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The Journal of Immunology, 2000, 164: 5398-5402.
Copyright © 2000 by The American Association of Immunologists

Cellular Regulation of Cytosolic Group IV Phospholipase A2 by Phosphatidylinositol Bisphosphate Levels1

Jesús Balsinde2,*, María A. Balboa*, Wen-Hong Li3, Juan Llopis4,{dagger} and Edward A. Dennis2,*

Departments of * Chemistry and Biochemistry and {dagger} Pharmacology, University of California at San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Cytosolic group IV phospholipase A2 (cPLA2)5 is a key effector of diverse pathways initiated by cytokines, growth factors, inflammatory mediators, hormones, and neurotransmitters (1, 2). The broad implication of cPLA2 in cellular signaling arises from the fact that this enzyme, once activated, specifically releases arachidonic acid (AA) from membrane phospholipids (1, 2). This essential role of cPLA2 in AA metabolism has been highlighted by recent experiments using cPLA2 knockout mice (3, 4). Cells obtained from these animals generate significantly less AA-derived metabolites (3, 4, 5). Aside from its key role in inflammatory reactions as a precursor of the biologically active eicosanoids, AA has been recognized as an intracellular second messenger on its own, capable of activating a number of targets, including protein kinases and ion channels (6).

Regulation of cPLA2 has been a key aspect in cellular signal transduction studies over the recent years. Results from these stud- ies suggest a scenario for activation of cPLA2 in which two dif-ferent kinds of signals act in concert to elicit full enzyme activation. On one hand, an increase in intracellular Ca2+ results in the enzyme being translocated from cytosol to membrane fractions, where its substrate resides (1, 2). This process is made possible by the existence in the N-terminal half of the protein of a C2 domain, similar to the one present in many other proteins with key roles in cellular signaling such as protein kinase C (7). Thus Ca2+ is required for the cPLA2 to act not because it is required for catalysis but because it appears to be essential for the enzyme to reach its substrate (8).

The second signal that is thought to act together with Ca2+ to promote full cPLA2 activation is mediated by direct phosphorylation at Ser505 of cPLA2 by members of the mitogen-activated protein kinase cascade (9). However, recent results have questioned the importance of mitogen-activated protein kinase-mediated phosphorylation of the cPLA2 in terms of AA mobilization by showing that the latter response may indeed take place under circumstances where phosphorylation of the cPLA2 at Ser505 is inhibited (10, 11, 12). Similarly, certain conditions that lead to full cPLA2 phosphorylation at Ser505 do not result in an increased AA release response (13).

We have recently demonstrated that phosphatidylinositol bisphosphate (PtdInsP2) strongly increases both cPLA2 binding and activity toward phospholipid vesicles and mixed micelles. Interestingly, polyphosphoinositides decrease the requirement of cPLA2 for Ca2+ such that under certain conditions cPLA2 activity is truly Ca2+ independent (14). This is a remarkable finding because, as indicated above, an increase in intracellular Ca2+ levels is traditionally assumed to be the signal that allows the cPLA2 to interact with the membrane and hence with its substrate (15). Our previous studies thus raised the very intriguing possibility that PtdInsP2 might regulate a novel route for activation of the cPLA2 in cells. These observations have prompted us to investigate the possible existence of such a route in cells. Data reported here demonstrate that cellular PtdInsP2 levels do regulate cPLA2 activation in a Ca2+-independent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents

The cell line used in this study, termed P388D1/MAB, is a subclone of the P388D1 cell line (TIB 63) obtained from the American Type Culture Collection (Manassas, VA), which was selected on the basis of high responsivity to LPS/platelet-activating factor (PAF) (16, 17). IMDM (endotoxin, <0.05 ng/ml) was obtained from BioWhittaker (Walkersville, MD). FBS was obtained from HyClone (Logan, UT). Nonessential amino acids were obtained from Irvine Scientific (Santa Ana, CA). [5,6,8,9,11,12,14,15-3H]AA (sp. act., 100 Ci/mmol) was obtained from New England Nuclear (Boston, MA). LPS Re595 and PAF were obtained from Sigma (St. Louis, MO). Bromoenol lactone (BEL) and methyl arachidonyl fluorophosphonate (MAFP) were obtained from Biomol (Plymouth Meeting, PA). Rac1 and GTP were obtained from Calbiochem (La Jolla, CA).

Cell culture and labeling conditions

P388D1 cells were maintained at 37°C in a humidified atmosphere at 80% air and 10% CO2 in IMDM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids. Cells were plated at 106 per well, allowed to adhere overnight, and used for experiments the following day. All experiments were conducted in serum-free IMDM.

Stimulation of P388D1 cells

Our standard regimen for short-term activation of the P388D1 cells has been described previously (18, 19). Briefly, radiolabeling of the cells with [3H]AA was achieved by including 0.5 µCi/ml [3H]AA during the overnight adherence period. The cells were placed in serum-free medium for 30–60 min before the addition of LPS (200 ng/ml) for 1 h. After the LPS incubation, the cells were exposed to UV light (mercury lamp at 366 nm; intensity, 9.6 mJ/s · cm2; Spectroline, Westbury, NY), PAF, or both for the time indicated in the presence of 0.1 mg/ml BSA. The supernatants were removed, cleared of detached cells by centrifugation, and assayed for radioactivity by liquid scintillation counting. More than 99% of the released radioactive material remains as unmetabolized AA under these experimental conditions.

Intracellular Ca2+ determination

The cells, either LPS primed or unprimed, were loaded in HBSS containing 0.01% pluronic and 0.5 µM fura-2/AM for 30 min at room temperature. Cells were then exposed to UV light and/or PAF as indicated. Fluorescence Ca2+ images were obtained and calibrated as previously described (20). For experiments using fluo-3, a protocol identical to that described by Li et al. (21) was followed.

Permeabilization studies

The cells were permeabilized using 30 µM digitonin in a buffer consisting of 120 mM KCl, 30 mM NaCl, 10 mM PIPES, 1 mM KH2PO4, 1.03 MgCl2, 0.0374 mM CaCl2, and 1 mM EGTA, pH 7.0, to give a final free Ca2+ concentration of 15 nM (22). Immediately after adding the digitonin, the GTPase protein Rac was added, and the reactions proceeded for up to 10 min. Rac was loaded with GTP exactly as described by Hartwig et al. (23). Cell permeabilization was conducted at 37°C using adherent cells, and the total incubation time with digitonin did not exceed 10 min, as longer incubations with digitonin induced excessive detachment of the cells from the plastic culture dishes.

Determination of phosphoinositides

Cells labeled with 100 µCi/ml myo[3H]inositol for 3 days were used. After the different treatments, the reactions were stopped and a lipidic fraction in chloroform was obtained as described (24). The chloroform was dried under a gentle stream of nitrogen, and the dried samples were applied to Silicagel G-60 TLC plates (Analtech, Newark, DE). The plates were coated with 1% potassium oxalate and heat-dried before sample application. Phospholipids were separated with chloroform/acetone/methanol/acetic acid/water (60:30:26:24:14) (25). The location of the different phosphoinositides was determined by running known standards on the same plate.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
P388D1 macrophages respond to LPS by releasing AA in a cPLA2-dependent manner in a process that takes several hours to develop (16, 17). This process can be greatly accelerated if a Ca2+-mobilizing stimulus such as PAF is added after 1 h of exposure to LPS. Then, a robust AA release response is observed within 15 min of addition of PAF (26, 27, 28). Our investigations into the molecular mechanisms involved in the immediate AA release have highlighted the requirement for a rise in intracellular Ca2+ levels—an event that occurs within seconds after PAF addition—to fully activate the cPLA2 (20, 28). Activation of the cPLA2 constitutes the key step and is thought to regulate the recruitment of a novel group V sectretory PLA2 (sPLA2) to the membrane, which ultimately results in an amplified release response (18, 19, 26).

We have now observed that AA release in LPS-treated macrophages could also be accelerated if the cells were briefly exposed to UV radiation (9.6 mJ/s · cm2; 4 s) (Fig. 1GoA). Exposure of the LPS-treated cells to both PAF and UV did not have any effect beyond what was already induced by either of them alone (Fig. 1GoA). These data suggest that the signaling step targeted by the UV is probably the same as the one targeted by PAF. In accord with these observations, the cPLA2 inhibitor MAFP completely abrogated the UV-induced AA release (Fig. 1GoB), indicating that the cPLA2 is also under these settings a key component of the signaling cascade. MAFP has recently been shown to inhibit another intracellular PLA2, i.e., the group VI Ca2+-independent PLA2 (iPLA2) (29). The iPLA2, but not the sPLA2, is also strongly inhibited by BEL (18), a compound that had no measurable effect on the UV-triggered response (Fig. 1GoB). Therefore, the MAFP effects on AA release reported above are attributed to inhibition of the cPLA2.



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  		FIGURE 1. AA release in P388D1 cells. A, The cells, labeled with [3H]AA, were treated with LPS (200 ng/ml) for 1 h, UV light for 4 s, LPS for 1 h, followed by UV light (Both) or neither (Ctrl) as indicated. Afterward, the cells were incubated in the absence ({square}) or presence () of 100 nM PAF for 10 min. The supernatants were then poured off and assayed for [3H]AA release. B, Effect of PLA2 inhibitors on AA release. The [3H]AA-labeled cells were incubated with 200 ng/ml LPS for 1 h. Afterward, MAFP (25 µM), BEL (25 µM), or neither (Ctrl) were added, as indicated. After 15 min, the cells were then exposed to UV light for 4 s in the presence of 0.1 mg/ml BSA. Extracellular AA release was quantified as described under Materials and Methods.

 
The above data indicate that both UV and PAF appear to signal through a common pathway involving cPLA2, and we have previously shown that PAF signaling requires elevated Ca2+ (20, 28). Therefore, we would expect for UV to induce a transient increase in the intracellular Ca2+ concentration as well. Exposure of the cells, either untreated or LPS-treated, to UV did not alter the intracellular Ca2+ levels; however, subsequent addition of PAF did induce large alterations in the intracellular Ca2+ concentration, as measured with fura-2-loaded cells (Fig. 2Go). Identical results were obtained when fluo-3-loaded cells were used (not shown). Thus, unlike PAF, UV signaling does not involve an increase in the intracellular Ca2+ concentration, which demonstrates that increased Ca2+ mobilization is not a prerequisite for AA release to occur.



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  		FIGURE 2. Effect of UV and PAF on intracellular Ca2+ rise. The LPS-treated cells, loaded with fura-2/AM, were exposed to UV for 4 s where indicated. Afterward, 100 nM PAF was added. The same profile was obtained when LPS-untreated cells were used.

 
Several enzymes with key roles in cellular signaling that act on lipid surfaces, namely protein kinase C and Raf-1 kinase, dock to membranes via the "lipid anchors" diacylglycerol and phosphatidic acid, respectively (30, 31). We have recently shown that, in vitro, the cPLA2 binds tightly and specifically to vesicles or micelles containing PtdInsP2, resulting in dramatic increases in enzyme activity even at nanomolar Ca2+ levels, i.e., those present in unstimulated cells (14). Thus, higher levels of PtdInsP2 in membranes targeted by the cPLA2 as a consequence of cellular activation could result in increased amounts of enzyme bound to the membrane as well as increased enzyme activity (14).

To test the possibility that increased PtdInsP2 levels could serve to anchor the cPLA2 to membranes at resting cytosolic Ca2+ levels, we took advantage of the use of permeabilized cells. A useful approach to increase cellular PtdInsP2 levels in the permeabilized cells is to add GTPase proteins that activate PtdInsP2 synthesis (23, 32). In agreement with these previous observations, the addition of the GTPase protein Rac1 to the digitonin-permeabilized cells increased the cellular levels of PtdInsP2 (Fig. 3GoA). Interestingly, this treatment also led to substantial release of AA to the incubation medium (Fig. 3GoB). The concentration-response curve of AA release corresponded well with that of PtdInsP2 production (Fig. 3Go). Thus, these results suggest that increasing the concentration of PtdInsP2 in the cells triggers an AA release response.



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  		FIGURE 3. Rac1 effects on PtdInsP2 production and AA mobilization. The GTPase protein Rac1 was added at the indicated concentrations to digitonin-permeabilized cells for 10 min. Afterward, PtdInsP2 production (A) and AA release (B) were quantified as described in Materials and Methods.

 
Fig. 4Go shows that Ca2+-independent activation of the macrophages by LPS and UV induced a time-dependent increase in both PtdInsP and PtdInsP2 levels in cells prelabeled with [3H]inositol (Fig. 4GoA). These changes were not observed if LPS-unprimed cells were used. Significant changes in the levels of phosphatidylinositol under these conditions could not be detected. Because under the LPS/UV stimulation conditions no Ca2+ mobilization occurs (see Fig. 2Go), such an elevation of PtdInsP2 levels is unlikely to reflect any compensatory mechanism, but synthesis "on demand," i.e., as a step of the LPS/UV signaling machinery. Increased PtdInsP2 synthesis in the absence of intracellular Ca2+ increases is known to occur in cells treated with phorbol esters (33), and, coincidentally, phorbol esters are able to trigger the Ca2+-independent activation of cPLA2 and concomitant AA release in certain cell types including macrophages (34). In this regard, the time course of AA release by LPS/UV, as shown in Fig. 4GoB, reflected well the time course of PtdInsP2 production.



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  		FIGURE 4. Phosphoinositide production and AA release by LPS/UV-treated cells. A, The LPS-treated cells, labeled with [3H]inositol, were exposed (closed symbols) or not (open symbols) to UV for 4 s, and the incubations were allowed to proceed for the times indicated. PtdInsP2 (circles) and PtdInsP (inverted triangles) levels were quantified by TLC. B, The LPS-treated cells, labeled with [3H]AA, were exposed (closed symbols) or not (open symbols) to UV for 4 s, and the incubations were allowed to proceed for the times indicated. AA release was quantified as described in Materials and Methods.

 
The UV-induced rises in PtdInsP2 and PtdInsP could be inhibited by 2,3-dihydroxybenzaldehyde (DBA), a compound that has been shown to inhibit phosphatidylinositol 4-kinase (35, 36) (Fig. 5Go). DBA also blunted the UV-induced AA release (Fig. 6Go). At concentrations up to 50 µM, DBA had no direct effect on cPLA2 activity from P388D1 cell homogenates as measured toward 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles in the presence of 50 µM BEL (to block endogenous iPLA2 activity (37)) and 2 mM 2-ME (to block endogenous sPLA2 activity). Thus, these results directly link PtdInsP2 levels with cPLA2-mediated AA release under Ca2+-independent activation conditions.



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  		FIGURE 5. Effect of DBA on phosphoinositide levels. The cells, primed with 200 ng/ml LPS for 1 h, were exposed to UV for 4 s in the absence or presence of 25 µM DBA as indicated. The drug was added to the cells 30 min before the UV treatment. The incubations were then allowed to proceed for 10 min. PtdInsP ({square}) and PtdInsP2 () levels were quantified by TLC. To highlight the activated phosphoinositide production, unstimulated basal levels have been subtracted. These levels were 400 ± 100 cpm for PtdInsP and 650 ± 100 cpm for PtdInsP2. DBA did not affect basal levels.

 


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  		FIGURE 6. Effect of DBA on AA release. The cells, primed with 200 ng/ml LPS for 1 h, were exposed () or not ({square}) to UV for 4 s in the absence or presence of 25 µM DBA, as indicated. The incubations were then allowed to proceed for 10 min. AA release was quantified as described in Materials and Methods.

 
Collectively, the current results place the cPLA2 among the growing list of proteins whose function and/or activity are regulated by PtdInsP2 (38). Evidence has been presented for the existence of a route for cPLA2 activation via PtdInsP2 in which the final message is the mobilization of AA with Ca2+ levels equaling those of a quiescent cell. It should be noted that although cPLA2 activation by UV light has previously been observed under biologically relevant settings (39), the use of UV light in our macrophage system should be contemplated as an experimental paradigm that allowed us to define a novel biochemical mechanism for AA mobilization. This mechanism allows one to explain the participation of cPLA2 in cell regulation not involving Ca2+ signaling and solves the paradox of the involvement of cPLA2 in the Ca2+-independent delayed phase (hours) of eicosanoid generation that is characteristic of immunoinflammatory cells (16, 40).


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HD 26,171 and GM 20,501. Back

2 Address correspondence to either Dr. Jesús Balsinde or Dr. Edward Dennis, Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601. Back

3 Current address: Beckman Institute 139-74, California Institute of Technology, Pasadena, CA 91125. Back

4 Current address: Department of Physiology, University of Castilla-La Mancha, Albacete, Spain. Back

5 Abbreviations used in this paper: cPLA2, group IV cytosolic phospholipase A2; AA, arachidonic acid; PtdInsP2, phosphatidylinositol bisphosphate; PtdInsP, phosphatidylinositol phosphate; DBA, 2,3-dihydroxybenzaldehyde; MAFP, methyl arachidonyl fluorophosphonate; PAF, platelet-activating factor; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; BEL, bromoenol lactone. Back

Received for publication October 14, 1999. Accepted for publication March 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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