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. 1991 Feb;87(2):476–481. doi: 10.1172/JCI115020

Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2.

K Satoh 1, T Imaizumi 1, Y Kawamura 1, H Yoshida 1, M Hiramoto 1, S Takamatsu 1, M Takamatsu 1
PMCID: PMC295106  PMID: 1846878

Abstract

The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.

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  1. Aden D. P., Fogel A., Plotkin S., Damjanov I., Knowles B. B. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature. 1979 Dec 6;282(5739):615–616. doi: 10.1038/282615a0. [DOI] [PubMed] [Google Scholar]
  2. Blank M. L., Hall M. N., Cress E. A., Snyder F. Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: higher activities in hypertensive rats. Biochem Biophys Res Commun. 1983 Jun 15;113(2):666–671. doi: 10.1016/0006-291x(83)91778-3. [DOI] [PubMed] [Google Scholar]
  3. Blank M. L., Lee T., Fitzgerald V., Snyder F. A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid). J Biol Chem. 1981 Jan 10;256(1):175–178. [PubMed] [Google Scholar]
  4. Chen C. H., Albers J. J. Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity. J Lipid Res. 1982 Jul;23(5):680–691. [PubMed] [Google Scholar]
  5. Chen C. H., Forte T. H., Cahoon B. E., Thrift R. N., Albers J. J. Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2. Biochim Biophys Acta. 1986 Jul 18;877(3):433–439. doi: 10.1016/0005-2760(86)90209-2. [DOI] [PubMed] [Google Scholar]
  6. Elstad M. R., Stafforini D. M., McIntyre T. M., Prescott S. M., Zimmerman G. A. Platelet-activating factor acetylhydrolase increases during macrophage differentiation. A novel mechanism that regulates accumulation of platelet-activating factor. J Biol Chem. 1989 May 25;264(15):8467–8470. [PubMed] [Google Scholar]
  7. Farr R. S., Cox C. P., Wardlow M. L., Jorgensen R. Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF). Clin Immunol Immunopathol. 1980 Mar;15(3):318–330. doi: 10.1016/0090-1229(80)90044-6. [DOI] [PubMed] [Google Scholar]
  8. Goding J. W. Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods. 1976;13(3-4):215–226. doi: 10.1016/0022-1759(76)90068-5. [DOI] [PubMed] [Google Scholar]
  9. Hanahan D. J., Demopoulos C. A., Liehr J., Pinckard R. N. Identification of platelet activating factor isolated from rabbit basophils as acetyl glyceryl ether phosphorylcholine. J Biol Chem. 1980 Jun 25;255(12):5514–5516. [PubMed] [Google Scholar]
  10. Hanahan D. J. Platelet activating factor: a biologically active phosphoglyceride. Annu Rev Biochem. 1986;55:483–509. doi: 10.1146/annurev.bi.55.070186.002411. [DOI] [PubMed] [Google Scholar]
  11. Khalil A., Farooqui J., Scanu A. M. Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A2 family. Biochim Biophys Acta. 1986 Aug 14;878(1):127–130. doi: 10.1016/0005-2760(86)90350-4. [DOI] [PubMed] [Google Scholar]
  12. Knowles B. B., Howe C. C., Aden D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. doi: 10.1126/science.6248960. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Maki N., Hoffman D. R., Johnston J. M. Platelet-activating factor acetylhydrolase activity in maternal, fetal, and newborn rabbit plasma during pregnancy and lactation. Proc Natl Acad Sci U S A. 1988 Feb;85(3):728–732. doi: 10.1073/pnas.85.3.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marinari L., Lenich C. M., Ross A. C. Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2. J Lipid Res. 1987 Aug;28(8):941–948. [PubMed] [Google Scholar]
  17. Miwa M., Miyake T., Yamanaka T., Sugatani J., Suzuki Y., Sakata S., Araki Y., Matsumoto M. Characterization of serum platelet-activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children. J Clin Invest. 1988 Dec;82(6):1983–1991. doi: 10.1172/JCI113818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morris K. M., Goldberger G., Colten H. R., Aden D. P., Knowles B. B. Biosynthesis and processing of a human precursor complement protein, pro-C3, in a hepatoma-derived cell line. Science. 1982 Jan 22;215(4531):399–400. doi: 10.1126/science.7199205. [DOI] [PubMed] [Google Scholar]
  19. Okazaki M., Ohno Y., Hara I. High-performance aqueous gel permeation chromatography of human serum lipoproteins. J Chromatogr. 1980 Dec 12;221(2):257–264. doi: 10.1016/s0378-4347(00)84310-8. [DOI] [PubMed] [Google Scholar]
  20. Ostermann G., Kertscher H. P., Winkler L., Schlag B., Rühling K., Till U. The role of lipoproteins in the degradation of platelet-activating factor. Thromb Res. 1986 Nov 1;44(3):303–314. doi: 10.1016/0049-3848(86)90005-8. [DOI] [PubMed] [Google Scholar]
  21. Ostermann G., Rühling K., Zabel-Langhennig R., Winkler L., Schlag B., Till U. Plasma from atherosclerotic patients exerts an increased degradation of platelet-activating factor. Thromb Res. 1987 Aug 1;47(3):279–285. doi: 10.1016/0049-3848(87)90141-1. [DOI] [PubMed] [Google Scholar]
  22. Patterson M. K., Jr Measurement of growth and viability of cells in culture. Methods Enzymol. 1979;58:141–152. doi: 10.1016/s0076-6879(79)58132-4. [DOI] [PubMed] [Google Scholar]
  23. Prescott S. M., Zimmerman G. A., McIntyre T. M. Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3534–3538. doi: 10.1073/pnas.81.11.3534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pritchard P. H., Chonn A., Yeung C. C. The degradation of platelet-activating factor in the plasma of a patient with familial high density lipoprotein deficiency (Tangier disease). Blood. 1985 Dec;66(6):1476–1478. [PubMed] [Google Scholar]
  25. Rash J. M., Rothblat G. H., Sparks C. E. Lipoprotein apolipoprotein synthesis by human hepatoma cells in culture. Biochim Biophys Acta. 1981 Nov 23;666(2):294–298. doi: 10.1016/0005-2760(81)90120-x. [DOI] [PubMed] [Google Scholar]
  26. Satoh K., Imaizumi T., Kawamura Y., Yoshida H., Takamatsu S., Mizono S., Shoji B., Takamatsu M. Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from patients with ischemic cerebrovascular disease. Prostaglandins. 1988 May;35(5):685–698. doi: 10.1016/0090-6980(88)90142-6. [DOI] [PubMed] [Google Scholar]
  27. Satoh K., Imaizumi T., Kawamura Y., Yoshida H., Takamatsu S., Takamatsu M. Increased activity of the platelet-activating factor acetylhydrolase in plasma low density lipoprotein from patients with essential hypertension. Prostaglandins. 1989 Jun;37(6):673–682. doi: 10.1016/0090-6980(89)90104-4. [DOI] [PubMed] [Google Scholar]
  28. Schwartz A. L., Fridovich S. E., Knowles B. B., Lodish H. F. Characterization of the asialoglycoprotein receptor in a continuous hepatoma line. J Biol Chem. 1981 Sep 10;256(17):8878–8881. [PubMed] [Google Scholar]
  29. Snyder F. Chemical and biochemical aspects of platelet activating factor: a novel class of acetylated ether-linked choline-phospholipids. Med Res Rev. 1985 Jan-Mar;5(1):107–140. doi: 10.1002/med.2610050105. [DOI] [PubMed] [Google Scholar]
  30. Stafforini D. M., McIntyre T. M., Carter M. E., Prescott S. M. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J Biol Chem. 1987 Mar 25;262(9):4215–4222. [PubMed] [Google Scholar]
  31. Stafforini D. M., Prescott S. M., McIntyre T. M. Human plasma platelet-activating factor acetylhydrolase. Purification and properties. J Biol Chem. 1987 Mar 25;262(9):4223–4230. [PubMed] [Google Scholar]
  32. Swenson T. L., Simmons J. S., Hesler C. B., Bisgaier C., Tall A. R. Cholesteryl ester transfer protein is secreted by Hep G2 cells and contains asparagine-linked carbohydrate and sialic acid. J Biol Chem. 1987 Dec 5;262(34):16271–16274. [PubMed] [Google Scholar]
  33. Terashita Z., Imura Y., Nishikawa K. Inhibition by CV-3988 of the binding of [3H]-platelet activating factor (PAF) to the platelet. Biochem Pharmacol. 1985 May 1;34(9):1491–1495. doi: 10.1016/0006-2952(85)90689-6. [DOI] [PubMed] [Google Scholar]
  34. Tolman K. G., Peterson P., Gray P., Hammar S. P. Hepatotoxicity of salicylates in monolayer cell cultures. Gastroenterology. 1978 Feb;74(2 Pt 1):205–208. [PubMed] [Google Scholar]
  35. Verdery R. B., 3rd, Gatt S. Assay for lecithin: cholesterol acyltransferase. Methods Enzymol. 1981;72:375–384. doi: 10.1016/s0076-6879(81)72027-5. [DOI] [PubMed] [Google Scholar]
  36. Volwerk J. J., Pieterson W. A., de Haas G. H. Histidine at the active site of phospholipase A2. Biochemistry. 1974 Mar 26;13(7):1446–1454. doi: 10.1021/bi00704a020. [DOI] [PubMed] [Google Scholar]
  37. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  38. Zannis V. I., Breslow J. L., SanGiacomo T. R., Aden D. P., Knowles B. B. Characterization of the major apolipoproteins secreted by two human hepatoma cell lines. Biochemistry. 1981 Dec 8;20(25):7089–7096. doi: 10.1021/bi00528a006. [DOI] [PubMed] [Google Scholar]

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