15-Deoxy-Δ12,14-prostaglandin J2 Impairs Phosphatidylcholine Synthesis and Induces Nuclear Accumulation of Thiol-modified Cytidylyltransferase

Alan J Ryan,Bill B Chen,Prashanth R Vennalaganti,Florita C Henderson,Linda A Tephly,A Brent Carter,Rama K Mallampalli

doi:10.1074/jbc.m801167200

Abstract

Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ2-treated cells. 15d-PGJ2 modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ2 into CCTalpha. These effects of 15d-PGJ2 were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.

Highlights

Phosphatidylcholine (PtdCho)2 is a critical phospholipid component of mammalian cell membranes and variably contributes to the makeup of secretory products such as bile, serum lipoproteins, and pulmonary surfactant
Cyclopentenone prostaglandins (CyPG) of the J series are synthesized from the parent compound, arachidonic acid, by a series of enzymatic steps using cyclooxygenase and prostaglandin D2 (PGD2) synthase and successive spontaneous dehydration to yield PGJ2, ⌬12-PGJ2, and 15-deoxy
15d-PGJ2 on PtdCho synthesis, we found that primary alveolar type II cells as well as MLE cells showed dose-dependent reductions in CTP:phosphocholine cytidylyltransferase (CCT) activity when cells were exposed to increasing concentrations of 15d-PGJ2 (Fig. 1, C and D)

Summary

EXPERIMENTAL PROCEDURES

The murine lung epithelial cell line (MLE-12) was obtained from ATCC. 15d-PGJ2, its biologically inactive analog 9,10-dihydro-15-deoxy-⌬12,14-prostaglandin J2 (CAY10410), and 15 deoxy-⌬12,14-prostaglandin J2-biotinamide (biotin-15d-PGJ2) were obtained from Cayman Chemicals. MLE cells were seeded on 60-mm dishes and grown to confluency in Hite’s medium plus 2% FBS. Incubations were conducted at 37 °C for 1–3 h in 50-␮l reaction mixtures containing 1.0 ␮g of purified rat CCT in Buffer A with ethanol vehicle or 15d-PGJ2. Transient Transfection—Transfections were conducted on subconfluent (ϳ80% confluency) cells for 4 h in serum-free Hite’s medium using FuGENE 6 (3 ␮l/␮g DNA) plus either a pCMV5-CCT␣-his expression vector (wild type or Cys 3 Ser mutant) or pCDNA3.1 (as control) at 8 ␮g/100-mm dish. After treatment with vehicle or 15d-PGJ2, cells were harvested in Buffer B (10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, 1% Triton X-100, plus protease inhibitor mixture) and briefly sonicated on ice, and cellular debris was pelleted at 12,000 ϫ g for 10 min at 4 °C. Zeiss LSM 510 confocal microscope using the 568 nm line of an argon laser (5 milliwatt nominal output, beam width at specimen 0.2 ␮m)

All experiments were done with a

RESULTS

Findings

DISCUSSION