Relationships between phosphatidic acid and cyclic nucleotide phosphodiesterases in activated human blood mononuclear cells

Abstract

Abstract:We have previously shown that mitogenic activation of human PBMC rapidly increases both the intracellular phosphatidic acid (PA) level and cyclic nucleotide phosphodiesterase (PDE) activity, with time-course responses, suggesting a causative relationship between the two events. PA also directly stimulated cAMP-PDE activity in acellular systems. Thus the mitogenic properties of PA might be due to its ability to lower the level of cAMP, a negative effector of lymphocyte activation, through PDE activation. In this study, human PBMC were stimulated either with the mitogenic lectin ConA, the anti-CD3 mAb OKT3, or the phorbol ester TPA. All three agonists increased the radiolabeled PA level and the PA mass in treated cells and simultaneously increased cytosolic and particulate cAMP-and cGMP-PDE activities, with significant positive correlations between PA accumulation and PDE activities. Furthermore, the ConAinduced PDE activation was dose-dependently reduced by treatment of PBMC with the diacylglycerol-kinase inhibitor R59022. This compound also dose-dependently lowered the PA level and inhibited the proliferative response to ConA. In addition, TPA-induced PDE activation was totally abolished by ethanol, which strongly reduced PA accumulation in response to the phorbol ester. These data suggest that PA increase may be linked to mitogen-induced PDE activation. Experiments performed in the presence of rolipram indicated that ConA and TPA stimulated both the rolipramsensitive PDE4 and the rolipram-insensitive PDE activities, OKT3 being more active on PDE4. All three agonists stimulated the cGMP-specific PDE5. These results suggest that PA is an important component of the mechanisms that maintain a low level of cyclic nucleotides, which is a prerequisite for an optimal lymphoproliferative response. J. Leukoc. Biol. 65: 381-390; 1999.

Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation

“…Our data also suggest that this mechanism is relevant to human physiology, given the significant correlations between adipose tissue lipin 1 expression and lipolytic rates and PKA signaling in obese human subjects. Previous work on PA-mediated regulation of intracellular signaling events, including allosteric activation of mTOR (22) and PDE4 (20,25), linked the activity of PLD and DAG kinase enzymes to controlling this "signaling pool" of PA. An important finding of the present study is that the pool of PA dephosphorylated by lipin 1 that is likely destined for phosphoglycerolipid synthesis can regulate mTOR and PDE4 activity as well. It should be noted that PA was recently found to suppress formation of the mTORC2 complex (26); thus, further work is needed to determine whether mTORC1 is specifically activated by this pool of PA. We also acknowledge that we cannot rule out the possibility that alterations in other lipid species upstream or downstream of PA in these pathways are mediating these signaling events.…”

The Mechanism of Docosahexaenoic Acid-induced Phospholipase D Activation in Human Lymphocytes Involves Exclusion of the Enzyme from Lipid Rafts

“…The sense and antisense primers were 5Ј-GGGATCCGTGTGAAGCGGGTCACTTCAGGACC-G-3Ј and 5Ј-GGGAATTCTCTGGTTTCCCCATGCAGCTCTCCCAC-3Ј, respectively. Primers to specifically amplify PLD2 transcripts were designed on the basis of published human sequences (27) Caldwell et al (28) as described previously (29). After glycerol treatment, the cells were pelleted at 900 ϫ g for 10 min, resuspended in lysis buffer (20 mM Hepes, 25 mM sucrose, 0.1 mM EGTA, 0.05 mM phenylmethylsulfonyl fluoride, 10 units/ml aprotinin, 2 g/ml pepstatin A, pH 7.4), and stored frozen at Ϫ80°C.…”

Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function

“…Although some reports have described an activation of PLD following ligation of the TCR/CD3 complex (6, 7), other studies failed to demonstrate efficient CD3 coupling to PLD, either in normal peripheral lymphocytes (8) or in T cell lines (9). It has been shown that TCR ligation was accompanied by a fast and large increase in PA level occurring in the first minutes of T cell activation (10,11). However, this early peak could be attributed to PA derived from DAG through the sequential activation of phospholipase C-␥ and DAG kinase because it was totally suppressed by DAG kinase inhibitors (10,11).…”

“…It has been shown that TCR ligation was accompanied by a fast and large increase in PA level occurring in the first minutes of T cell activation (10,11). However, this early peak could be attributed to PA derived from DAG through the sequential activation of phospholipase C-␥ and DAG kinase because it was totally suppressed by DAG kinase inhibitors (10,11). Only when phospholipids of lymphocyte membrane were modified by enrichment with DHA (5,12) or the monohydroxylated derivative of arachidonic acid, 12-HETE (13), were we able to observe PLD activation following mitogenic stimulation.…”