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Differential modulation and subsequent blockade of mitogenic signaling and cell cycle progression by Pasteurella
B A Wilson1, L R Aminova, V G Ponferrada
1Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. bawilson@life.uiuc.edu
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)alpha-protein, which stimulates phospholipase C (PLC) beta1. 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.
Insights
Pasteurella multocida toxin (PMT) activates cell signaling pathways, causing initial proliferation but ultimately cell cycle arrest. Cell type influences PMT
Area of Science:
- Cell Biology
- Molecular Biology
- Toxicology
Background:
- Pasteurella multocida toxin (PMT) is an intracellular protein known to affect various cellular processes.
- PMT activates the G(q/11)-alpha-protein, leading to phospholipase C (PLC) beta1 stimulation and inositol 1,4,5-trisphosphate (IP(3)) pathway activation.
- The link between PMT's IP(3) pathway activation and its mitogenic/proliferative effects remains unclear.
Purpose of the Study:
- To investigate the downstream effects of PMT on mitogenesis and cell cycle progression.
- To characterize the differential cellular responses to PMT in Swiss 3T3 and Vero cells.
- To elucidate the signaling pathways involved in PMT-induced cell cycle regulation.
Main Methods:
- Treatment of Swiss 3T3 and Vero cells with PMT.
- Morphological analysis and assessment of cell cycle progression using cell cycle analysis.
- Western blot analysis to detect the expression of key cell cycle regulatory proteins (e.g., c-Myc, cyclins, PCNA, Rb proteins).
Main Results:
- PMT induced significant morphological changes in both cell lines, with Vero cells showing multinucleation and disrupted cytokinesis.
- A strong, but transient, mitogenic response was observed in Swiss 3T3 cells, while Vero cells showed a limited response.
- Both cell types arrested in the G(1) phase after initial PMT exposure and became unresponsive.
- PMT differentially regulated cell cycle proteins; Swiss 3T3 cells showed up-regulation of cyclins and proliferation markers, which was not sustained.
- Vero cells failed to up-regulate certain key proteins like PCNA and cyclin E, correlating with their limited proliferative response.
Conclusions:
- PMT triggers an initial mitogenic response followed by cell cycle arrest, indicating complex regulatory mechanisms.
- The differential expression of cell cycle proteins and signaling pathways in response to PMT contributes to varying cellular outcomes.
- Understanding these cell-type-specific responses is crucial for comprehending the overall impact of PMT toxicity.