Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts

Abstract

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.

FIGURE 1. Comparison of fatty acids released from IMLF^+/+ and IMLF ^−/−

IMLF^+/+ and IMLF^−/− were incubated overnight in medium containing various radiolabeled fatty acids and then either left unstimulated (US, white bar) or stimulated with A23187 (2 μg/ml) (black bar) for 45 min (A) or with 10% mouse serum (MS, black bar) for 30 min (B). The amount of fatty acids released into the medium is expressed as a percentage of the total cellular incorporated radioactivity. Results are the average ± S.E. of four independent experiments.

FIGURE 2. Effect of PLA₂ inhibitors on arachidonic acid release fromIMLF^+/+ and IMLF ^−/−

A, [³H]arachidonic acid-labeled IMLF^−/− were treated with PLA₂ inhibitors (10 μM) or Me₂SO (vehicle control) for 15 min and then left unstimulated (US, light gray bars) or incubated with A23187 (2 μg/ml) (dark gray bars) for 45 min. The amount of [³H]arachidonic acid released into the medium is expressed as a percentage of the total cellular incorporated radioactivity. IMLF^+/+ and IMLF^−/− were preincubated with the indicated amounts of pyrrolidine-2 (B) or Wyeth-1 (C) for 15 min and then stimulated with 2 μg/ml A23187. The amount of [³H]arachidonic acid released is expressed as a percentage of the total incorporated radioactivity after subtracting the % release from unstimulated cells treated with inhibitors. Data shown are the average ± S.E. of three independent experiments.

FIGURE 3. Real-time PCR of GIV cPLA₂s in IMLF ^+/+ and IMLF^−/−

A, real-time PCR was carried out using Taqman gene expression assays for mouse GIV cPLA₂s using iScript cDNA from IMLF^+/+ and IMLF^−/−. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a cDNA template control. The data are represented as cycle threshold values, which is the cycle number that a signal was detected for a specific cDNA and is inversely proportional to the abundance of the transcript. Results are the average ± S.E. of six independent experiments. B, amino acid alignment of human cPLA₂β1 and mouse cPLA₂β. The N-terminal extension containing the truncated JmjC domain (highlighted, with double overline) in human cPLA₂β1 is not present in mouse cPLA₂β. The calcium-binding loops (CBL) in the C2 domains (highlighted and boxed) of human and mouse cPLA₂β are shown. Residues required for catalytic activity, including the active site Ser (human Ser-566; mouse Ser-333) and Asp (human Asp-846; mouse Asp-613) catalytic dyad and Arg (human Arg-538; mouse Arg-305) are shown in open boxes. The region in the catalytic domain of human cPLA₂β1 that is deleted in the human cPLA₂β3 splice variant (22) is highlighted in gray.

FIGURE 4. cPLA₂β has low enzymatic activity and is insensitive to pyrrolidine-2 and Wyeth-1

A, lysophospholipase activity of affinity purified His₆-cPLA₂β (1 μg) was assayed using 1-[¹⁴C]palmitoyl-2-lyso-PC for the indicated times. Expression of His₆-cPLA₂β in Sf9 cells infected with increasing volumes (μl) of baculovirus is shown in the inset. B, PLA₂ activity of His₆-cPLA₂β (1 μg) was measured at the indicated time points using 1-palmitoyl-[¹⁴C]arachidonyl-PE or -PC as described under “Experimental Procedures.” C, the effect of pyrrolidine-2 and Wyeth-1 on lysophospholipase activity of cPLA₂β (1 μg) or cPLA₂ζ (50 ng) was studied by incubating purified enzyme at 37 °C for 2 min with different doses of inhibitors followed by the addition of the substrate. Results shown are the average ± S.D. of two independent experiments.

FIGURE 5. Enzymatic properties of cPLA₂ζ and inhibition by pyrrolidine-2 and Wyeth-1

A, lysophospholipase activity of affinity-purified His₆-cPLA₂ζ (50 ng) was assayed using 1-[¹⁴C]palmitoyl-2-lyso-PC. Expression of His₆-cPLA₂ζ in Sf9 cells infected with increasing volumes (μl) of baculovirus is shown in the inset. B and C, PLA₂ activity (150 ng) was assayed using 1-palmitoyl-2-[¹⁴C]arachidonyl-PC (P*APC) and 1-palmitoyl-2-[¹⁴C]oleoyl-PC (P*OPC) (B) and 1-palmitoyl-[¹⁴C]arachidonyl-PE (P*APE) (C) for the indicated times. D, the effect of pyrrolidine-2 and Wyeth-1 on PLA₂ activity of His₆-cPLA₂ζ (150 ng) was studied by incubating the purified enzyme at 37 °C for 2 min with different amount of inhibitors followed by the addition of the substrate (P*APE). Results shown are the average ± S.D. of two independent experiments.

FIGURE 6. A23187 stimulates arachidonic acid release from Sf9 cells expressing His-cPLA₂ζ

A, Sf9 cells were infected with different amounts of recombinant baculoviruses for expression of glutathione S-transferase-His (pAcGHLT, vector control), mouse His₆-cPLA₂β, or mouse His₆-cPLA₂ζ. [³H]Arachidonic acid-labeled Sf9 cells were stimulated with A23187 (2 μg/ml) for 45 min. The amount of [³H]arachidonic acid released into the medium from unstimulated (US) or A23187-stimulated cells is expressed as a percentage of the total incorporated radioactivity. The bottom panels are representative immunoblots of Sf9 lysates showing levels of expression of glutathione S-transferase-His₆ (vector control), His₆-cPLA₂β and His₆-cPLA₂ζ using antibody to His₆. B, [³H]arachidonic acid-labeled Sf9 cells expressing His₆-cPLA₂ζ were treated with the indicated amounts of pyrrolidine-2 or Wyeth-1 for 15 min prior to stimulation with A23187. Arachidonic acid release was measured similarly as described in A. Arachidonic acid release from Sf9 cells infected with control vector treated similarly was subtracted as background. Results shown are the average ± S.D. of two independent experiments.

FIGURE 7. Calcium dependence of cPLA₂ζ activity

A, PLA₂ activity of His₆-cPLA₂ζ was measured with 1-palmitoyl-[¹⁴C]arachidonyl-PE in the absence (1 mM EGTA) (open bars) or presence (1 mM EGTA with 5 mM CaCl₂) of calcium (gray bars). Results are the average ± S.D. of two independent experiments. B, cPLA₂α and His₆-cPLA₂ζ were assayed using 1-palmitoyl-2-[¹⁴C]arachidonyl-PC vesicles as a function of the concentration of free calcium. A representative experiment of triplicate analyses is shown.

FIGURE 8. Calcium-induced translocation of EGFP-cPLA₂ζ and EGFP- cPLA₂α in IMLF ^−/−

Ionomycin (0.5 μM) was added to IMLF^−/− expressing EGFP-cPLA₂ζ (A) or EGFP-cPLA₂α (B), and images were taken every 5 s. The arrows in A, a–f, point to vesicles with EGFP-cPLA₂ζ. A movie of this cell can be found in the supplemental data (http://www.jbc.org). An enlarged portion of the cell showing translocation of EGFP-cPLA₂ζ to vesicles 3.3 min after adding ionomycin is shown in A, panel g. Another cell depicting tranlocation of EGFP-cPLA₂ζ to ruffles is shown in A, h–j.