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. 1998 May 15;332(Pt 1):213–221. doi: 10.1042/bj3320213

Characterization of the stress-inducing effects of homocysteine.

P A Outinen 1, S K Sood 1, P C Liaw 1, K D Sarge 1, N Maeda 1, J Hirsh 1, J Ribau 1, T J Podor 1, J I Weitz 1, R C Austin 1
PMCID: PMC1219470  PMID: 9576870

Abstract

The mechanism by which homocysteine causes endothelial cell (EC) injury and/or dysfunction is not fully understood. To examine the stress-inducing effects of homocysteine on ECs, mRNA differential display and cDNA microarrays were used to evaluate changes in gene expression in cultured human umbilical-vein endothelial cells (HUVEC) exposed to homocysteine. Here we show that homocysteine increases the expression of GRP78 and GADD153, stress-response genes induced by agents or conditions that adversely affect the function of the endoplasmic reticulum (ER). Induction of GRP78 was specific for homocysteine because other thiol-containing amino acids, heat shock or H2O2 did not appreciably increase GRP78 mRNA levels. Homocysteine failed to elicit an oxidative stress response in HUVEC because it had no effect on the expression of heat shock proteins (HSPs) including HSP70, nor did it activate heat shock transcription factor 1. Furthermore homocysteine blocked the H2O2-induced expression of HSP70. In support of our findings in vitro, steady-state mRNA levels of GRP78, but not HSP70, were elevated in the livers of cystathionine beta-synthase-deficient mice with hyperhomocysteinaemia. These studies indicate that the activation of stress response genes by homocysteine involves reductive stress leading to altered ER function and is in contrast with that of most other EC perturbants. The observation that homocysteine also decreases the expression of the antioxidant enzymes glutathione peroxidase and natural killer-enhancing factor B suggests that homocysteine could potentially enhance the cytotoxic effect of agents or conditions known to cause oxidative stress.

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Selected References

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  1. Austin R. C., Howard P. L., D'Souza V. N., Klamut H. J., Ray P. N. Cloning and characterization of alternatively spliced isoforms of Dp71. Hum Mol Genet. 1995 Sep;4(9):1475–1483. doi: 10.1093/hmg/4.9.1475. [DOI] [PubMed] [Google Scholar]
  2. Braakman I., Helenius J., Helenius A. Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature. 1992 Mar 19;356(6366):260–262. doi: 10.1038/356260a0. [DOI] [PubMed] [Google Scholar]
  3. Chen Q., Yu K., Holbrook N. J., Stevens J. L. Activation of the growth arrest and DNA damage-inducible gene gadd 153 by nephrotoxic cysteine conjugates and dithiothreitol. J Biol Chem. 1992 Apr 25;267(12):8207–8212. [PubMed] [Google Scholar]
  4. Dorner A. J., Wasley L. C., Kaufman R. J. Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells. EMBO J. 1992 Apr;11(4):1563–1571. doi: 10.1002/j.1460-2075.1992.tb05201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dorner A. J., Wasley L. C., Kaufman R. J. Protein dissociation from GRP78 and secretion are blocked by depletion of cellular ATP levels. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7429–7432. doi: 10.1073/pnas.87.19.7429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dudman N. P., Hicks C., Wang J., Wilcken D. E. Human arterial endothelial cell detachment in vitro: its promotion by homocysteine and cysteine. Atherosclerosis. 1991 Nov;91(1-2):77–83. doi: 10.1016/0021-9150(91)90189-a. [DOI] [PubMed] [Google Scholar]
  7. Fortin L. J., Genest J., Jr Measurement of homocyst(e)ine in the prediction of arteriosclerosis. Clin Biochem. 1995 Apr;28(2):155–162. doi: 10.1016/0009-9120(94)00073-5. [DOI] [PubMed] [Google Scholar]
  8. Fryer R. H., Wilson B. D., Gubler D. B., Fitzgerald L. A., Rodgers G. M. Homocysteine, a risk factor for premature vascular disease and thrombosis, induces tissue factor activity in endothelial cells. Arterioscler Thromb. 1993 Sep;13(9):1327–1333. doi: 10.1161/01.atv.13.9.1327. [DOI] [PubMed] [Google Scholar]
  9. Georgopoulos C., Welch W. J. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol. 1993;9:601–634. doi: 10.1146/annurev.cb.09.110193.003125. [DOI] [PubMed] [Google Scholar]
  10. Graven K. K., Zimmerman L. H., Dickson E. W., Weinhouse G. L., Farber H. W. Endothelial cell hypoxia associated proteins are cell and stress specific. J Cell Physiol. 1993 Dec;157(3):544–554. doi: 10.1002/jcp.1041570314. [DOI] [PubMed] [Google Scholar]
  11. Hajjar K. A. Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor. J Clin Invest. 1993 Jun;91(6):2873–2879. doi: 10.1172/JCI116532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Halleck M. M., Holbrook N. J., Skinner J., Liu H., Stevens J. L. The molecular response to reductive stress in LLC-PK1 renal epithelial cells: coordinate transcriptional regulation of gadd153 and grp78 genes by thiols. Cell Stress Chaperones. 1997 Mar;2(1):31–40. doi: 10.1379/1466-1268(1997)002<0031:tmrtrs>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harker L. A., Harlan J. M., Ross R. Effect of sulfinpyrazone on homocysteine-induced endothelial injury and arteriosclerosis in baboons. Circ Res. 1983 Dec;53(6):731–739. doi: 10.1161/01.res.53.6.731. [DOI] [PubMed] [Google Scholar]
  14. Harker L. A., Slichter S. J., Scott C. R., Ross R. Homocystinemia. Vascular injury and arterial thrombosis. N Engl J Med. 1974 Sep 12;291(11):537–543. doi: 10.1056/NEJM197409122911101. [DOI] [PubMed] [Google Scholar]
  15. Hightower L. E. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell. 1991 Jul 26;66(2):191–197. doi: 10.1016/0092-8674(91)90611-2. [DOI] [PubMed] [Google Scholar]
  16. Hill S. A., Shaughnessy S. G., Joshua P., Ribau J., Austin R. C., Podor T. J. Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line. Blood. 1996 Jun 15;87(12):5061–5073. [PubMed] [Google Scholar]
  17. Hladovec J. Experimental homocystinemia, endothelial lesions and thrombosis. Blood Vessels. 1979;16(4):202–205. doi: 10.1159/000158207. [DOI] [PubMed] [Google Scholar]
  18. Huang L. E., Zhang H., Bae S. W., Liu A. Y. Thiol reducing reagents inhibit the heat shock response. Involvement of a redox mechanism in the heat shock signal transduction pathway. J Biol Chem. 1994 Dec 2;269(48):30718–30725. [PubMed] [Google Scholar]
  19. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jornot L., Mirault M. E., Junod A. F. Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide. Am J Respir Cell Mol Biol. 1991 Sep;5(3):265–275. doi: 10.1165/ajrcmb/5.3.265. [DOI] [PubMed] [Google Scholar]
  21. Jornot L., Mirault M. E., Junod A. F. Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide. Am J Respir Cell Mol Biol. 1991 Sep;5(3):265–275. doi: 10.1165/ajrcmb/5.3.265. [DOI] [PubMed] [Google Scholar]
  22. Kang S. S., Wong P. W., Malinow M. R. Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr. 1992;12:279–298. doi: 10.1146/annurev.nu.12.070192.001431. [DOI] [PubMed] [Google Scholar]
  23. Kassis J., Hirsh J., Podor T. J. Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis. Blood. 1992 Oct 1;80(7):1758–1764. [PubMed] [Google Scholar]
  24. Kim A. T., Sarafian T. A., Shau H. Characterization of antioxidant properties of natural killer-enhancing factor-B and induction of its expression by hydrogen peroxide. Toxicol Appl Pharmacol. 1997 Nov;147(1):135–142. doi: 10.1006/taap.1997.8270. [DOI] [PubMed] [Google Scholar]
  25. Kokame K., Kato H., Miyata T. Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/BiP and novel genes. J Biol Chem. 1996 Nov 22;271(47):29659–29665. doi: 10.1074/jbc.271.47.29659. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lautier D., Luscher P., Tyrrell R. M. Endogenous glutathione levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygenase gene. Carcinogenesis. 1992 Feb;13(2):227–232. doi: 10.1093/carcin/13.2.227. [DOI] [PubMed] [Google Scholar]
  28. Lentz S. R., Sadler J. E. Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum. Blood. 1993 Feb 1;81(3):683–689. [PubMed] [Google Scholar]
  29. Lentz S. R., Sadler J. E. Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest. 1991 Dec;88(6):1906–1914. doi: 10.1172/JCI115514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lentz S. R., Sobey C. G., Piegors D. J., Bhopatkar M. Y., Faraci F. M., Malinow M. R., Heistad D. D. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest. 1996 Jul 1;98(1):24–29. doi: 10.1172/JCI118771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  32. Liu H., Bowes R. C., 3rd, van de Water B., Sillence C., Nagelkerke J. F., Stevens J. L. Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J Biol Chem. 1997 Aug 29;272(35):21751–21759. doi: 10.1074/jbc.272.35.21751. [DOI] [PubMed] [Google Scholar]
  33. Loscalzo J. The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest. 1996 Jul 1;98(1):5–7. doi: 10.1172/JCI118776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Loven D. P. A role for reduced oxygen species in heat induced cell killing and the induction of thermotolerance. Med Hypotheses. 1988 May;26(1):39–50. doi: 10.1016/0306-9877(88)90111-9. [DOI] [PubMed] [Google Scholar]
  35. Lu D., Maulik N., Moraru I. I., Kreutzer D. L., Das D. K. Molecular adaptation of vascular endothelial cells to oxidative stress. Am J Physiol. 1993 Mar;264(3 Pt 1):C715–C722. doi: 10.1152/ajpcell.1993.264.3.C715. [DOI] [PubMed] [Google Scholar]
  36. Lüscher T. F., Tanner F. C., Tschudi M. R., Noll G. Endothelial dysfunction in coronary artery disease. Annu Rev Med. 1993;44:395–418. doi: 10.1146/annurev.me.44.020193.002143. [DOI] [PubMed] [Google Scholar]
  37. Mayer E. L., Jacobsen D. W., Robinson K. Homocysteine and coronary atherosclerosis. J Am Coll Cardiol. 1996 Mar 1;27(3):517–527. doi: 10.1016/0735-1097(95)00508-0. [DOI] [PubMed] [Google Scholar]
  38. McCully K. S. Homocysteine and vascular disease. Nat Med. 1996 Apr;2(4):386–389. doi: 10.1038/nm0496-386. [DOI] [PubMed] [Google Scholar]
  39. Morris J. A., Dorner A. J., Edwards C. A., Hendershot L. M., Kaufman R. J. Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins. J Biol Chem. 1997 Feb 14;272(7):4327–4334. doi: 10.1074/jbc.272.7.4327. [DOI] [PubMed] [Google Scholar]
  40. Navab M., Berliner J. A., Watson A. D., Hama S. Y., Territo M. C., Lusis A. J., Shih D. M., Van Lenten B. J., Frank J. S., Demer L. L. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb Vasc Biol. 1996 Jul;16(7):831–842. doi: 10.1161/01.atv.16.7.831. [DOI] [PubMed] [Google Scholar]
  41. Navab M., Fogelman A. M., Berliner J. A., Territo M. C., Demer L. L., Frank J. S., Watson A. D., Edwards P. A., Lusis A. J. Pathogenesis of atherosclerosis. Am J Cardiol. 1995 Sep 28;76(9):18C–23C. doi: 10.1016/s0002-9149(99)80466-4. [DOI] [PubMed] [Google Scholar]
  42. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  43. Pinkus R., Weiner L. M., Daniel V. Role of quinone-mediated generation of hydroxyl radicals in the induction of glutathione S-transferase gene expression. Biochemistry. 1995 Jan 10;34(1):81–88. doi: 10.1021/bi00001a010. [DOI] [PubMed] [Google Scholar]
  44. Rodgers G. M., Conn M. T. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood. 1990 Feb 15;75(4):895–901. [PubMed] [Google Scholar]
  45. Rodgers G. M., Kane W. H. Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J Clin Invest. 1986 Jun;77(6):1909–1916. doi: 10.1172/JCI112519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rolland P. H., Friggi A., Barlatier A., Piquet P., Latrille V., Faye M. M., Guillou J., Charpiot P., Bodard H., Ghiringhelli O. Hyperhomocysteinemia-induced vascular damage in the minipig. Captopril-hydrochlorothiazide combination prevents elastic alterations. Circulation. 1995 Feb 15;91(4):1161–1174. doi: 10.1161/01.cir.91.4.1161. [DOI] [PubMed] [Google Scholar]
  47. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801–809. doi: 10.1038/362801a0. [DOI] [PubMed] [Google Scholar]
  48. Sarge K. D., Murphy S. P., Morimoto R. I. Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol Cell Biol. 1993 Mar;13(3):1392–1407. doi: 10.1128/mcb.13.3.1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Selhub J., Jacques P. F., Bostom A. G., D'Agostino R. B., Wilson P. W., Belanger A. J., O'Leary D. H., Wolf P. A., Schaefer E. J., Rosenberg I. H. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med. 1995 Feb 2;332(5):286–291. doi: 10.1056/NEJM199502023320502. [DOI] [PubMed] [Google Scholar]
  50. Stamler J. S., Osborne J. A., Jaraki O., Rabbani L. E., Mullins M., Singel D., Loscalzo J. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest. 1993 Jan;91(1):308–318. doi: 10.1172/JCI116187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Starkebaum G., Harlan J. M. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest. 1986 Apr;77(4):1370–1376. doi: 10.1172/JCI112442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tsai J. C., Perrella M. A., Yoshizumi M., Hsieh C. M., Haber E., Schlegel R., Lee M. E. Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6369–6373. doi: 10.1073/pnas.91.14.6369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Upchurch G. R., Jr, Welch G. N., Fabian A. J., Freedman J. E., Johnson J. L., Keaney J. F., Jr, Loscalzo J. Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem. 1997 Jul 4;272(27):17012–17017. doi: 10.1074/jbc.272.27.17012. [DOI] [PubMed] [Google Scholar]
  54. Wall R. T., Harlan J. M., Harker L. A., Striker G. E. Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb Res. 1980 Apr 1;18(1-2):113–121. doi: 10.1016/0049-3848(80)90175-9. [DOI] [PubMed] [Google Scholar]
  55. Wang X. Z., Lawson B., Brewer J. W., Zinszner H., Sanjay A., Mi L. J., Boorstein R., Kreibich G., Hendershot L. M., Ron D. Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153). Mol Cell Biol. 1996 Aug;16(8):4273–4280. doi: 10.1128/mcb.16.8.4273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Watanabe M., Osada J., Aratani Y., Kluckman K., Reddick R., Malinow M. R., Maeda N. Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1585–1589. doi: 10.1073/pnas.92.5.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Watowich S. S., Morimoto R. I. Complex regulation of heat shock- and glucose-responsive genes in human cells. Mol Cell Biol. 1988 Jan;8(1):393–405. doi: 10.1128/mcb.8.1.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Whelan S. A., Hightower L. E. Differential induction of glucose-regulated and heat shock proteins: effects of pH and sulfhydryl-reducing agents on chicken embryo cells. J Cell Physiol. 1985 Nov;125(2):251–258. doi: 10.1002/jcp.1041250212. [DOI] [PubMed] [Google Scholar]
  59. Yellon D. M., Latchman D. S. Stress proteins and myocardial protection. J Mol Cell Cardiol. 1992 Feb;24(2):113–124. doi: 10.1016/0022-2828(92)93148-d. [DOI] [PubMed] [Google Scholar]
  60. Zhu W., Roma P., Pellegatta F., Catapano A. L. Oxidized-LDL induce the expression of heat shock protein 70 in human endothelial cells. Biochem Biophys Res Commun. 1994 Apr 15;200(1):389–394. doi: 10.1006/bbrc.1994.1461. [DOI] [PubMed] [Google Scholar]
  61. Zhu W., Roma P., Pirillo A., Pellegatta F., Catapano A. L. Human endothelial cells exposed to oxidized LDL express hsp70 only when proliferating. Arterioscler Thromb Vasc Biol. 1996 Sep;16(9):1104–1111. doi: 10.1161/01.atv.16.9.1104. [DOI] [PubMed] [Google Scholar]
  62. de Groot P. G., Willems C., Boers G. H., Gonsalves M. D., van Aken W. G., van Mourik J. A. Endothelial cell dysfunction in homocystinuria. Eur J Clin Invest. 1983 Oct;13(5):405–410. doi: 10.1111/j.1365-2362.1983.tb00121.x. [DOI] [PubMed] [Google Scholar]

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