Kathryn E. Meier

Role for 18:1 Lysophosphatidic Acid as an Autocrine Mediator in Prostate Cancer Cells

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.

Expression of phospholipase D isoforms in mammalian cells

Two mammalian isoforms of phospholipase D, PLD1 and PLD2, have recently been characterized at the molecular level. Effects of physiologic agonists on PLD activity in intact cells, as characterized in earlier studies, have generally not been attributed to specific PLD isoforms. Recent work has established that expression of PLD1 and PLD2 varies within tissues and between cell lines. A single cell type can express one, both, or neither isoform, although most cells co-express PLD1 and PLD2. Lymphocytes often lack expression of one or both isoforms of PLD. Relative levels of PLD mRNA expression vary considerably between established cell lines. Expression of transcripts for both PLD1 and PLD2 can be regulated at the transcriptional level by growth and differentiation factors in cultured cells. Thus, it is apparent that the known mammalian PLD isoforms are subject to regulation at the transcriptional level. The available data do not conclusively establish whether PLD1 and PLD2 are the only isoforms responsible for agonist-mediated PLD activation. Further studies of the regulation of expression of PLD isoforms should provide insight into the roles of PLD1 and PLD2 in physiologic responses, and may suggest whether additional forms of PLD remain to be characterized.

Oleic Acid–Induced Mitogenic Signaling in Vascular Smooth Muscle Cells A Role for Protein Kinase C

As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon cis-monounsaturated fatty acid (18:1 [cis]), from 25 to 200 mumol/L significantly increased [3H]thymidine uptake in RASMCs with an EC50 of 41.0 mumol/L and a maximal response of 196 +/- 15% of control (P < .01). Oleic acid from 25 to 200 mumol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210 +/- 13% of control at 100 mumol/L (P < .001). PKC inhibition with 4 mumol/L bisindolyImaleimide I and PKC depletion (alpha, mu, iota, and zeta) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328 +/- 8% of control (P < .001), but did not enhance PKC activity in the membrane fraction (105 +/- 4%, P = NS). The oleic acid-induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca2+, phosphatidylserine, and diolein (maximum response, 360 +/- 4% versus 342 +/- 9% of control, P = NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 mumol/L stearic (18:0) and elaidic (18:1 [trans]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca(2+)-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.

Lysophosphatidic acid as an autocrine and paracrine mediator

Recent studies have established that lysophosphatidic acid (LPA) is produced by a wide variety of cell types, and that most mammalian cells express receptors for LPA. These findings raise the hypothesis that LPA acts as an autocrine mediator to initiate signaling in the cells where it is produced, as well as a paracrine mediator to affect neighboring cells. The extent to which these scenarios occur will depend on the species of LPA generated, the LPA receptors expressed, and the ability of these receptors to bind to the LPA produced. The enzymes involved in LPA synthesis and their cellular localization in relationship to LPA receptors are also likely to be important. Studies addressing these issues with respect to the potential roles of LPA as an autocrine and paracrine mediator are reviewed, with examples.

Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells.

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.

Lysophosphatidic acid stimulates phospholipase D activity and cell proliferation in PC-3 human prostate cancer cells

Phospholipase D (PLD) is activated in mammalian cells in response to a variety of growth factors and may play a role in cell proliferation. Lysophosphatidic acid (LPA) is a bioactive metabolite potentially generated as a result of PLD activation. Two human prostate cancer cell lines, PC‐3 and LNCaP, express membrane PLD activity. The effects of LPA on PLD activity and proliferation were examined in PC‐3 cells, which express hPLD1a/1b. Phorbol 12‐myristate 13‐acetate (PMA) induced a prolonged activation of PLD, as detected in both intact cells and membranes. LPA induced a transient activation of PLD that was maximal by 10 minutes. The EC50 for LPA‐induced PLD activation was approximately 1 μM. Pertussis toxin did not inhibit activation of PLD by LPA or PMA. Ro‐31‐8220 and bisindolylmaleimide I, inhibitors of protein kinase C, blocked activation by PLD by both PMA and LPA. PMA‐induced activation of PLD did not appear to require translocation of PLDs from cytosol to membrane. LPA stimulated proliferation of PC‐3 cells with an EC50 of approximately 0.2 μM; this response was not inhibited by pertussis toxin. Perillyl alcohol, an anti‐cancer drug, reversibly inhibited proliferation in response to either serum or LPA but did not inhibit activation of PLD by PMA or LPA. These data establish that LPA activates PLD and stimulates proliferation via Gi‐independent pathways in a human prostate cancer cell line. J. Cell. Physiol. 174:261–272, 1998.

Expression and regulation of phospholipase D isoforms in mammalian cell lines.

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G‐protein‐coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL‐60 (human myeloid leukemia); Jurkat (human leukemia); PC‐3 (human prostate adenocarcinoma); PC‐12K (rat phaeochromocytoma); and Rat‐1 HIR (rat fibroblast). All, with the exception of EL4, express agonist‐activated PLD activity. PLD1 is expressed in A7r5, HL‐60, PC‐3, and Rat‐1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC‐3, and Rat‐1. Neither isoform is expressed in EL4. Guanine nucleotide‐independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6–12 h. A corresponding increase is observed in membrane PLD activity and in phorbol‐12‐myristate‐13‐acetate (PMA)‐stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists. J. Cell. Physiol. 182:77–87, 2000.

Oleic Acid and Angiotensin II Induce a Synergistic Mitogenic Response in Vascular Smooth Muscle Cells

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.

Omega-3 Fatty Acids and Other FFA4 Agonists Inhibit Growth Factor Signaling in Human Prostate Cancer Cells

Omega-3 fatty acids (n-3 FAs) are proposed to have many beneficial effects on human health. However, the mechanisms underlying their potential cancer preventative effects are unclear. G protein–coupled receptors (GPCRs) of the free fatty acid receptor (FFAR) family, FFA1/GPR40 and FFA4/GPR120, specifically bind n-3 FAs as agonist ligands. In this study, we examined the effects of n-3 FAs in human prostate cancer cell lines. Initial studies established that the long-chain n-3 FAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid, inhibit proliferation of DU145 cells in response to lysophosphatidic acid (LPA), a mitogenic lipid mediator. When added alone to serum-starved DU145 cells, EPA transiently activates signaling events, including p70S6K phosphorylation. However, when added 15 minutes prior to LPA, EPA suppresses LPA-induced activating phosphorylations of ERK, FAK, and p70S6K, and expression of the matricellular protein CCN1. The rapid onset of the inhibitory action of EPA suggested involvement of a GPCR. Further studies showed that DU145 and PC-3 cells express mRNA and protein for both FFA4 and FFA1. TUG-891 (4-[(4-fluoro-4′-methyl[1,1′-biphenyl]-2-yl)methoxy]-benzenepropanoic acid), a selective agonist for FFA4, exerts inhibitory effects on LPA- and epidermal growth factor–induced proliferation and migration, similar to EPA, in DU145 and PC-3 cells. The effects of TUG-891 and EPA are readily reversible. The FFA1/FFA4 agonist GW9508 (4-[[(3-phenoxyphenyl)methyl]amino]-benzenepropranoic acid) likewise inhibits proliferation at doses that block FFA4. Knockdown of FFA4 expression prevents EPA- and TUG-891–induced inhibition of growth and migration. Together, these results indicate that activation of FFA4 initiates signaling events that can inhibit growth factor–induced signaling, providing a novel mechanism for suppression of cancer cell proliferation.

Endotoxin activation of mitogen-activated protein kinase in THP-1 cells; diminished activation following endotoxin desensitization.

The signal transduction events occurring in monocytes in response to endotoxin (LPS) stimulation are incompletely delineated, although pertussis toxin (PT)‐sensitive G proteins and the mitogen‐activated protein kinase (MAPK) cascade have been implicated. Cellular desensitization in response to 18‐h pre‐exposure to 1 μg/mL LPS alters signal transduction pathways of cellular activation and decreases production of certain inflammatory mediators such as thromboxane (Tx)B2, the stable metabolite of TxA2. We hypothesized that LPS stimulation of the human monocyte cell line THP‐1 occurs via MAPK activation, and that LPS desensitization, induced by pre‐exposure to LPS, is associated with altered signaling through the MAPK cascade. Involvement of a specific MAPK, ERK, in LPS‐stimulated TxB2 production was further tested using a specific MAPK cascade inhibitor, PD98059 (PD). PD inhibited LPS and phorbol myristate acetate (PMA)‐stimulated ERK activation as demonstrated by immunoblots using anti‐activated ERK antibodies. PD significantly inhibited LPS and PMA‐stimulated TxB2 synthesis to non‐detectable levels, suggesting an involvement of MAPK in LPS‐stimulated activation. Because PT‐sensitive G proteins mediate LPS‐stimulated signal transduction, their role in MAPK activation was tested. Pretreatment with PT inhibited basal and LPS‐stimulated, but not PMA‐stimulated ERK activation. Activation of ERK after LPS desensitization was also assessed. LPS pre‐exposure resulted in a profound decrease in LPS‐stimulated activation of ERK, but did not affect PMA activation of ERK. These data implicate the involvement of the MAPK cascade in LPS‐stimulated activation of THP‐1 cells and suggest coupling of Gi proteins and MAPKs in LPS‐stimulated events. LPS desensitization is associated with decreased MAPK activation, but does not impair MAPK activation by PMA. Thus, LPS desensitization appears to selectively alter signal transduction upstream of ERK. J. Leukoc. Biol. 64: 259–264; 1998.