Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri
Submitted 29 July 2004 ; accepted in final form 27 September 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
lysophospholipids; cell signaling; phospholipid metabolism; arachidonic acid
The isoforms of PKC can be divided into three classes on the basis of primary structure and biochemical properties. The classic or conventional isoforms include PKC-, -
I, -
II, and -
. Classic isoforms are activated by diacylglycerol (DAG) or phorbol esters (e.g., PMA) in the presence of Ca2+ and anionic phospholipids. PKC-
, -
, -
, and -
are novel PKCs. Similarly to the classic isoforms, novel isoforms can be activated by DAG or phorbol esters in the presence of anionic phospholipids; however, this activation occurs through a Ca2+-independent mechanism. The third class of PKC isoforms is composed of the atypical isoforms PKC-
and -
/
. Activation of atypical isoforms is independent of both DAG or phorbol esters and Ca2+ (18).
PKCs have been shown to be intricately regulated kinases with multiple modes of activation and inactivation. With proteolytic cleavage of PKC, two functional domains are generated: one is hydrophobic and the other has full catalytic activity that is suppressed in the proenzyme form of the kinase by self-binding to a pseudosubstrate domain (18). In classic PKC isoforms, the proenzyme is activated in a manner that is dependent on Ca2+, anionic phospholipids, and phospholipase C-generated DAG, which functions to reduce the Ca2+ concentration required for the activation of PKC (22). Castagna et al. (3) demonstrated the ability of PMA to activate PKC by substituting for DAG. Studies by several groups (4, 17, 19) have shown that phosphorylation and membrane targeting, coupled with binding to second messengers, play critical roles in the activation of classic and novel PKC isoforms. PKC is a serine/threonine kinase, and a large number of proteins have been identified as possible substrates for PKC (18). At this time, however, the physiological purpose for many of these phosphorylation events is yet to be determined.
To participate in signal transduction, all signaling proteins must be subject to strict regulation. In the case of PLA2, the enzyme must be in contact with its membrane substrate and its catalytic activity must be modulated via posttranslational mechanisms such as phosphorylation. In EC, the majority of iPLA2 is constitutively membrane associated (14) and thus presumably is in constant contact with its endogenous phospholipid substrate. However, the role of phosphorylation of iPLA2 by upstream kinases has not been studied extensively. The PKC isoforms present in EC that may activate iPLA2 also have not been identified to date. However, amino acid analysis of the sequences of known human EC iPLA2 isoforms demonstrates the presence of several potential PKC phosphorylation sites, suggesting that this mechanism may play a role in iPLA2 activation.
In preliminary studies conducted in our laboratory, the potential regulation of membrane-associated iPLA2 activity by PKC has been examined in isolated rabbit ventricular myocytes. In Ca2+-free medium, thrombin or PMA stimulation of isolated membrane fractions from myocytes led to a two- to threefold increase in membrane-associated iPLA2 activity (21). This increase in iPLA2 activity was accompanied by accelerated hydrolysis of membrane plasmalogen phospholipids and increased production of arachidonic acid and lysoplasmenylcholine. Pretreatment with GF-109203X, a PKC inhibitor, blocked thrombin-induced iPLA2 activation. Activation of iPLA2 was observed when isolated membrane fractions were incubated with PMA in the absence of Ca2+. Immunoblot analysis demonstrated the presence of PKC- as the only isoform detectable in the isolated membrane fraction containing iPLA2 activity. Together these data demonstrate that a membrane-associated novel PKC isoform likely mediates activation of membrane-associated iPLA2 in thrombin-stimulated rabbit ventricular myocytes (21).
In the present study, we have demonstrated that thrombin stimulation of membrane fractions isolated from human coronary artery endothelial cells (HCAEC) resulted in activation of iPLA2. This observation suggests that the protease-activated receptor (PAR) and the G proteins and G protein-coupled effector molecules required for iPLA2 activation are all colocalized in the isolated membrane fraction, which comprises both cell membrane and endoplasmic reticulum (8). In addition, because iPLA2 in the membrane fraction is activated by PMA in the absence of Ca2+, this suggests a role for a novel PKC isoforms in HCAEC.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurement of membrane-associated PLA2 activity. Surrounding medium was removed from confluent monolayers of HCAEC, and then the cells were quickly washed with ice-cold PBS and removed from the tissue culture plate in ice-cold buffer containing (in mmol/l) 250 sucrose, 10 KCL, 10 imidazole, 5 EDTA, 2 dithiothreitol (DTT), and 10% glycerol (pH 7.8; PLA2 assay buffer). The suspension was sonicated on ice three times for 10 s each, and the sonicate was centrifuged at 14,000 g for 10 min. The supernatant was then centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). The membrane fraction, consisting of microsomes including vesicles of cellular membrane and endoplasmic reticulum (8), was washed twice by resuspending in PLA2 assay buffer and centrifuging at 100,000 g for 60 min. Isolated membrane fractions were incubated with thrombin or PMA immediately before assay of iPLA2 activity. PLA2 activity in the membrane fraction was assessed by incubating enzyme (8 µg membrane protein) with 100 µM (16:0, [3H]18:1) plasmenylcholine substrate in assay buffer containing 10 mM Tris, 4 mM EGTA, and 10% glycerol, pH 7.0, at 37°C for 5 min in a total volume of 200 µl. Reactions were initiated by adding the radiolabeled phospholipid substrate as a concentrated stock solution in ethanol. Reactions were terminated by the addition of 100 µl butanol, and released radiolabeled fatty acid was isolated by application of 25 µl of the butanol phase to channeled silica gel G plates, development in petroleum ether/diethyl ether/acetic acid (70:30:1 vol/vol) and subsequent quantification by liquid scintillation spectrometry with activity normalized to protein content as described previously (11, 12).
Separation and quantification of individual choline and ethanolamine glycerophospholipid molecular species. Cellular phospholipids were extracted from HCAEC by using the method of Bligh and Dyer (1). The chloroform layer was dried under N2, and the lipid residue was resuspended in chloroform-methanol (1:1 vol/vol). Phospholipids were separated into different classes by performing HPLC using gradient elution with a mobile phase composed of hexane/isopropanol/water (14). Individual choline and ethanolamine glycerophospholipid molecular species were separated by performing reverse-phase HPLC using a gradient elution system with a mobile phase composed of acetonitrile/methanol/water with 20 mM choline chloride as described previously (5, 14). Quantification of individual phospholipid molecular species was achieved by determination of lipid phosphorus in reverse-phase HPLC column effluents (14).
Arachidonic acid release. The extent of arachidonic acid release was determined by measuring the amount of [3H]arachidonic acid released into the surrounding medium from HCAEC monolayers prelabeled with 3 µCi of [3H]arachidonic acid per 35-mm culture dish for 18 h. After incubation, HCAEC were washed three times with Tyrode's solution containing 3.6% bovine serum albumin to remove unincorporated [3H]arachidonic acid. EC were incubated at 37°C for 15 min before implementation of the experimental conditions. At the end of the stimulation period, the surrounding medium was removed to a scintillation vial and represented the amount of radiolabeled arachidonic acid released from HCAEC during the stimulation interval. The amount of radiolabeled arachidonic acid remaining in the endothelial monolayer was measured by adding 1 ml of 10% sodium dodecyl sulfate (SDS), removing the cells from the culture well by scraping, and then adding them to a scintillation vial. Radioactivity in both the surrounding medium and the HCAEC was quantified by performing liquid scintillation spectrometry.
Platelet-activating factor production. Confluent HCAEC monolayers were washed twice with Hanks' balanced salt solution containing (in mM) 135 NaCl, 0.8 MgSO4, 10 HEPES (pH 7.4), 1.2 CaCl2, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, and 6.6 glucose and incubated with 50 µCi of [3H]acetic acid for 20 min. After thrombin stimulation for the selected interval, lipids were extracted from the cells by using the method of Bligh and Dyer (1). The chloroform layer was concentrated by evaporation under N2, applied to a silica gel 60 thin-layer chromatography (TLC) plate, and developed in chloroform/methanol/acetic acid/water (50:25:8:4 vol/vol). The region corresponding to platelet-activating factor (PAF) was scraped, and radioactivity was quantified using liquid scintillaiton spectrometry. Loss of PAF during extraction and chromatography was corrected by adding a known amount of [14C]PAF.
Choline lysophospholipid production. Lysophosphatidylcholine and lysoplasmenylcholine were measured using a modification of a radiometric assay method developed previously (12). Lipids were extracted from HCAEC and the surrounding medium by using the method of Bligh and Dyer (1), and lysophospholipids were separated from other phospholipids by performing HPLC. The purified lysophosphatidylcholine and lysoplasmenylcholine fractions were acetylated with [3H]acetic anhydride using 0.33 M (dimethylamino)-pyridine as a catalyst. The acetylated lysophospholipid was then separated by performing TLC, and radioactivity was quantified by using liquid scintillation spectrometry. Standard curves were constructed, and the lysophosphatidylcholine and lysoplasmenylcholine contents were derived for all samples and normalized according to the protein content of HCAEC. [14C]Lysophosphatidylcholine was added as an internal standard to all samples to correct for the loss of sample that occurred during extraction, purification, and acetylation.
Detection of PKC isoforms by PCR analysis. Total RNA was isolated from HCAEC using the PureScript RNA isolation kit (Gentra, Minneapolis, MN). cDNA was synthesized using random hexamer priming of RNA and amplified with isoform-specific oligonucleotide primers flanking 1001,200 bp of guanine/cytosine (GC)-rich cDNA for each of the PKC isoforms. Amplicons recovered from each PCR mixture were separated by performing agarose gel electrophoresis and visualized using ethidium bromide. PCR products excised from the gel or isolated from the PCR mixture were then subjected to automated sequencing (using an Applied Biosystems 377 sequencer) and compared with the cDNA sequences published in the GenBank database.
Immunoblot analysis.
HCAEC were removed from the tissue culture plate in lysis buffer containing 20 mM HEPES, 250 mM sucrose, 2 mM DTT, 2 mM EDTA, 2 mM EGTA, 10 mM -glycerophosphate, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 5 µg/ml pepstatin A (pH 7.6). Cells were sonicated on ice for six bursts of 10 s each and centrifuged at 14,000 g at 4°C for 10 min to remove cellular debris, nuclei, and mitochondria. Cytosolic and membrane fractions were separated by centrifuging the supernatant at 100,000 g for 60 min. The membrane fraction pellet was washed twice by resuspension in lysis buffer, and the suspension was centrifuged at 100,000 g for 60 min to minimize contamination of the membrane fraction with cytosolic protein. The final pellet was resuspended in lysis buffer containing 0.1% Triton X-100. Protein from the membrane fraction was mixed with an equal volume of SDS sample buffer and heated at 95°C for 5 min before loading onto a 10% polyacrylamide gel. Protein was separated by SDS-PAGE at 200 V for 40 min and electrophoretically transferred to polyvinylidene difluoride (PVDF-Plus) membranes (Micron Separations, Westborough, MA) at 100 V for 1 h. Nonspecific sites were blocked by incubating the membranes with Tris buffer solution containing 0.05% vol/vol Tween 20 (TBST) and 5% (wt/vol) nonfat milk for 1 h at room temperature. The blocked PVDF membrane was incubated with primary antibodies to PKC isoforms for 2 h at room temperature. Unbound antibodies were removed with five washes with TBST solution, and membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. After five washes with TBST, regions of antibody binding were detected using enhanced chemiluminescence (SuperSignal; Pierce, Rockford, IL) and exposure to film (Hyperfilm; Amersham).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To examine the regulation of membrane-associated iPLA2 activity, subcellular fractions were isolated from HCAEC, resuspended in a Ca2+-free buffer, and incubated with thrombin (0.05 IU/ml; 1 min) or PMA (100 nm; 10 min) in the presence or absence of 200 nmol ATP (Fig. 1). The increase in PLA2 activity after thrombin treatment in the absence of Ca2+ demonstrates the presence of a functional PAR and all of the required, coupled effector molecules needed for iPLA2 activation in the isolated membrane fraction. PMA stimulation of isolated membrane fractions in the presence of ATP also resulted in a significant increase in membrane-associated iPLA2 activity. The magnitude of the increase in iPLA2 was similar to that observed after thrombin stimulation (Fig. 1), suggesting that iPLA2 activation by thrombin proceeds predominantly, if not exclusively, through a pathway that is dependent on one or more PKC isoforms activated by PMA in a Ca2+-independent fashion. Although there are no published studies demonstrating activation of HCAEC membrane-associated iPLA2 by PKC, the amino acid analysis of the sequences of known human EC iPLA2 isoforms demonstrates the presence of several potential PKC phosphorylation sites, including sites near or within the pleckstrin homology (PH) domain of iPLA2, which may affect the interaction of iPLA2 with putative regulatory proteins (2).
|
|
|
Because we observed a similar increase (i.e., relative increase over baseline) in lysoplasmenylcholine and arachidonic acid production in HCAEC incubated with PMA or thrombin over a similar time course, we performed additional studies to determine whether thrombin- or PMA-induced activation of iPLA2 is accompanied by selective, stoichiometric hydrolysis of plasmalogen phospholipid substrates. For these studies, we quantified individual choline and ethanolamine phospholipid molecular species in unstimulated HCAEC and in cells incubated with thrombin or PMA. Finally, we pretreated HCAEC with the selective, catalytic site-directed, inhibitor of iPLA2 bromoenol lactone (BEL) before incubation with PMA or thrombin to demonstrate that PKC-stimulated phospholipid hydrolysis was a direct result of iPLA2 activation.
After demonstrating the production of both arachidonic acid and lysophospholipids upon thrombin stimulation, we investigated the downstream conversion of the lysophospholipids to PAF (Fig. 4). HCAEC monolayers were either stimulated with thrombin or pretreated with PMA (5 min or 24 h) before thrombin stimulation. PAF production was increased in cells either stimulated with thrombin alone or pretreated with PMA for 5 min before thrombin stimulation, corresponding to increases in lysophospholipid production under identical experimental conditions (Fig. 4). Pretreatment with PMA for 24 h to downregulate PKC resulted in complete inhibition of thrombin-stimulated PAF production (Fig. 4). These results support the hypothesis that increased lysophospholipids produced upon thrombin stimulation are readily converted to their downstream metabolite, PAF.
|
|
|
To identify and localize the PKC isoforms modulating iPLA2 activity, we determined the presence of mRNA transcripts for each of the PKC isoforms in HCAEC. As described in MATERIALS AND METHODS, mRNA was isolated from HCAEC, and cDNA was synthesized. RT-PCR was then used to determine whether mRNA transcripts for previously identified PKC isoforms were present in these cells. Limited sequence analysis of cDNA corresponding to the bands in each of the lanes in Fig. 7 confirms the presence of mRNA transcripts in HCAEC for eight of the known PKC isoforms. We were unable to detect the classic isoforms of PKC-I and -
II. In a separate experiment, we were able to detect PKC isoforms -µ and -
(data not shown); however, others now think that these are truly protein kinase D isoforms (18).
|
|
|
Previously, we examined the role of PKC activity in the regulation of iPLA2 in isolated rabbit ventricular myocytes. Similarly to the present study, in our previous work, we demonstrated that cardiac myocyte iPLA2 activity was regulated by a membrane-associated novel PKC isoform. Immunoblot analysis of each of the novel PKC isoforms demonstrated the presence of PKC- as the only isoform present in isolated cardiac myocyte membrane fractions. Other novel PKC isoforms were not detectable by this method, however, these isoforms may be present in sufficient amounts to activate iPLA2 but not sufficient enough to be detected by immunoblot analysis. In this study, we were able to detect a significant portion of each of the novel PKC isoforms present in the membrane fraction of HCAEC, suggesting that there is a greater diversity of membrane-associated PKC isoforms in EC compared with cardiac myocytes.
As we have demonstrated, the presence of thrombin leads to an increase in the membrane-associated iPLA2, which was associated with an increase in arachidonic acid and lysoplasmenylcholine production accompanied by downstream conversion to PAF and other inflammatory metabolites. Increases in EC PAF expression and alterations in the phospholipid composition of the EC membrane enhance inflammatory cell adherence and transmigration, respectively, allowing them access to sites of vascular injury (15). Increased production of eicosanoids synthesized from arachidonic acid is involved in both of these entities' regulating the body's response to inflammation (7, 16). Overstimulation of the inflammatory response through enhanced or extended production of specific inflammatory mediators often leads to further injury. Accordingly, identification of the PKC isoform responsible for activation of iPLA2 could lead to the production of a specific inhibitor with potential as an anti-inflammatory agent capable of reducing the cell injury caused by an overactive inflammatory response.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Blom N, Gammeltoft S, and Brunak S. Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294: 13511362, 1999.[CrossRef][ISI][Medline]
3. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, and Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257: 78477851, 1982.
4. Cho W. Membrane targeting by C1 and C2 domains. J Biol Chem 276: 3240732410, 2001.
5. Creer MH and Gross RW. Reverse-phase HPLC separation of molecular species of alkyl ether, vinyl ether and monoacyl lysophospholipids. J Chromatogr A 338: 6169, 1985.[CrossRef]
6. Creer MH and McHowat J. Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation. Am J Physiol Cell Physiol 275: C1498C1507, 1998.
7. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 18711875, 2001.
8. Gross RW and Sobel BE. Lysophosphatidylcholine metabolism in the rabbit heart: characterization of metabolic pathways and partial purification of myocardial lysophospholipase-transacylase. J Biol Chem 257: 67026708, 1982.
9. Hazen SL and Gross RW. ATP-dependent regulation of rabbit myocardial cytosolic calcium-independent phospholipase A2. J Biol Chem 266: 1452614534, 1991.
10. Kell PJ, Creer MH, Crown KN, Wirsig K, and McHowat J. Inhibition of platelet-activating factor (PAF) acetylhydrolase by methylarchidonyl flurophosphonate potentates PAF synthesis in thrombin stimulated human coronary artery endothelial cells. J Pharmacol Exp Ther 307: 11631170, 2003.
11. McHowat J and Creer MH. Lysophosphatidylcholine accumulation in cardiomyocytes requires thrombin activation of Ca2+-independent PLA2. Am J Physiol Heart Circ Physiol 272: H1972H1980, 1997.
12. McHowat J and Creer MH. Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes. Am J Physiol Cell Physiol 274: C447C454, 1998.
13. McHowat J, Creer MH, and Rickard A. Stimulation of protease activated receptors on RT4 cells mediates arachidonic acid release via Ca2+ independent phospholipase A2. J Urol 165: 20632067, 2001.[CrossRef][ISI][Medline]
14. McHowat J, Kell PJ, O'Neill HB, and Creer MH. Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca2+-independent phospholipase A2. Biochemistry 40: 1492114931, 2001.[CrossRef][ISI][Medline]
15. Meyer M and McHowat J. The role of platelet-activating factor in the adherence of circulating cells to the endothelium. In: Recent Research Developments in Physiology, edited by Pandalai SG. Kerala, India: Research Signpost, 2004, vol. 2, p. 129147.
16. Murakami M, Kambe T, Shimbara S, and Kudo I. Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways. J Biol Chem 274: 31033115, 1999.
17. Newton AC. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101: 23532364, 2001.[CrossRef][ISI][Medline]
18. Ohno S and Nishizuka Y. Protein kinase C isoforms and their specific functions: prologue. J Biochem (Tokyo) 132: 509511, 2002.[ISI][Medline]
19. Parekh DB, Ziegler W, and Parker PJ. Multiple pathways control protein kinase C phosphorylation. EMBO J 19: 496503, 2000.
20. Pike LJ, Han X, Chung KN, and Gross RW. Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis. Biochemistry 1: 20752088, 2002.[CrossRef]
21. Steer SA, Wirsig KC, Creer MH, Ford DA, and McHowat J. Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes. Am J Physiol Cell Physiol 283: C1621C1626, 2002.
22. Takai Y, Kishimoto A, Kikkawa U, Mori T, and Nishizuka Y. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun 91: 12181224, 1979.[CrossRef][ISI][Medline]
23. Toker A. Phosphoinositides and signal transduction. Cell Mol Life Sci 59: 761779, 2002.[CrossRef][ISI][Medline]
24. Wolf MJ, Wang J, Turk J, and Gross RW. Depletion of intracellular calcium stores activates smooth muscle cell calcium-independent phospholipase A2: a novel mechanism underlying arachidonic acid mobilization. J Biol Chem 272: 15221526, 1997.
25. Ye K and Snyder SH. PIKE GTPase: a novel mediator of phosphoinositide signaling. J Cell Sci 117: 155161, 2004.