally induces all of the major symptoms of atrophic rhinitis in animals (2, 23). PMT appears to bind to and enter mammalian cells via receptor-mediated endocytosis (30, 33), although the details of this process are still unclear. In fibroblasts and osteoblasts, PMT acts intracellularly to enhance inositolphospholipid hydrolysis (25, 26), mobilize intracellular Ca 2ϩ pools (40), increase protein phosphorylation (41), stimulate cytoskeletal changes such as stress fiber formation and focal adhesion assembly (5, 21), and initiate DNA synthesis (33). In contrast to the case for fibroblast cells, PMT exerts cytotoxic or cytopathic effects on Vero cells (2, 28), embryonic bovine lung cells (34), and canine osteosarcoma cells (30).PMT exerts its effects on these cellular processes by acting on the free, monomeric ␣ subunit of the G q/11 family of heterotrimeric G-proteins (46), which stimulates phospholipase C (PLC) ␤1 to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Accordingly, the releas...

Cell Surface-localized Nucleolin Is a Eukaryotic Receptor for the Adhesin Intimin-γ of Enterohemorrhagic Escherichia coliO157:H7

“…For subconfluent HEp-2 cell monolayers, 8-well chamber slides (Lab-Tek) were seeded with 6 ϫ 10 4 cells/well in complete EMEM and incubated for no more than 24 h (ϳ80% confluent) before use. Confluent HEp-2 cell monolayers were prepared according to a method previously described for Vero cells (40). HEp-2 cells were seeded into 8-well chamber slides at a density of 3 ϫ 10 4 cells/well and incubated for 72 h. The medium in each well was then removed and replaced with complete EMEM that contained 1% fetal calf serum.…”

Pasteurella multocida: from Zoonosis to Cellular Microbiology

“…Recent studies have revealed that in addition to the ␣ subunit of the heterotrimeric G q protein (488)(489)(490), PMT also acts on other G proteins, i.e., G 11 , G i , and G 12/13 (488)(489)(490)(491)(492)(493)(494). A number of studies have implicated PMT as a modulator of host immunity (475,(495)(496)(497)(498) and cellular differentiation and proliferation (476,477,485,(499)(500)(501)(502)(503)(504)(505)(506)(507). Interestingly, because of its potent mitogenic and proliferative properties, there has been speculation that exposure to PMT might play a role in cancer predisposition as a longterm consequence of infection with toxinogenic P. multocida (485,495,499,504,505,(508)(509)(510).…”

Activation of Gαi and Subsequent Uncoupling of Receptor-Gαi Signaling by Pasteurella multocida Toxin

“…First, PMT-induced stimulation of PLC␤ signaling pathways were described (9), which were then shown to be caused by activation of G␣ q (10). G␣ q activation leads to mitogenic signaling through mitogen-activated protein kinase phosphorylation (11)(12)(13) and to stimulation of signal transducer and activator of transcription 1, 3, and 5 through Janus kinase phosphorylation (14). Gene knock-out and reconstitution experiments using G␣ q/11 -deficient mouse embryonic fibroblasts (MEFs) indicated that only G␣ q , and not the closely related G␣ 11 , is activated by PMT (15,16).…”