©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Fatty Acid Bimodal Action on Superoxide Anion Production by Human Adherent Monocytes under Phorbol 12-Myristate 13-Acetate or Diacylglycerol Activation Can Be Explained by the Modulation of Protein Kinase C and p47 Translocation (*)

Najib Kadri-Hassani , Claude L. Léger (§) , Bernard Descomps

From the (1)Laboratoire de Biologie et Biochimie des Lipides, Institut de Biologie, Unité de Formation et de Recherche de Médecine, Montpellier I, 34000 Montpellier, and Centre de Recherche, INSERM, 70 rue de Navacelles, 34090 Montpellier, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We studied the translocation of protein kinase C (PKC), the endogenous phosphorylation and presence in the membrane fraction of p47 (the 47-kDa cytosolic component of the phagocyte NADPH oxidase), and the O production in human adherent monocytes (HAMs). This was performed under phorbol myristate acetate (PMA) or diacylglycerol stimulation after cell preincubation in the presence of either 13-methyltetradecanoate or arachidonate. At 3 nM and 30 µM, both fatty acids had enhancing and depressing effects, respectively, on PKC translocation and O production strictly depending on the PMA- or diacylglycerol-stimulated state of the cell. Endogenous phosphorylation and membrane presence of p47 were markedly reinforced in PMA-stimulated HAMs in the presence as compared to the absence of 13-methyltetradecanoate. These results emphasize the fact that in intact cells the capacity of both FAs to potentiate or depress the HAM O production is mediated by a direct action on the PKC membrane translocation leading to a simultaneous endogenous phosphorylation and membrane translocation of p47. They confirm the recent findings (Kadri-Hassani, N., Léger, C. L., and Descomps, B.(1995) J. Lipid Med. Cell Signal. 11, 159-173) on the PKC-mediated, adherent monocyte-specific capacity of these fatty acids and others (with the exception of linear saturated fatty acids) to enhance the PMA-stimulated O production at nanomolar concentrations and to depress it at micromolar concentrations.


INTRODUCTION

Almost all tissues contain PKC,()a phosphorylating and membrane-translocatable enzyme which plays a very important role in cell functions, i.e. secretion, proliferation and differentiation(1) . There are several isozymes of PKC having four conserved and five variable regions in their single polypeptide chain (C-C and V-V, respectively). The N-terminal part of the polypeptide designated as the regulatory domain is composed of two conserved sequences functionally characterized. The C region binds activatory lipids (DAG) or tumor promoters (PMA, for example), whereas the C region binds Ca and PtdSer. Other Ca binding sites exist and probably depend on PKC-phospholipid interactions(2) . It is now well established that the C region contains two cysteine-rich zinc-finger-like sequences probably needed for a strong association of PKC with particular lipid-phase conformational forms of biomembranes(3) . Moreover, the presence of at least one cysteine-rich sequence is required for the phorbol-ester binding(4, 5) , an assumption that is generally thought to be extended to the binding of DAG. The PMA activation of membrane-associated PKC does not imply an increasing penetration of protein further into a lipid monolayer system(6) , suggesting that activation may affect the lipid-protein interactions at the surface of the lipid bilayer rather in the depth of it, as proposed by the unfolding of PKC after its DAG or PMA activation at the surface of the cytosolic leaflet of plasma membranes(3) .

The PKC isozymes belong to conventional, non-conventional, or atypical PKCs, so-called c-PKCs, n-PKC, and a-PKC, respectively. The c-PKCs require Ca and DAG (or PMA) to translocate from the cytoplasm to the cell plasma membrane and become activated, the n-PKCs are independent of Ca and the a-PKCs are dependent only on PtdSer and independent of DAG, PMA and Ca for activation (for exhaustive description, see Ref. 7).

It has been shown that FAs themselves are able to modulate PKC activity in reconstituted membrane systems (for review, see Ref. 8). Early observations were made with unsaturated FAs and requirement for their ``cis'' configuration (9) in a monomeric (non-micellar) solution (10) was recognized. It was also established that (i) they activate PKC in the absence of the usual cofactors PtdSer and Ca, these latter being needed, however, in the case of DAG or PMA activation, and (ii) they are able in particular to activate the cytosolic form of PKC(11) . In a cell-free system and in the presence of phospholipids and Ca, evidence for a synergistic action of DAG and unsaturated FAs was provided(12) . The potential action of free FAs on the different PKC isozymes was established (for review, see Ref. 7). In vivo, Lester et al.(13) demonstrated that unsaturated FAs regulate a PKC-dependent function. Despite these findings, free FA action remains unclear and controversial (14, 15) in physiological conditions. It is of utmost importance to obtain more information on the ability of FAs to translocate and therefore activate PKC in intact cells.

We recently found that the preincubation of HAMs in the presence of certain FAs brought about a potentiating effect at nanomolar concentrations and a depressing effect at micromolar concentrations on the NADPH oxidase-mediated production of O(16) . This novel bimodal action was assumed to be linked to PKC activity, and its magnitude was found to be largely dependent on the FA chain structure. Moreover, it did not occur in non-adherent monocytes and other leukocytes such as neutrophils. In other words, this novel non-conventional action of FAs seems to be a specific property of HAMs.

In this study, we investigated how translocation of PKC, endogenous phosphorylation and presence in membrane fraction of p47(the 47-kDa cytosolic phagocyte oxidase factor) are modified by iso15:0 and 20:4n-6 in order to explain the bimodal action of FAs in HAMs. These two FAs were chosen because of their marked bimodal action and their particular physiological significance, respectively. We concluded that FAs act on PKC and p47 translocation and endogenous p47phosphorylation in a manner that accounts for their concentration-dependent bimodal action on the PMA-stimulated O production.


EXPERIMENTAL PROCEDURES

Materials

PtdSer, DAG (1,2-didecanoyl-sn-glycerol), oleate (18:1n-9), arachidonate (20:4n-6), histone III-S, EGTA, EDTA, dithiothreitol, antipain, E-64, leupeptin, pepstatin, PMA, superoxide dismutase, and the luminescent probe, lucigenin (10,10`dimethyl-9,9`-bisacridinium dinitrate), were purchased from Sigma (St. Quentin Fallavier, France) and 13-methyltetradecanoate (iso15:0) from Larodan Fine Chemicals (Malmö, Sweden). Percoll was from Pharmacia (St. Quentin en Yvelines, France); the nutritive mediums and the fetal calf serum were from Life Technologies, Inc. (Glasgow, Scotland). R59022 was from TEBU (Le Parray en Yvelines, France), and GF109203X was kindly provided by Dr. J. Kirilovsky. [-P]ATP, P, ECL-Western blotting detection reagents, anti-rabbit IgGs, nitrocellulose membranes for hybridization transfer, and Hyper MP film were from Amersham (Les Ulis, France). The molecular weight markers for electrophoresis were purchased from Bio-Rad S.A. (Ivry sur Seine, France). The rabbit polyclonal anti-peptide SPLEEERQTQRSK corresponding to residues 348-360 of p47 was kindly provided by M.-C. Billoud-Dagher from the Laboratoire de Biochimie (Centre d'Etudes Nucléaires, Grenoble, France).

Preparation of Human Adherent Monocytes

Human monocytes were isolated as reported previously(17) . Briefly, peripheral blood was recovered from healthy volunteers by venipuncture using heparin. Mononuclear cells were obtained by centrifugation of blood samples over a discontinuous isotonic Percoll gradient (1.086/1.097 gml) in order to separate neutrophils from monocytes. Erythrocytes were discarded by lysis, and the mononuclear cell suspension was then washed with RPMI 1640 medium. After evaluating monocyte number, cells (15 10) were incubated for 2 h at 37 °C in a humid atmosphere with 5% CO in Petri dishes (35 10 mm) with 10 ml of RPMI 1640 containing 20% of fetal calf serum. Dishes were washed three times with the same medium to eliminate non-adherent cells. The purity of the remaining HAMs was >95% as assessed by May-Grunwald-Giemsa staining.

Incubation Conditions of Adherent Monocytes

HAMs (15 10) were preincubated with FAs added in ethanol (final concentration inferior to 0.1%) for 30 min (unless otherwise indicated), and PMA or DAG was added for activation. For the translocation assessments, supernatants were discarded after a 10- or 30-min incubation time for PMA or DAG, respectively, and HAMs were washed twice with 5 ml of RPMI 1640 and then once with 5 ml of PBS. The viability (>95%) was determined by trypan blue exclusion. For the assessment of NADPH oxidase activity through the lucigenin-detected superoxide anion production, cells (2 10 in RPMI 1640) were preincubated as above. A first measurement of chemiluminescence (CL) was performed immediately after adding 10M lucigenin in order to obtain the level of O production in the absence of PMA or DAG. PMA or DAG was then added for PKC stimulation (i.e. 30 min after adding FAs) and incubations were routinely stopped after 60 min, the time needed for CL peak to be reached in the presence of PMA. For a more exhaustive study of PMA and DAG stimulation, the incubation was stopped after 4 h. When used, the inhibitors of PKC activity were added just before PMA or DAG.

Purification of Soluble and Particulate PKC Fractions

HAMs (15 10) were scraped and resuspended in 5 ml of the ice-cold buffer A (20 mM Tris/HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.3 M sucrose, 2 mM dithiothreithol, and 2 mM phenylmethylsulfonyl fluoride) containing leupeptin, E-64, antipain, and pepstatin (50 µg/ml each) and sonicated by two 30-s pulse treatments with 1-min interval by means of a cell disrupter (Bronson B30 Sonic Power OSI, Paris, France). Soluble (cytosolic) and particulate (membrane) fractions were separated by ultracentrifugation for 40 min at 4 °C, using a Ti70 rotor at 40,000 rpm (Beckman Instruments). The supernatant (cytosolic fraction) was kept at 4 °C, whereas the pellet (membrane fraction) was suspended in 5 ml of buffer A containing leupeptin, E-64, antipain, and pepstatin plus 1% Triton X-100, sonicated, and then kept for 1 h at 4 °C. Each fraction was partially purified through a DE52 column (a siliconized Pasteur's pipette), first using buffer B (20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, and 1 mM dithiothreithol) and then the same solution with a 100-350 mM NaCl elution gradient in order to obtain PKC without its partially proteolyzed form PKM.

Assay of PKC

PKC was assessed through the histone kinase activity, i.e. by measuring the incorporation of [-P]ATP into 0.5 mg of histone type III-S/ml. Fractions of elution (50 µl) were simultaneously assayed in a 96 Multiwell plate 25860B (Corning Glass Works, Poly Labo, Strasbourg, France) for 5 min at room temperature in the presence or absence of 1 mM CaCl, 80 µg of PtdSer, and 8 µg of DAG/ml. The reaction was stopped after 5 min of incubation by adding 100 µl of 40% trichloroacetic acid and 20 µl of a solution containing 50 mM ATP and 5 mg of bovine serum albumin/ml. The precipitated proteins were then collected on glass fiber filters using a Skatron cell harvester (Lier, Norway). Radioactivity retained on the filters was determined by counting in the presence of 2 ml of scintillation fluid. PKC activity was assessed by subtracting the results obtained in the absence of Ca, PtdSer and DAG from those obtained in their presence. It was expressed as pmol of P phosphorylating histone III-S/min/10 cells. Total PKC activity was the sum of those of cytosol plus membrane.

Chemiluminescence Assays

O production was specifically measured using lucigenin as an enhancer of CL as described previously(18, 19) . This procedure was chosen because experiments clearly show results similar to those obtained by the superoxide dismutase-inhibitable reduction of ferricytochrome c(17) .

P Labeling of Monocytes and Measurement of Endogenous Phosphorylation

Monocytes (2 10 cell in 1 ml) were suspended in RPMI 1640 in the presence of 500 µCi of P (370 MBq/ml) and incubated at 37 °C for 30 min. They were then incubated in the same conditions but in the presence of the tested FA. Finally, cells were stimulated by adding 30 nM PMA in the incubation medium. After 5 min the cells were scraped, transferred into a microcentrifugation tube containing ice-cold 50% trichloracetic acid, and rapidly mixed. Pellets obtained after centrifugation (12,000 g, 5 min) of trichloroacetic acid-precipitated material were rapidly washed with 1 ml of distilled water and solubilized in 30 µl of Tris-HCl buffer (0.01 M, pH 6.8) containing 0.01% bromphenol blue, 3% urea, 1% dithiothreitol, and 1% SDS. After immersion in a boiling water bath for 5 min, solubilized proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 0.1% SDS and 12% acrylamide gel using the Laemmli solution as buffer(20) . After staining with Coomassie Blue, gels were destained, dried, and exposed to Hyper MP film for 40-80 h. The films were finally scanned by means of a Chromoscan 3 densitometer (Joyce Loebl, Gateshead, United Kingdom).

p47Translocation Assessment

Aliquots (20 µg of protein) of the membrane fraction provided by activated HAMs were submitted to SDS-PAGE (0.1% SDS, 10% acrylamide gel) according to the Laemmli method. Proteins were transferred to nitrocellulose membranes using a Mini Trans-Blot electrophoretic transfer cell apparatus (Bio-Rad) in 25 mM Tris-HCl, 192 mM glycine, and 20% (v/v) methanol. Blots were incubated for 60 min in TBS (50 mM Tris-HCl, 130 mM NaCl, pH 7.4) containing 15% milk powder and then for 120 min in the same medium containing the rabbit anti-peptide SPLEEERQTQRSK corresponding to residues 348-360 of p47. The antipeptide antibody was used with a 1:1000 dilution. Blots were rinsed several times in TTBS (TBS + 0.1% Tween 20) and then incubated for 60 min at room temperature in the same medium containing the peroxidase-labeled anti-rabbit IgGs with a 1:5000 dilution and 0.1% milk powder. After washing, bound antibodies were revealed by enhanced CL detection reagents. Blots were dried and exposed to Hyper MP film for 5 min. Films were finally scanned as reported above.

Fatty Acid Incorporation in Monocyte and Lipid Extraction

HAMs (2 10) were incubated in 2 ml of RPMI with [C]20:4n-6 or [C] 18:1n-9 (50 µCi/ml, specific activity 55 mCi/mmol) for 30, 60, and 90 min. The incubating medium was discarded for measuring the non-incorporated radioactivity. The remaining cells were scraped, suspended in 1 ml of 0.9% NaCl (pH 2.8), thoroughly agitated in 5 ml of chloroform/methanol (2:1, v/v)(21) , and centrifuged at 3000 g for 20 min. The lower phase (total lipids) was recovered, dried under nitrogen, and resuspended in chloroform. One part of the chloroform solution was used for assessing the total lipid-incorporated radioactivity. The other part was submitted to lipid class TLC separation on plates (2.5 20 cm, with concentrating zone) coated with Silica Gel 60 previously activated at 115 °C for 60 min. The solvent of migration was petroleum ether/diethyl ether/acetic acid (90:10:1, v/v/v). The determination of radioactivity under the spots representing phospholipids, free FAs, and triglycerides was carried out by using a Chromolec 101 Numelec scanner (La Verrière, France).

Statistical Analysis

Values were reported as mean ± S.E.. Data were analyzed using ANOVA-repeated measures.


RESULTS

Effect of PMA on the Translocation of PKC

Fig. 1A shows the PKC activity of membrane and cytosol fractions of human monocytes after different incubation times. Before adding PMA, more than 90% of the activity was localized in the cytosol, whereas adding 100 nM PMA brought about a redistribution of the PKC activity resulting in a substantial increase in the proportion of the activity recovered in the membrane fraction (up to 80% for a 10-min incubation). This is indicative of a rapid translocation of PKC. Fig. 1B illustrates that the percentage of PKC activity recovered in the membrane fraction after a 10-min incubation time was only multiplied by 1.5 from 30 nM to 100 nM PMA, whereas it was multiplied by 7.9 between 0 and 30 nM, showing a saturation phenomenon near the 100 nM PMA concentration. The half-translocation was obtained for about 30 nM. Therefore, given these results, the PMA concentrations of 30 and 100 nM were chosen for the respective FA potentiation and inhibition of the 10-min incubated PMA stimulating effect on translocation.


Figure 1: Effect of PMA on the translocation of PKC in human adherent monocytes. The Ca/PtdSer-dependent PKC activity was measured in cytosolic and particulate (membrane) fractions and expressed as pmol of P-phosphorylating histone III-S/min/10 cells. We only show here the percent distribution of recovered activity found in either fractions. A, distribution versus the incubation time after adding 100 nM PMA. B, distribution versus PMA concentration after 10-min incubation. The total (cytosolic plus particulate) activity expressed as indicated above was 8.0 ± 0.2 for A and 8.7 ± 0.3 for B (means ± S.E. from two separate preparations). , cytosolic fraction; , particulate (membrane) fraction.



Effect of Nanomolar and Micromolar Concentrations of Iso15:0 or 20:4n-6 in the Presence or Absence of PMA

Fig. 2A shows that 3 nM iso15:0 alone did not produce changes in the distribution of PKC between membrane and cytosol compared to the control without FA. On the other hand, adding 30 nM PMA to the preincubated iso15:0 clearly led to an increased translocation compared to that obtained with PMA alone. It is noteworthy that translocation with 30 nM PMA + 3 nM iso15:0 was similar to that with 100 nM PMA alone. These data provide evidence for a 3 nM iso15:0-potentiating action on the PKC-translocating effect of PMA, whereas iso15:0 alone was not able to promote any translocation. Contrasting with that, 30 µM iso15:0 totally blocked the translocation provided by 100 nM PMA. Exposure of HAMs to 20:4n-6 in the same conditions (Fig. 2B) brought about similar results. However, 20:4n-6 appears to be less effective at enhancing translocation at 3 nM and depressing translocation at 30 µM compared to iso15:0.


Figure 2: Effect of nanomolar and micromolar concentrations of either iso15:0 or 20:4n-6 on the translocation of PMA-stimulated PKC. The PKC activity was measured and expressed as indicated in Fig. 1. A and B, results obtained with 30-min preincubation in the presence of iso15:0 and 20:4n-6, respectively, before adding PMA. The total (cytosolic plus particulate) PKC activity expressed as indicated in Fig. 1 was 8.7 ± 0.2 for A and 7.5 ± 0.1 for B (mean ± S.E. from three separate preparations). All conditions were statistically (p < 0.01) different from the control, except for the micromolar concentration of iso15:0. For more details, see the p values above the different bars.



Effect of DAG on the Translocation of PKC

Fig. 3shows the effect of increasing concentrations of DAG on the PKC translocation after a 30-min incubation time. The following two points need to be made. First, the concentration of DAG for a half-translocation was approximately 10 times higher than that of PMA (25 µMversus 30 nM); Second, it clearly appears that the concentrations of DAG higher than 25 µM were unable to translocate more than 50% of the PKC activity, contrasting with the almost complete translocation brought about by PMA. R59022, an inhibitor of the DAG kinase, was used to determine whether this incomplete translocation was due to a rapid conversion of DAG into phosphatidic acid. If so, we would have expected R59022 to reinforce the DAG translocation of PKC. R59022 was found to be ineffective for promoting translocation further. On the contrary, it totally canceled the PKC translocation (not shown).


Figure 3: Effect of DAG on the translocation of PKC in human adherent monocytes. For legend, see Fig. 1, PMA being replaced by DAG. The total (cytosolic plus particulate) PKC activity expressed as indicated was 7.7 ± 0.2 (mean ± S.E. from two separate preparations).



Effect of Nanomolar and Micromolar Concentrations of Iso15:0 and 20:4n-6 in the Presence of DAG

Using the same rationale as above, we chose two concentrations of DAG, i.e. 15 and 25 µM, for testing the respective potentiating and depressing FA effects. Fig. 4(A and B) illustrates very clearly that the responses to iso15:0 and 20:4n-6 were similar to those obtained in the presence of PMA. It is particularly striking that the PKC translocation responses at both nanomolar and micromolar concentrations of 20:4n-6 were attenuated as compared to those obtained at the same concentrations of iso15:0. This was the same phenomenon as that typically observed for PMA.


Figure 4: Effect of nanomolar and micromolar concentrations of either iso15:0 or 20:4n-6 on the translocation of DAG-stimulated PKC. For legend, see Fig. 2. The total (cytosolic plus particulate) PKC activity expressed as indicate was 7.7 ± 0.3 for A and 7.5 ± 0.2 for B (mean ± S.E. from three separate preparations). All conditions were statistically (p < 0.01) different from the control, except for 15 µM DAG (p < 0.05). For more details, see the p values above the different bars.



Effect of Iso15:0 and 20:4n-6 on the O Production in the Presence or Absence of PMA or DAG

Fig. 5shows that, in the absence of PMA or DAG, neither iso15:0 nor 20:4n-6 brought about a modification in the O production. Conversely, in the presence of PMA both FAs were able to potentiate or depress the production at 3 nM or 30 µM, respectively. However, it clearly appears that both the potentiating and depressing effects were much more pronounced in the case of iso15:0. GF109203X, a selective and potent inhibitor of PKC, totally abolished the PMA-stimulated O production enhanced by 3 nM iso15:0 or 20:4n-6. Thus, such results correlated with the translocation data. As shown in Fig. 6A, the peak O production was shifted from 60 min for 5 and 10 µM DAG to 120 min for 20 µM DAG. In spite of this, 3 nM iso15:0 was able to potentiate the O production stimulated by 10 and 20 µM DAG. It was without effect in the presence of 5 µM DAG. Similar results were obtained with 20:4n-6 in the presence of 10 µM or 20 µM DAG (Fig. 6, C and D). However, when studied in greater detail, the results showed that 20:4n-6 produced a potentiation of the effect of 20 µM DAG at 60 min, and not at 120 min as was reported for iso15:0. In order to determine whether the last peak could be attributed to a direct effect of DAG on NADPH oxidase, i.e. to an effect non-mediated by PKC, we successively examined the actions of R59022 and R59022 + GF109203X on the 10 µM DAG-stimulated O production. Fig. 6E demonstrates that R59022 considerably potentiated the stimulating effect of 10 µM DAG as expected by its available-DAG enhancing effect. Moreover, this action was totally inhibited by GF109203X, providing evidence for a strictly PKC-mediated effect of DAG. Fig. 6F also shows that the peak PMA-stimulated O production was obtained at the same time (60 min) as the peak production stimulated by 5 or 10 µM DAG. We also verified (Fig. 6F) that R59022 itself did not possess a delaying effect on the peak O production.


Figure 5: Effect of iso15:0 or 20:4n-6 on the lucigenin-detected superoxide anion production in human adherent monocytes in the presence or absence of 30 nM PMA. Data were recorded by means of an ultrasensitive photon-counting imaging camera equipped with a computer-assisted image processor (Argus 100; Hamamatsu Photonics, Japan). Lucigenin (10M) chemiluminescence assays were performed every minute in the dark at room temperature. The curve obtained for each condition represents the number of photons/min/mg of protein against the elapsed time. The peak of the lucigenin-CL was reached and measured 60 min after adding 30 nM PMA, i.e. 90 min after adding FAs taking into account the 30-min preincubation in the presence of FAs. Because each volunteer appeared to be constitutively different for the PMA-stimulated adherent-monocyte CL (139,200 ± 15,000, mean ± S.E., 52% of coefficient of variation), we chose to compare the CL values obtained for a given subject to the arbitrary value of 100 obtained in the presence of 30 nM PMA alone for the same subject. GFX, 100 nM GF109203X. The preparation number was 14 for monocytes alone, monocytes plus PMA, and monocytes plus PMA plus 3 nM iso15:0, and was 2 or 3 for the remaining conditions.




Figure 6: Effect of iso15:0 or 20:4n-6 on the lucigenin-detected superoxide anion production in human adherent monocytes in the presence or absence of DAG. Recording, measures and expression of results were the same as in Fig. 5. In this case, however, the values were given for different times from 0 to 4 h after adding DAG. A-D, the time course of the superoxide anion production for different concentrations of DAG in the presence of 3 nM (A) and 30 µM (B) iso15:0; of 3 nM (C) and 30 µM (D) 20:4n-6. E, the production time course for monocytes (MC) or monocytes plus 10 µM DAG in the presence or absence of 5 µM R59022 (DAG+R) or 5 µM R59022 plus 100 nM GF109203X (DAG+R+G). F, the production time course for monocytes (MC), monocytes plus R59022 (+R), or monocytes plus 30 nM PMA (+PMA) in the presence of R59022 (PMA+R), GF109203X (PMA+G), or R59022 plus GF109203X (PMA+R+G). The value of 100 on the vertical axes has the same meaning as in Fig. 5 and corresponds to 167,500 ± 17,500 photons/min/mg of protein. Results were given from one typical experiment in A-E and from 2 separate preparations in F.



Effect of Incubation Time on O Production and Lipid FA Incorporation

It has already been demonstrated by our group that the potentiating effect of certain FAs is specifically obtained in adherent monocytes within the 3-300 nM concentration range. Therefore, in this study we used a FA concentration of 60 nM to obtain information on the influence of the FA preincubation time on both the O production and the FA cell incorporation into phospholipids and triglycerides. Because radiolabeled iso15:0 was not available for this experiment, we only used 18:n-9 and 20:4n-6, as it can be estimated that their physical and metabolic membrane properties are close to those of iso15:0. Fig. 7A shows that the potentiating effect of the FAs was not immediate since no activation took place when they were added just before PMA. On the other hand, the potentiating effect was about maximal from the 5th min of incubation. Such a time course appears to be completely different from that of cell lipid incorporation (Fig. 7B). It can also be noted (Fig. 7, C and D) that FA distribution into phospholipid, triglyceride, and free FA fractions was not greatly modified, except for the part of the FAs incorporated into the free FA fraction that tended to steadily decline in relation to time, whereas the triglyceride or phospholipid incorporation was enhanced for 20:4n-6 or 18:1n-9, respectively. Interestingly, there was an immediately maximal FA potentiation when the O production was stimulated by DAG (not shown), contrary to that observed for PMA stimulation.


Figure 7: Comparative influence of the fatty acid preincubation time on their capacity to potentiate the lucigenin-detected superoxide anion production and their cell incorporation. A, superoxide anion production versus the FA preincubation time before adding 30 nM PMA; for meaning of the vertical axes units, see Fig. 5. B, cell lipid incorporation of 18:1n-9 and 20:4n-6 versus their preincubation time. HAMs (2 10) were incubated in the presence of [C] 20:4n-6 or [C] 18:1n-9 (50 µCi/ml, 55 mCi/mmol). An aliquot of the extracted lipids was used for assessing the 20:4n-6 and 18:1n-9 lipid incorporation. The other one was submitted to the TLC separation of lipid classes. C and D, incorporation of 20:4n-6 and 18:1n-9, respectively, in phospholipid (PL) triglyceride (TG), and free fatty acid (FFA) fractions. Results were given as follows: A, from 2 separate preparations for 18:1n-9 and 20:4n-6 and from one single preparation for iso15:0; B-D, from one single preparation.



Enhanced Phosphorylation and Membrane Translocation of p47in the Presence of Iso15:0

Fig. 8shows the enhanced endogenous phosphorylating activity of monocytes following PMA stimulation after iso15:0 preincubation as compared to monocytes stimulated by PMA in the absence of iso15:0. It also shows that the phosphorylation of the band corresponding to p47 was enhanced. This enhancing effect was very low in the presence of iso15:0 and absence of PMA. On the other hand, the anti-peptide antibody directed toward the residues 348-360 of p47 highlighted that 3 nM and 30 µM iso15:0 were able to increase and decrease, respectively, the p47 membrane concentration after PMA cell treatment as compared to that obtained in the presence of PMA alone (Fig. 9). This led to the conclusion that iso15:0 is readily able to modulate NADPH oxidase activity through the phosphorylation-dependent modulation of p47translocation.


Figure 8: Phosphorylation of the HAM proteins during stimulation by iso15:0, PMA, or iso15:0 plus PMA. A, complete autoradiograms of P-phosphoproteins from cells in the following conditions: a, unstimulated; b, stimulated by 3 nM iso15:0; c, stimulated by 30 nM PMA; d, stimulated by 3 nM iso15:0 plus 30 nM PMA; e, stimulated by 30 µM iso15:0 plus 30 nM PMA. The visualized phosphoproteins correspond to 2 10 cells for each lane. FA preincubation and PMA stimulation lasted 30 and 5 min, respectively. B, densitometric scans of autoradiograms; inset shows the calibration curve for molecular weight determinations obtained with phosphorylase b (M 94,000), albumin (M 67,000), ovalbumin (M 43,000), carbonic anhydrase (M 30,000), and trypsin inhibitor (M 20,000). Three other experiments showed similar results.




Figure 9: Presence of p47phox in the membrane fraction of HAMs in unstimulated and diversely stimulated cells. A, Western blot analysis of p47 in the particulate fraction obtained after utracentrifugation at 40,000 rpm for 40 min. Membrane (particulate) proteins (20 µg) were submitted to SDS-PAGE, transferred to the nitrocellulose membrane, immunoblotted with anti-rabbit anti-peptide (residue 348-360 of p47) antibodies, and revealed by peroxidase-labeled anti-rabbit IgGs. Cells were: a, unstimulated; b, stimulated by 30 nM PMA; c, stimulated by 30 nM PMA plus 30 µM iso15:0; d, stimulated by 30 nM PMA plus 3 nM iso15:0. B, densitometric scans obtained for each condition. Two other experiments showed similar results.




DISCUSSION

Our results demonstrate the fact that, in intact cells, the capacity of both iso15:0 and 20:4n-6 to potentiate or depress the HAM O production is mediated by an action on the PKC translocation, leading to a simultaneous endogenous phosphorylation and translocation of p47. They fully account for the concentration-dependent bimodal action of these two FAs and others (with the exception of linear saturated ones) on the PKC-mediated O production that we recently established in HAMs(16) . They are in line with the literature reporting the implication of PKC in the activation of the respiratory burst in neutrophils (22, 23, 24) and the capacity of the cytosolic p47to be an endogenous phosphorylation substrate for PKC(25, 26) , this phosphorylation being in its turn definitely involved in the p47 translocation from cytosol to plasma membrane and in the resulting activation of the assembled multicomponent NADPH oxidase complex(27) . The above explanation is also supported by the fact that (i) we were unable to highlight any direct effect of either iso15:0 or 20:4n-6 on the NADPH oxidase activity as shown by the absence of O production without PMA or with both PMA and GF109203X, and (ii) both FAs were totally unable to modulate the translocation of PKC in the absence of PMA or DAG. In this respect, it is also worth noting that iso15:0 alone did not produce any appreciable action on the endogenous phosphorylation of p47 as compared with the basal condition, contrary to what was found in the presence of PMA.

The evidence for FA modulation of the O production through a strictly PKC-dependent route is not inconsistent with the conclusion of several authors claiming a direct activating effect of arachidonate (28, 29, 30) or other cis-unsaturated FAs (31) on the NADPH oxidase of neutrophils. It has more recently been proposed that phosphorylation and translocation of p47, although necessary, are insufficient for oxidase activity in these cells(32) . FAs could specifically and differently act on different leukocyte lines. We advance that FA modulation exclusively mediated by PKC we describe may only take place in adherent monocytes(16) .

The finding that both iso15:0 and 20:4n-6 alone were unable to trigger the PKC translocation requires distinguishing between the cell-free PKC studies and those on the intact cell PKC activity or translocation. The first ones have demonstrated a direct FA action (33) or a synergistic FA intervention with DAG (34) on the PKC activity. FA action more strictly depends on the presence of DAG for the - and -isotypes and not for the -isotype of c-PKC(34) . In the intact cells studied until now, only a synergistic FA stimulation of PKC activity has clearly been shown with DAG(13, 35) . This is, to some extent, in line with the present results.

In any case, however, these types of action took place at micromolar FA concentrations. In addition to the FA action on PKC translocation strictly depending on DAG or PMA, the present results confirm both the FA stimulating action at nanomolar concentrations and the depressing action at the micromolar concentrations (usually considered as stimulating) upon O production. They also establish that either action is mediated by phosphorylating and translocating processes. Considering the opposite FA responses of circulating and adherent monocytes (16) and that little is known on monocyte PKC(36) , the question of whether and how the adherence plays a role first in changing the distribution of PKC isotypes, and presumably (i) in the properties of the protein components of the NADPH oxidase (especially p47), or (ii) in the lipid environment of PKC and/or NADPH oxidase in the plasma membrane remains to be addressed.

An apparently contradictory finding is that R59022 did not potentiate (it did inhibit) the PKC translocation, whereas it greatly enhanced the NADPH oxidase activity in the presence of DAG (the activity was not enhanced in the presence of PMA). This can easily be explained by the view that the increasingly available amounts of DAG (due to the inhibition of DAG phosphorylation) are able to directly activate NADPH oxidase(37, 38) . The fact that this effect was totally canceled in the presence of GF109203X emphasizes the crucial role of a preliminary phosphorylation of translocatable subunit(s) in the oxidase activation by DAG, but also the priming effect of large amounts of DAG probably through their action on the membrane transfer of p47 and p67 (another translocatable subunit of NADPH oxidase) as already described in neutrophils on a cell-free system(39) .

It is worth noting that preincubation in the presence of FAs was needed for FA potentiation of PMA-stimulated, but not DAG-stimulated O production. We previously established that FA modulation is due to an action located at the membrane-interacting PMA-binding site of PKC and is probably mediated by the FA membrane-perturbing properties(16) . It can now be advanced that PMA and DAG PKC stimulations are not equivalent and therefore probably lead to different lipid-protein interactions and different lipid organization around the cysteine-rich zinc-finger-like region of the regulatory domain of PKC. This is consistent with recent results suggesting that PMA and DAG bind at different sites on PKC and induce distinct activated conformational forms of the protein(40) . The discrepancy (see Fig. 7) between both the free FA incorporation time course and the comparative capacity of these FAs to be incorporated, on the one hand, and the preincubation time minimally needed for potentiation by these same FAs (except for iso15:0, which is still not available as radiolabeled molecule), on the other, supports the view that FAs act through their free form rather than through their phospholipid-esterified form in cell membranes. In this respect, it is striking that 20:4n-6 was slightly incorporated into lipids and that its free form declined from the 100% value at zero time over the whole period studied, although the maximal potentiating effect was reached for (less than) 5 min of preincubation.

Such opposite FA effects can be considered as a priming or a desensibilizing effect on HAMs, rendering cells more or less reactive to mediators. They may be of utmost importance, given the nanomolar and micromolar plasma concentrations of minor (typically iso15:0) and major (typically 20:4n-6) free FAs, respectively, in healthy and pathological conditions.


FOOTNOTES

*
This work was supported by Grant 91111/91112 from the Ministère de l'Agriculture et des Forts, France and a grant from the Groupe Lipides et Nutrition. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Present address: Centre de Recherche, INSERM, 70 rue de Navacelles, 34090 Montpellier, France. Tel.: 33-67-04-37-19; Fax: 33-67-52-06-77.

The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; PtdSer, phosphatidylserine; FA, fatty acid; HAM, human adherent monocyte; PAGE: polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; CL, chemiluminescence; iso15:0, 13-methyltetradecanoic acid; 20:4n-6, arachidonic acid; 18:1n-9, oleic acid.


ACKNOWLEDGEMENTS

We thank Dr. M.-C. Billoud-Dagher and Professor V. Vignais for the rabbit anti-peptide SPLEEERQTQRSK (residues 348-360 of p47) polyclonal antibody.


REFERENCES
  1. Nishizuka, Y.(1989) JAMA (J. Am. Med. Assoc.) 262, 1826-1833 [Abstract]
  2. Bazzi, M. D., and Nelsestuen, G. L.(1991) Biochemistry30, 971-979 [Medline] [Order article via Infotrieve]
  3. Zidovetzki, R., and Lester, D. S.(1992) Biochim. Biophys. Acta1134, 261-272 [Medline] [Order article via Infotrieve]
  4. Burns, D. J., and Bell, R. M.(1991) J. Biol. Chem.266, 18330-18338 [Abstract/Free Full Text]
  5. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y.(1990) Proc. Natl. Acad. Sci. U. S. A.86, 4868-4871
  6. Souvignet, C., Pelosin, J.-M., Daniel, S., Chambaz, E. M., Ransac, S., and Verger, R.(1991) J. Biol. Chem.266, 40-44 [Abstract/Free Full Text]
  7. Nishizuka, Y.(1992) Science258, 607-614 [Medline] [Order article via Infotrieve]
  8. Léger, C. L.(1993) Prostagland. Leukotrienes Essent. Fatty Acids48, 17-21 [Medline] [Order article via Infotrieve]
  9. Murakami, K., and Routtenberg, A.(1985) FEBS Lett.192, 189-193 [CrossRef][Medline] [Order article via Infotrieve]
  10. Murakami, K., Chan, S. Y., and Routtenberg, A.(1986) J. Biol. Chem.261, 15424-15429 [Abstract/Free Full Text]
  11. Khan, W. A., Blobe, G. C., and Hannun, Y. A.(1992) J. Biol. Chem.267, 3605-3612 [Abstract/Free Full Text]
  12. Shinomura, T., Asaoka, Y., Oka, M., Yoshida, K., and Nishizuka, Y. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 5149-5153 [Abstract]
  13. Lester, D. S., Collin, C., Etcheberrigaray, R., and Alkon, D. L.(1991) Biochem. Biophys. Res. Commun.179, 1522-1528 [Medline] [Order article via Infotrieve]
  14. El Touny, S., Khan, W., and Hannun, Y.(1990) J. Biol. Chem.265, 16437-16443 [Abstract/Free Full Text]
  15. Huang, X. P., Da Silva, C., Fan, X. T., and Castagna, M.(1993) Biochim. Biophys. Acta1175, 351-356 [CrossRef][Medline] [Order article via Infotrieve]
  16. Kadri-Hassani, N., Leger, C. L., Vachier, I., and Descomps, B.(1995) J. Lipid Med. Cell Signal.11, 159-173 [CrossRef][Medline] [Order article via Infotrieve]
  17. Vachier, I., Damon, M., Le Doucen, C., Crastes de Paulet, A., Chanez, P., Michel, F. B., and Godard, P.(1992) Am. Rev. Respir. Dis.146, 1161-1166 [Medline] [Order article via Infotrieve]
  18. Gyllenhammar, H.(1987) J. Immunol. Methods97, 209-215 [CrossRef][Medline] [Order article via Infotrieve]
  19. Allen, R. C.(1986) Methods Enzymol.133, 449-493 [Medline] [Order article via Infotrieve]
  20. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  21. Folch, J., Lees, M., and Sloan-Stanley, G. H.(1957) J. Biol. Chem.226, 497-509 [Free Full Text]
  22. Wolfson, M., McPhail, L. C., and Nasrallah, V. N.(1985) J. Immunol.135, 2057-2062 [Abstract/Free Full Text]
  23. Pentremoli, S., Melloni, E., and Michetti, M.(1986) Biochem. Biophys. Res. Commun.136, 228-234 [Medline] [Order article via Infotrieve]
  24. Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D.(1986) J. Biol. Chem.261, 12616-12623 [Abstract/Free Full Text]
  25. Kramer, I. M., Verhoeven, A. J., Vanderbend, R. L., Weening, R. S., and Roos, D.(1988) J. Biol. Chem.263, 2352-2357 [Abstract/Free Full Text]
  26. Majumdar, S., Rossi, M. W., Fujiki, T., Phillips, W. A., Disa, S., Queen, C. F., Johnston, R. B., Jr., Rosen, O. M., Corkey, B. E., and Korchak, H. M.(1991) J. Biol. Chem.266, 9285-9294 [Abstract/Free Full Text]
  27. Morel, F., Doussiere, J., and Vignais, P. V.(1991) Eur. J. Biochem.201, 523-546 [Abstract]
  28. Henderson, L. M., Moule, S. K., and Chappell, J. B.(1993) Eur. J. Biochem.211, 157-162 [Abstract]
  29. Rubinek, T., and Levy, R.(1993) Biochim. Biophys. Acta1176, 51-58 [Medline] [Order article via Infotrieve]
  30. Curnutte, J. T, Badwey, J. A., Robinson, J. M., Karnovsky, M. J., and Karnovsky, M. L.(1984) J. Biol. Chem.259, 11851-11857 [Abstract/Free Full Text]
  31. Badwey, J. A., Curnutte, J. T., Robinson, J. M., Berde, C. B., Karnovsky, J. M., and Karnovsky, M. L.(1984) J. Biol. Chem.259, 7870-7877 [Abstract/Free Full Text]
  32. Dana, R., Malech, M. H. L., and Levy, R.(1994) Biochem. J.297, 217-223 [Medline] [Order article via Infotrieve]
  33. Khan, W., El Touny, S., and Hannun, Y. A.(1991) FEBS Lett.292, 98-102 [CrossRef][Medline] [Order article via Infotrieve]
  34. Shinomura, T., Asaoka, Y., Oka, M., Yoshida, K., and Nishizuka, Y. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 5149-5153 [Abstract]
  35. Yoshida, K., Asaoka, Y., and Nishizuka, Y.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 6443-6446 [Abstract]
  36. Chang, Z. L., and Beezhold, D. H.(1993) Immunology80, 360-366 [Medline] [Order article via Infotrieve]
  37. Tyagi, S. R., Neckelmann, N., Uhlinger, D. J., Burnham, D. N., and Lambeth, J. D.(1992) Biochemistry31, 2765-2774 [Medline] [Order article via Infotrieve]
  38. Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D.(1990) J. Biol. Chem.265, 17550-17559 [Abstract/Free Full Text]
  39. Park, J.-W., and Babior B. M.(1992) J. Biol. Chem.267, 19901-19906 [Abstract/Free Full Text]
  40. Slater, S., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem.269, 17160-17165 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.