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. 2002 Jan 15;361(Pt 2):267–276. doi: 10.1042/0264-6021:3610267

Expression of 5-lipoxygenase in pulmonary artery endothelial cells.

Ying-Yi Zhang 1, Jennifer L Walker 1, Annong Huang 1, John F Keaney 1, Clary B Clish 1, Charles N Serhan 1, Joseph Loscalzo 1
PMCID: PMC1222306  PMID: 11772398

Abstract

Increased expression of 5-lipoxygenase (5LO) in pulmonary artery endothelial cells (PAECs) has been observed in disease states such as pulmonary hypertension and allergen challenge. To understand the function of endothelial 5LO, we examined the expression of this enzyme in normally cultured human PAECs and its characteristics when overexpressed. A small amount of 5LO message and protein was detected by reverse-transcriptase-mediated PCR (RT-PCR) and Western blotting in PAECs. Sequencing of the RT-PCR products that overlapped the entire coding region of 5LO mRNA indicated that the sequence of PAEC 5LO was identical with that of leucocyte 5LO. Incubation of the PAECs with A23187 and arachidonic acid led to a small production of 5-hydroxyeicosatetraenoic acid (5-HETE) (46-98 pmol/4x10(6) cells) but no leukotrienes. Overexpression of 5LO in PAECs by adenovirus-mediated gene transfer revealed that the enzyme was localized in the nucleus. Incubation of the transduced cells with A23187 (5 microM) caused the production of both 5LO products and downstream leukotrienes. The proportions of the produced leukotriene A(4) (LTA(4)) hydrolates (sum of 6-trans-LTB(4) and 12-epi-6-trans-LTB(4)), LTB(4) and cysteinyl leukotriene were approx. 17:14:10. cGMP production in the 5LO-transduced PAECs was decreased by 33+/-14% on stimulation with A23187. These results show that cultured PAECs express a minimal amount of 5LO, which can generate some 5-HETE, but not leukotrienes. However, increased expression of 5LO in PAECs can lead to the production of all downstream leukotrienes, which could potentially cause endothelial dysfunction in the pulmonary vasculature.

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Selected References

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  1. Anderson K. M., Seed T., Vos M., Mulshine J., Meng J., Alrefai W., Ou D., Harris J. E. 5-Lipoxygenase inhibitors reduce PC-3 cell proliferation and initiate nonnecrotic cell death. Prostate. 1998 Nov 1;37(3):161–173. doi: 10.1002/(sici)1097-0045(19981101)37:3<161::aid-pros5>3.0.co;2-d. [DOI] [PubMed] [Google Scholar]
  2. Avis I. M., Jett M., Boyle T., Vos M. D., Moody T., Treston A. M., Martínez A., Mulshine J. L. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest. 1996 Feb 1;97(3):806–813. doi: 10.1172/JCI118480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkowitz B. A., Zabko-Potapovich B., Valocik R., Gleason J. G. Effects of the leukotrienes on the vasculature and blood pressure of different species. J Pharmacol Exp Ther. 1984 Apr;229(1):105–112. [PubMed] [Google Scholar]
  4. Boado R. J., Pardridge W. M., Vinters H. V., Black K. L. Differential expression of arachidonate 5-lipoxygenase transcripts in human brain tumors: evidence for the expression of a multitranscript family. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9044–9048. doi: 10.1073/pnas.89.19.9044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brock T. G., McNish R. W., Peters-Golden M. Capacity for repeatable leukotriene generation after transient stimulation of mast cells and macrophages. Biochem J. 1998 Feb 1;329(Pt 3):519–525. doi: 10.1042/bj3290519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bäck M., Norel X., Walch L., Gascard J., de Montpreville V., Dahlén S., Brink C. Prostacyclin modulation of contractions of the human pulmonary artery by cysteinyl-leukotrienes. Eur J Pharmacol. 2000 Aug 11;401(3):389–395. doi: 10.1016/s0014-2999(00)00453-2. [DOI] [PubMed] [Google Scholar]
  7. Chartier C., Degryse E., Gantzer M., Dieterle A., Pavirani A., Mehtali M. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol. 1996 Jul;70(7):4805–4810. doi: 10.1128/jvi.70.7.4805-4810.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen X. S., Naumann T. A., Kurre U., Jenkins N. A., Copeland N. G., Funk C. D. cDNA cloning, expression, mutagenesis, intracellular localization, and gene chromosomal assignment of mouse 5-lipoxygenase. J Biol Chem. 1995 Jul 28;270(30):17993–17999. doi: 10.1074/jbc.270.30.17993. [DOI] [PubMed] [Google Scholar]
  9. Chu S. J., Tang L. O., Watney E., Chi E. Y., Henderson W. R., Jr In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade. J Immunol. 2000 Oct 15;165(8):4640–4648. doi: 10.4049/jimmunol.165.8.4640. [DOI] [PubMed] [Google Scholar]
  10. Claesson H. E., Haeggström J. Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4. Eur J Biochem. 1988 Apr 5;173(1):93–100. doi: 10.1111/j.1432-1033.1988.tb13971.x. [DOI] [PubMed] [Google Scholar]
  11. Claesson H. E., Jakobsson P. J., Steinhilber D., Odlander B., Samuelsson B. Expression of 5-lipoxygenase and biosynthesis of leukotriene B4 in human monomorphonuclear leukocytes. J Lipid Mediat. 1993 Mar-Apr;6(1-3):15–22. [PubMed] [Google Scholar]
  12. Clish C. B., O'Brien J. A., Gronert K., Stahl G. L., Petasis N. A., Serhan C. N. Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitment in vivo. Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):8247–8252. doi: 10.1073/pnas.96.14.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dahlén S. E., Hedqvist P., Hammarström S., Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature. 1980 Dec 4;288(5790):484–486. doi: 10.1038/288484a0. [DOI] [PubMed] [Google Scholar]
  14. Devchand P. R., Keller H., Peters J. M., Vazquez M., Gonzalez F. J., Wahli W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature. 1996 Nov 7;384(6604):39–43. doi: 10.1038/384039a0. [DOI] [PubMed] [Google Scholar]
  15. Ding X. Z., Iversen P., Cluck M. W., Knezetic J. A., Adrian T. E. Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells. Biochem Biophys Res Commun. 1999 Jul 22;261(1):218–223. doi: 10.1006/bbrc.1999.1012. [DOI] [PubMed] [Google Scholar]
  16. Feinmark S. J., Cannon P. J. Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes. J Biol Chem. 1986 Dec 15;261(35):16466–16472. [PubMed] [Google Scholar]
  17. Ford-Hutchinson A. W., Bray M. A., Doig M. V., Shipley M. E., Smith M. J. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature. 1980 Jul 17;286(5770):264–265. doi: 10.1038/286264a0. [DOI] [PubMed] [Google Scholar]
  18. Ghosh J., Myers C. E. Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13182–13187. doi: 10.1073/pnas.95.22.13182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gupta S., Srivastava M., Ahmad N., Sakamoto K., Bostwick D. G., Mukhtar H. Lipoxygenase-5 is overexpressed in prostate adenocarcinoma. Cancer. 2001 Feb 15;91(4):737–743. doi: 10.1002/1097-0142(20010215)91:4<737::aid-cncr1059>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
  20. Hill E., Maclouf J., Murphy R. C., Henson P. M. Reversible membrane association of neutrophil 5-lipoxygenase is accompanied by retention of activity and a change in substrate specificity. J Biol Chem. 1992 Nov 5;267(31):22048–22053. [PubMed] [Google Scholar]
  21. Hong S. H., Avis I., Vos M. D., Martínez A., Treston A. M., Mulshine J. L. Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors. Cancer Res. 1999 May 1;59(9):2223–2228. [PubMed] [Google Scholar]
  22. Hong S. H., Avis I., Vos M. D., Martínez A., Treston A. M., Mulshine J. L. Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors. Cancer Res. 1999 May 1;59(9):2223–2228. [PubMed] [Google Scholar]
  23. Ibe B. O., Anderson J. M., Raj J. U. Leukotriene synthesis by isolated perinatal ovine intrapulmonary vessels correlates with age-related changes in 5-lipoxygenase protein. Biochem Mol Med. 1997 Jun;61(1):63–71. doi: 10.1006/bmme.1997.2579. [DOI] [PubMed] [Google Scholar]
  24. Janssen-Timmen U., Vickers P. J., Wittig U., Lehmann W. D., Stark H. J., Fusenig N. E., Rosenbach T., Rådmark O., Samuelsson B., Habenicht A. J. Expression of 5-lipoxygenase in differentiating human skin keratinocytes. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6966–6970. doi: 10.1073/pnas.92.15.6966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lammers C. H., Schweitzer P., Facchinetti P., Arrang J. M., Madamba S. G., Siggins G. R., Piomelli D. Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling. J Neurochem. 1996 Jan;66(1):147–152. doi: 10.1046/j.1471-4159.1996.66010147.x. [DOI] [PubMed] [Google Scholar]
  26. Lepley R. A., Fitzpatrick F. A. 5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins. J Biol Chem. 1994 Sep 30;269(39):24163–24168. [PubMed] [Google Scholar]
  27. Maccarrone M., Bari M., Corasaniti M. T., Nisticó R., Bagetta G., Finazzi-Agrò A. HIV-1 coat glycoprotein gp120 induces apoptosis in rat brain neocortex by deranging the arachidonate cascade in favor of prostanoids. J Neurochem. 2000 Jul;75(1):196–203. doi: 10.1046/j.1471-4159.2000.0750196.x. [DOI] [PubMed] [Google Scholar]
  28. Malaviya R., Malaviya R., Jakschik B. A. Reversible translocation of 5-lipoxygenase in mast cells upon IgE/antigen stimulation. J Biol Chem. 1993 Mar 5;268(7):4939–4944. [PubMed] [Google Scholar]
  29. Murphy R. C., Hammarström S., Samuelsson B. Leukotriene C: a slow-reacting substance from murine mastocytoma cells. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4275–4279. doi: 10.1073/pnas.76.9.4275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. O'Donnell V. B., Chumley P. H., Hogg N., Bloodsworth A., Darley-Usmar V. M., Freeman B. A. Nitric oxide inhibition of lipid peroxidation: kinetics of reaction with lipid peroxyl radicals and comparison with alpha-tocopherol. Biochemistry. 1997 Dec 9;36(49):15216–15223. doi: 10.1021/bi971891z. [DOI] [PubMed] [Google Scholar]
  31. O'Donnell V. B., Taylor K. B., Parthasarathy S., Kühn H., Koesling D., Friebe A., Bloodsworth A., Darley-Usmar V. M., Freeman B. A. 15-Lipoxygenase catalytically consumes nitric oxide and impairs activation of guanylate cyclase. J Biol Chem. 1999 Jul 16;274(29):20083–20091. doi: 10.1074/jbc.274.29.20083. [DOI] [PubMed] [Google Scholar]
  32. Ohtaka H., Tsang J. Y., Foster A., Hogg J. C., Schellenberg R. R. Comparative effects of leukotrienes on porcine pulmonary circulation in vitro and in vivo. J Appl Physiol (1985) 1987 Aug;63(2):582–588. doi: 10.1152/jappl.1987.63.2.582. [DOI] [PubMed] [Google Scholar]
  33. Ohtsuki T., Matsumoto M., Hayashi Y., Yamamoto K., Kitagawa K., Ogawa S., Yamamoto S., Kamada T. Reperfusion induces 5-lipoxygenase translocation and leukotriene C4 production in ischemic brain. Am J Physiol. 1995 Mar;268(3 Pt 2):H1249–H1257. doi: 10.1152/ajpheart.1995.268.3.H1249. [DOI] [PubMed] [Google Scholar]
  34. Peppelenbosch M. P., Tertoolen L. G., Hage W. J., de Laat S. W. Epidermal growth factor-induced actin remodeling is regulated by 5-lipoxygenase and cyclooxygenase products. Cell. 1993 Aug 13;74(3):565–575. doi: 10.1016/0092-8674(93)80057-l. [DOI] [PubMed] [Google Scholar]
  35. Provost P., Doucet J., Hammarberg T., Gerisch G., Samuelsson B., Radmark O. 5-Lipoxygenase interacts with coactosin-like protein. J Biol Chem. 2001 Jan 31;276(19):16520–16527. doi: 10.1074/jbc.M011205200. [DOI] [PubMed] [Google Scholar]
  36. Riddick C. A., Ring W. L., Baker J. R., Hodulik C. R., Bigby T. D. Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells. Eur J Biochem. 1997 May 15;246(1):112–118. doi: 10.1111/j.1432-1033.1997.00112.x. [DOI] [PubMed] [Google Scholar]
  37. Rubbo H., Radi R., Trujillo M., Telleri R., Kalyanaraman B., Barnes S., Kirk M., Freeman B. A. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem. 1994 Oct 21;269(42):26066–26075. [PubMed] [Google Scholar]
  38. Rybina I. V., Liu H., Gor Y., Feinmark S. J. Regulation of leukotriene A4 hydrolase activity in endothelial cells by phosphorylation. J Biol Chem. 1997 Dec 12;272(50):31865–31871. doi: 10.1074/jbc.272.50.31865. [DOI] [PubMed] [Google Scholar]
  39. Schellenberg R. R., Foster A. Differential activity of leukotrienes upon human pulmonary vein and artery. Prostaglandins. 1984 Mar;27(3):475–482. doi: 10.1016/0090-6980(84)90205-3. [DOI] [PubMed] [Google Scholar]
  40. Shimada K., Navarro J., Goeger D. E., Mustafa S. B., Weigel P. H., Weinman S. A. Expression and regulation of leukotriene-synthesis enzymes in rat liver cells. Hepatology. 1998 Nov;28(5):1275–1281. doi: 10.1002/hep.510280516. [DOI] [PubMed] [Google Scholar]
  41. Shiratori Y., Moriwaki H., Muto Y., Onishi H., Kato M., Asano F. Production of leukotriene B4 in parenchymal and sinusoidal cells of the liver in rats treated simultaneously with D-galactosamine and endotoxin. Gastroenterol Jpn. 1989 Dec;24(6):640–645. doi: 10.1007/BF02774162. [DOI] [PubMed] [Google Scholar]
  42. Smedegård G., Hedqvist P., Dahlén S. E., Revenäs B., Hammarström S., Samuelsson B. Leukotriene C4 affects pulmonary and cardiovascular dynamics in monkey. Nature. 1982 Jan 28;295(5847):327–329. doi: 10.1038/295327a0. [DOI] [PubMed] [Google Scholar]
  43. Teifel M., Heine L. T., Milbredt S., Friedl P. Optimization of transfection of human endothelial cells. Endothelium. 1997;5(1):21–35. doi: 10.3109/10623329709044156. [DOI] [PubMed] [Google Scholar]
  44. Uz T., Manev H. Circadian expression of pineal 5-lipoxygenase mRNA. Neuroreport. 1998 Mar 30;9(5):783–786. doi: 10.1097/00001756-199803300-00003. [DOI] [PubMed] [Google Scholar]
  45. Voelkel N. F., Tuder R. M., Wade K., Höper M., Lepley R. A., Goulet J. L., Koller B. H., Fitzpatrick F. Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest. 1996 Jun 1;97(11):2491–2498. doi: 10.1172/JCI118696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Woods J. W., Coffey M. J., Brock T. G., Singer I. I., Peters-Golden M. 5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation. J Clin Invest. 1995 May;95(5):2035–2046. doi: 10.1172/JCI117889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wright L., Tuder R. M., Wang J., Cool C. D., Lepley R. A., Voelkel N. F. 5-Lipoxygenase and 5-lipoxygenase activating protein (FLAP) immunoreactivity in lungs from patients with primary pulmonary hypertension. Am J Respir Crit Care Med. 1998 Jan;157(1):219–229. doi: 10.1164/ajrccm.157.1.9704003. [DOI] [PubMed] [Google Scholar]
  48. Wächtershäuser A., Steinhilber D., Loitsch S. M., Stein J. Expression of 5-lipoxygenase by human colorectal carcinoma Caco-2 cells during butyrate-induced cell differentiation. Biochem Biophys Res Commun. 2000 Feb 24;268(3):778–783. doi: 10.1006/bbrc.2000.2213. [DOI] [PubMed] [Google Scholar]
  49. Zhang Y. Y., Hamberg M., Rådmark O., Samuelsson B. Stabilization of purified human 5-lipoxygenase with glutathione peroxidase and superoxide dismutase. Anal Biochem. 1994 Jul;220(1):28–35. doi: 10.1006/abio.1994.1294. [DOI] [PubMed] [Google Scholar]
  50. Zhang Y. Y., Hammarberg T., Radmark O., Samuelsson B., Ng C. F., Funk C. D., Loscalzo J. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences. Biochem J. 2000 Nov 1;351(Pt 3):697–707. [PMC free article] [PubMed] [Google Scholar]
  51. Zhang Y. Y., Rådmark O., Samuelsson B. Mutagenesis of some conserved residues in human 5-lipoxygenase: effects on enzyme activity. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):485–489. doi: 10.1073/pnas.89.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]

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