Effects of Active and Inactive Phospholipase D2 on Signal Transduction, Adhesion, Migration, Invasion, and Metastasis in EL4 Lymphoma Cells

Abstract

The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.

Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

“…In addition, actin polymerization and stress fiber formation are tightly coupled to the activation of PLD (14). The formation of lamellipodium structures and membrane ruffles is blocked by PLD inhibition (53,60), and PLD activity is critical for epithelial cell, leukocyte, and neutrophil adhesion and migration (41,43,52). Furthermore, we have previously shown that the activity of PLD is upregulated, and that the activated PLD is translocated to lamellipodia, after integrin activation (3).…”

The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

“…PA mediates chemotaxis, as increasing concentrations of PA enhanced the rate of cell migration of phagocytes (35). In the murine lymphoma cell line EL4, Knoepp et al found that activated PLD2 promotes phosphorylation of FAK and Akt, leading to cell-substrate adhesion (7,29). However, while inactivated PLD2 inhibits adhesion, migration, proliferation, and tumor invasion, it does not alter the basal level of FAK and Akt phosphorylation (29).…”

“…In the murine lymphoma cell line EL4, Knoepp et al found that activated PLD2 promotes phosphorylation of FAK and Akt, leading to cell-substrate adhesion (7,29). However, while inactivated PLD2 inhibits adhesion, migration, proliferation, and tumor invasion, it does not alter the basal level of FAK and Akt phosphorylation (29). Although PA does play a role in cell migration, the specific mechanisms involved are not completely understood, and more precise structurefunction studies are needed.…”

Phospholipase D Meets Wnt Signaling: A New Target for Cancer Therapy

“…PLD is upregulated at the protein and/or activity levels in various cancers, including breast, colon, gastric, kidney, and thyroid cancer (6). Overexpression of PLD isozymes has been reported to induce anchorage-independent growth, tumor cell invasion, and the formation of metastases in syngeneic mice (7,8). Significant correlation of PLD2 expression levels with tumor size and patient survival in colorectal carcinoma has been reported by recent clinical studies (6).…”