Lipid Phosphate Phosphatase-2 Activity Regulates S-phase Entry of the Cell Cycle in Rat2 Fibroblasts

Abstract

Lipid phosphates are potent mediators of cell signaling and control processes including development, cell migration and division, blood vessel formation, wound repair, and tumor progression. Lipid phosphate phosphatases (LPPs) regulate the dephosphorylation of lipid phosphates, thus modulating their signals and producing new bioactive compounds both at the cell surface and in intracellular compartments. Knock-down of endogenous LPP2 in fibroblasts delayed cyclin A accumulation and entry into S-phase of the cell cycle. Conversely, overexpression of LPP2, but not a catalytically inactive mutant, caused premature S-phase entry, accompanied by premature cyclin A accumulation. At high passage, many LPP2 overexpressing cells arrested in G 2 /M and the rate of proliferation declined severely. This was accompanied by changes in proteins and lipids characteristic of senescence. Additionally, arrested LPP2 cells contained decreased lysophosphatidate concentrations and increased ceramide. These effects of LPP2 activity were not reproduced by overexpression or knock-down of LPP1 or LPP3. This work identifies a novel and specific role for LPP2 activity and bioactive lipids in regulating cell cycle progression.The lipid phosphates, lysophosphatidate (LPA) 4 and sphingosine 1-phosphate (S1P) are present in biological fluids and activate cells through families of four G-protein-coupled receptors for LPA and five receptors for S1P (1). These receptors are coupled through G␣ i that decreases cAMP concentrations; G 12/13 that stimulates phospholipase D and Rho leading to stress fiber formation; and G q that activates phospholipase C, Ca 2ϩ transients, and protein kinase C isoforms (1). LPA and S1P receptors also transactivate epidermal growth factor and platelet-derived growth factor receptors (2, 3).Intracellular lipid phosphates also act as signaling molecules. For example, PA stimulates NADPH oxidase, protein kinase C-, phosphatidylinositol 4-kinase, phospholipase C-␥, and sphingosine kinase-1, increases Ras-GTP and inhibits protein phosphatase-1 (4 -6). PA can increase proliferation through the mammalian target of rapamycin (7) and PA stimulates stress fiber formation (8). The relative concentrations of LPA and PA in biological membranes control their curvature and vesicle budding (9). C1P is the sphingolipid analogue of PA and is thought to be involved in synaptic vesicle movement and transport (10). It is formed during neutrophil phagocytosis and it is involved in liposome fusion (11). C1P binds to and activates cytosolic phospholipase A 2 , thereby increasing arachidonate and prostaglandin E 2 production (12). C1P also blocks activation of apoptosis in macrophages by inhibiting acidic sphingomyelinase activity (13).Intracellular LPA can signal through the peroxisome proliferatoractivated receptor-␥ receptor (14) and a nuclear receptor, LPA 1 , that regulates proinflammatory gene expression (15). Intracellular S1P stimulates ERK giving a mitogenic or anti-apoptotic response, it mobilizes intracellular Ca 2ϩ and increases act...

Genomics screen in transformed stem cells reveals RNASEH2A, PPAP2C, and ADARB1 as putative anticancer drug targets

“…In rat fibroblasts, the homologous gene, Ppap2c, caused a premature entry into S phase when overexpressed and delayed S-phase entry when knocked down (25). We have confirmed this in the human breast cancer cell line, MCF7, which expresses the highest level of PPAP2C and showed a high degree of inhibition of in vitro proliferation.…”

Lipid phosphate phosphatases and signaling

“…Increasing LPP2 activity in fibroblasts increases entry into S-phase of the cell cycle, whereas decreasing the activity has the opposite effect (48). LPP1 and LPP3 did not modify S-phase entry, demonstrating the specificity of the LPP2 effect.…”

“…The premature entry into S-phase caused by increased LPP2 activity resulted in G2/M arrest after 15 to 35 passages. These cells eventually exited the cell cycle, and they exhibited a senescent phenotype (48). Some oncogenes induce premature senescence after initially stimulating cell proliferation, and this may prevent malignancy (48).…”

Lipid Phosphate Phosphatase-1 Regulates Lysophosphatidate-induced Fibroblast Migration by Controlling Phospholipase D2-dependent Phosphatidate Generation

“…Endothelin-To determine how LPP1 activity alters migration to various agonists, we established mixed populations of Rat2 fibroblasts that stably overexpressed LPP1 mRNA by about 12-fold (41). This previous work also demonstrated that overexpression, or knockdown of LPP1 mRNA, did not change mRNA expression for LPP2 and LPP3.…”

“…Detection of mRNA Expression of LPPs-Changes in mRNA concentrations in fibroblasts that were left over from the migration assay were measured by real time reverse transcriptase-PCR using appropriate controls and primers as described previously (41).…”