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. 1988 Mar 1;106(3):893–904. doi: 10.1083/jcb.106.3.893

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture

PMCID: PMC2115104  PMID: 3279055

Abstract

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.

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Selected References

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  1. Ashburner M., Bonner J. J. The induction of gene activity in drosophilia by heat shock. Cell. 1979 Jun;17(2):241–254. doi: 10.1016/0092-8674(79)90150-8. [DOI] [PubMed] [Google Scholar]
  2. Bensaude O., Babinet C., Morange M., Jacob F. Heat shock proteins, first major products of zygotic gene activity in mouse embryo. Nature. 1983 Sep 22;305(5932):331–333. doi: 10.1038/305331a0. [DOI] [PubMed] [Google Scholar]
  3. Bensaude O., Morange M. Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo. EMBO J. 1983;2(2):173–177. doi: 10.1002/j.1460-2075.1983.tb01401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Booyse F. M., Sedlak B. J., Rafelson M. E., Jr Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Haemorrh. 1975 Dec 15;34(3):825–839. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chappell T. G., Welch W. J., Schlossman D. M., Palter K. B., Schlesinger M. J., Rothman J. E. Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell. 1986 Apr 11;45(1):3–13. doi: 10.1016/0092-8674(86)90532-5. [DOI] [PubMed] [Google Scholar]
  7. Craig E. A. The heat shock response. CRC Crit Rev Biochem. 1985;18(3):239–280. doi: 10.3109/10409238509085135. [DOI] [PubMed] [Google Scholar]
  8. Falkner F. G., Saumweber H., Biessmann H. Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells. J Cell Biol. 1981 Oct;91(1):175–183. doi: 10.1083/jcb.91.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. George J. N., Lyons R. M., Morgan R. K. Membrane changes associated with platelet activation. Exposure of actin on the platelet surface after thrombin-induced secretion. J Clin Invest. 1980 Jul;66(1):1–9. doi: 10.1172/JCI109821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glass J. R., DeWitt R. G., Cress A. E. Rapid loss of stress fibers in Chinese hamster ovary cells after hyperthermia. Cancer Res. 1985 Jan;45(1):258–262. [PubMed] [Google Scholar]
  11. Heacock C. S., Sutherland R. M. Induction characteristics of oxygen regulated proteins. Int J Radiat Oncol Biol Phys. 1986 Aug;12(8):1287–1290. doi: 10.1016/0360-3016(86)90155-0. [DOI] [PubMed] [Google Scholar]
  12. Hickey E., Brandon S. E., Sadis S., Smale G., Weber L. A. Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia. Gene. 1986;43(1-2):147–154. doi: 10.1016/0378-1119(86)90018-1. [DOI] [PubMed] [Google Scholar]
  13. Hightower L. E. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. J Cell Physiol. 1980 Mar;102(3):407–427. doi: 10.1002/jcp.1041020315. [DOI] [PubMed] [Google Scholar]
  14. Jaffe E. A., Ruggiero J. T., Leung L. K., Doyle M. J., McKeown-Longo P. J., Mosher D. F. Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix. Proc Natl Acad Sci U S A. 1983 Feb;80(4):998–1002. doi: 10.1073/pnas.80.4.998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnston D., Oppermann H., Jackson J., Levinson W. Induction of four proteins in chick embryo cells by sodium arsenite. J Biol Chem. 1980 Jul 25;255(14):6975–6980. [PubMed] [Google Scholar]
  16. Kelley P. M., Schlesinger M. J. The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell. 1978 Dec;15(4):1277–1286. doi: 10.1016/0092-8674(78)90053-3. [DOI] [PubMed] [Google Scholar]
  17. Krüger C., Benecke B. J. In vitro translation of Drosophila heat-shock and non--heat-shock mRNAs in heterologous and homologous cell-free systems. Cell. 1981 Feb;23(2):595–603. doi: 10.1016/0092-8674(81)90155-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lahav J., Schwartz M. A., Hynes R. O. Analysis of platelet adhesion with a radioactive chemical crosslinking reagent: interaction of thrombospondin with fibronectin and collagen. Cell. 1982 Nov;31(1):253–262. doi: 10.1016/0092-8674(82)90425-1. [DOI] [PubMed] [Google Scholar]
  20. Lawler J. W., Slayter H. S., Coligan J. E. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J Biol Chem. 1978 Dec 10;253(23):8609–8616. [PubMed] [Google Scholar]
  21. Lawler J. W., Slayter H. S. The release of heparin binding peptides from platelet thrombospondin by proteolytic action of thrombin, plasmin and trypsin. Thromb Res. 1981 May 1;22(3):267–279. doi: 10.1016/0049-3848(81)90119-5. [DOI] [PubMed] [Google Scholar]
  22. Leung L. L., Nachman R. L. Complex formation of platelet thrombospondin with fibrinogen. J Clin Invest. 1982 Sep;70(3):542–549. doi: 10.1172/JCI110646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Levinson W., Oppermann H., Jackson J. Transition series metals and sulfhydryl reagents induce the synthesis of four proteins in eukaryotic cells. Biochim Biophys Acta. 1980;606(1):170–180. doi: 10.1016/0005-2787(80)90108-2. [DOI] [PubMed] [Google Scholar]
  24. Majack R. A., Cook S. C., Bornstein P. Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J Cell Biol. 1985 Sep;101(3):1059–1070. doi: 10.1083/jcb.101.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McKeown-Longo P. J., Hanning R., Mosher D. F. Binding and degradation of platelet thrombospondin by cultured fibroblasts. J Cell Biol. 1984 Jan;98(1):22–28. doi: 10.1083/jcb.98.1.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morange M., Diu A., Bensaude O., Babinet C. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells. Mol Cell Biol. 1984 Apr;4(4):730–735. doi: 10.1128/mcb.4.4.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morimoto R., Fodor E. Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes. J Cell Biol. 1984 Oct;99(4 Pt 1):1316–1323. doi: 10.1083/jcb.99.4.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mumby S. M., Abbott-Brown D., Raugi G. J., Bornstein P. Regulation of thrombospondin secretion by cells in culture. J Cell Physiol. 1984 Sep;120(3):280–288. doi: 10.1002/jcp.1041200304. [DOI] [PubMed] [Google Scholar]
  29. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  30. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  31. Phillips D. R., Jennings L. K., Prasanna H. R. Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets. J Biol Chem. 1980 Dec 25;255(24):11629–11632. [PubMed] [Google Scholar]
  32. Raugi G. J., Mumby S. M., Abbott-Brown D., Bornstein P. Thrombospondin: synthesis and secretion by cells in culture. J Cell Biol. 1982 Oct;95(1):351–354. doi: 10.1083/jcb.95.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scott M. P., Pardue M. L. Translational control in lysates of Drosophila melanogaster cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3353–3357. doi: 10.1073/pnas.78.6.3353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spatz M., Bembry J., Dodson R. F., Hervonen H., Murray M. R. Endothelial cell cultures derived from isolated cerebral microvessels. Brain Res. 1980 Jun 9;191(2):577–582. doi: 10.1016/0006-8993(80)91310-4. [DOI] [PubMed] [Google Scholar]
  35. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  36. Wagner R. C., Matthews M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc Res. 1975 Nov;10(3):286–297. doi: 10.1016/0026-2862(75)90033-3. [DOI] [PubMed] [Google Scholar]
  37. Welch W. J., Feramisco J. R. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem. 1984 Apr 10;259(7):4501–4513. [PubMed] [Google Scholar]
  38. Welch W. J., Suhan J. P. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol. 1985 Oct;101(4):1198–1211. doi: 10.1083/jcb.101.4.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zimmerman J. L., Petri W., Meselson M. Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock. Cell. 1983 Apr;32(4):1161–1170. doi: 10.1016/0092-8674(83)90299-4. [DOI] [PubMed] [Google Scholar]

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