Advertisement

Cell Stress and Chaperones

, Volume 24, Issue 1, pp 273–282 | Cite as

Cell-surface HSP70 associates with thrombomodulin in endothelial cells

  • Thaís L. S. AraujoEmail author
  • Gabriela Venturini
  • Ana I. S. Moretti
  • Leonardo Y. Tanaka
  • Alexandre Costa Pereira
  • Francisco R. M. Laurindo
Original Paper

Abstract

Heat shock protein-70 (HSP70) is crucial for proteostasis and displays cell-protective effects. Meanwhile, enhanced levels of cell-surface (cs) and secreted HSP70 paradoxically associate with pathologic cardiovascular conditions. However, mechanisms regulating csHSP70 pool are unknown. We hypothesized that total and csHSP70 expressions are modulated by hemodynamic forces, major contributors to endothelial pathophysiology. We also investigated whether thrombomodulin, a crucial thromboresistance cell-surface protein, is a csHSP70 target. We used proteomic/western analysis, confocal microscopy, and cs-biotinylation to analyze the pattern and specific characteristics of intracellular and csHSP70. HSP70 interaction with thrombomodulin was investigated by confocal colocalization, en face immunofluorescence, proximity assay, and immunoprecipitation. Thrombomodulin activity was assessed by measured protein C activation two-step assay. Our results show that csHSP70 pool in endothelial cells (EC) exhibits a peculiar cluster-like pattern and undergoes enhanced expression by physiological arterial-level laminar shear stress. Conversely, total and csHSP70 expressions were diminished under low shear stress, a known proatherogenic hemodynamic pattern. Furthermore, total HSP70 levels were decreased in aortic arch (associated with proatherogenic turbulent flow) compared with thoracic aorta (associated with atheroprotective laminar flow). Importantly, csHSP70 co-localized with thrombomodulin in cultured EC and aorta endothelium; proximity ligation assays and immunoprecipitation confirmed their physical interaction in EC. Remarkably, immunoneutralization of csHSP70 enhanced thrombomodulin activity in EC and aorta ex vivo. Overall, proatherogenic hemodynamic forces promote reduced total HSP70 expression, which might implicate in disturbed proteostasis; meanwhile, the associated decrease in cs-HSP70 pool associates with thromboresistance signaling. Cell-surface HSP70 (csHSP70) expression regulation and csHSP70 targets in vascular cells are unknown. We showed that HSP70 levels are shear stress-modulated and decreased under proatherogenic conditions. Remarkably, csHSP70 binds thrombomodulin and inhibits its activity in endothelial cells. This mechanism can potentially explain some deleterious effects previously associated with high extracellular HSP70 levels, as csHSP70 potentially could restrict thromboresistance and support thrombosis/inflammation in stress situations.

Keywords

Endothelial cells Atherosclerosis HSP70 Thrombomodulin Cell stress Shear stress 

Abbreviations

HSP70

70 kDa heat shock protein family

HSC70

Constitutive HSP70

HSP701A/1B

Stress-inducible HSP70

Cs

Cell surface

TM

Thrombomodulin

PLA

Proximity ligation assay

PCa

Protein C activated

VEGF

Vascular endothelial growth factor

LSS

Low laminar shear stress

EC

Endothelial cells

PDI, PDIA1

Protein disulfide isomerase A1

Notes

Acknowledgements

We thank Ana L. Garippo and Laura Ventura for technical support. We are grateful to Prof. Marcelo L. Santoro, from Instituto Butantan, for advice and reagents.

Author contributions

T.L.S.A. conceived the project, designed and performed most experiments, analyzed data, and wrote the manuscript; G.V. and A.C.P. designed, performed, and discussed shear stress-associated proteomic analysis; L.Y.T. performed en face immunofluorescence experiments; A.I.S.M. performed DUOLINK and immunoprecipitation experiments; and F.R.M.L. conceived the project, analysis results, discussed experiments, and wrote the manuscript.

Funding information

Work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (Process number 15/06210-2), Centro de Pesquisa, Inovação e Difusão FAPESP (CEPID “Processos Redox em Biomedicina—Redoxoma,” Grant 13/07937-8), and Fundação Zerbini.

Compliance with ethical standards

Animal studies were performed in male C57BL/6 6-week old following approval from to Ethics Committee of the Heart Institute and School of Medicine from University of São Paulo, Brazil.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Allende M, Molina E, Guruceaga E, Tamayo I, González-Porras JR, Gonzalez-López TJ, Toledo E, Rabal O, Ugarte A, Roldán V, Rivera J, Oyarzabal J, Montes R, Hermida J (2016) Hsp70 protects from stroke in atrial fibrillation patients by preventing thrombosis without increased bleeding risk. Cardiovasc Res 110(3):309–318.  https://doi.org/10.1093/cvr/cvw049 Google Scholar
  2. Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT (2011) Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost 9(9):1795–1803.  https://doi.org/10.1111/j.1538-7836.2011.04422.x Google Scholar
  3. Araujo TL, Zeidler JD, Oliveira PV, Dias MH, Armelin HA, Laurindo FR (2016) Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes. Free Radic Biol Med 103:199–208.  https://doi.org/10.1016/j.freeradbiomed.2016.12.021 Google Scholar
  4. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK (2000) HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 6(4):435–442.  https://doi.org/10.1038/74697 Google Scholar
  5. Bobkova NV, Evgen’ev M, Garbuz DG, Kulikov AM, Morozov A, Samokhin A et al (2015) Exogenous Hsp70 delays senescence and improves cognitive function in aging mice. Proc Natl Acad Sci U S A 112(52):16006–16011.  https://doi.org/10.1073/pnas.1516131112 Google Scholar
  6. Borges JC, Ramos CH (2005) Protein folding assisted by chaperones. Protein Pept Lett 12(3):257–261Google Scholar
  7. Calderwood SK, Mambula SS, Gray PJ, Theriault JR (2007) Extracellular heat shock proteins in cell signaling. FEBS Lett 581(19):3689–3694.  https://doi.org/10.1016/j.febslet.2007.04.044 Google Scholar
  8. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372.  https://doi.org/10.1038/nbt.1511 Google Scholar
  9. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10(4):1794–1805.  https://doi.org/10.1021/pr101065j Google Scholar
  10. De Maio A, Santoro MG, Tanguay RM, Hightower LE (2012) Ferruccio Ritossa’s scientific legacy 50 years after his discovery of the heat shock response: a new view of biology, a new society, and a new journal. Cell Stress Chaperones 17(2):139–143.  https://doi.org/10.1007/s12192-012-0320-z Google Scholar
  11. Essex DW, Wu Y (2018) Multiple protein disulfide isomerases support thrombosis. Curr Opin Hematol 25(5):395–402.  https://doi.org/10.1097/MOH.0000000000000449. Google Scholar
  12. Fernandez-Funez P, Sanchez-Garcia J, de Mena L, Zhang Y, Levites Y, Khare S, Golde TE, Rincon-Limas DE (2016) Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila. Proc Natl Acad Sci U S A 113(35):E5212–E5221.  https://doi.org/10.1073/pnas.1608045113 Google Scholar
  13. Flaumenhaft R, Furie B (2016) Vascular thiol isomerases. Blood 128(7):893–901.  https://doi.org/10.1182/blood-2016-04-636456 Google Scholar
  14. Fong JJ, Sreedhara K, Deng L, Varki NM, Angata T, Liu Q, Nizet V, Varki A (2015) Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec-5 and Siglec-14. EMBO J 34(22):2775–2788.  https://doi.org/10.15252/embj.201591407 Google Scholar
  15. Gutiérrez M, Isa P, Sánchez-San Martin C, Pérez-Vargas J, Espinosa R, Arias CF et al (2010) Different rotavirus strains enter MA104 cells through different endocytic pathways: the role of clathrin-mediated endocytosis. J Virol 84(18):9161–9169.  https://doi.org/10.1128/JVI.00731-10 Google Scholar
  16. Henderson B, Pockley AG (2012) Proteotoxic stress and circulating cell stress proteins in the cardiovascular diseases. Cell Stress Chaperones 17(3):303–311.  https://doi.org/10.1007/s12192-011-0318-y Google Scholar
  17. Jang J, Kim MR, Kim TK, Lee WR, Kim JH, Heo K, Lee S (2017) CLEC14a-HSP70-1A interaction regulates HSP70-1A-induced angiogenesis. Sci Rep 7(1):10666.  https://doi.org/10.1038/s41598-017-11118-y. Google Scholar
  18. Jenei ZM, Gombos T, Förhécz Z, Pozsonyi Z, Karádi I, Jánoskuti L, Prohászka Z (2013) Elevated extracellular HSP70 (HSPA1A) level as an independent prognostic marker of mortality in patients with heart failure. Cell Stress Chaperones 18(6):809–813.  https://doi.org/10.1007/s12192-013-0425-z Google Scholar
  19. Kim TK, Na HJ, Lee WR, Jeoung MH, Lee S (2016) Heat shock protein 70-1A is a novel angiogenic regulator. Biochem Biophys Res Commun 469(2):222–228.  https://doi.org/10.1016/j.bbrc.2015.11.125 Google Scholar
  20. Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem de Bittencourt PI (2015) The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediat Inflamm 2015:249205.  https://doi.org/10.1155/2015/249205 Google Scholar
  21. Krepuska M, Szeberin Z, Sótonyi P, Sarkadi H, Fehérvári M, Apor A, Rimely E, Prohászka Z, Acsády G (2011) Serum level of soluble Hsp70 is associated with vascular calcification. Cell Stress Chaperones 16(3):257–265.  https://doi.org/10.1007/s12192-010-0237-3 Google Scholar
  22. Leng X, Wang X, Pang W, Zhan R, Zhang Z, Wang L, Gao X, Qian L (2013) Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis. Cell Stress Chaperones 18(4):483–493.  https://doi.org/10.1007/s12192-013-0404-4 Google Scholar
  23. Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7(12):952–958.  https://doi.org/10.1038/nrm2067 Google Scholar
  24. Martin FA, Murphy RP, Cummins PM (2013) Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects. Am J Physiol Heart Circ Physiol 304(12):H1585–H1597.  https://doi.org/10.1152/ajpheart.00096.2013 Google Scholar
  25. Mayer MP (2018) Intra-molecular pathways of allosteric control in Hsp70s. Philos Trans R Soc Lond Ser B Biol Sci 373(1749):20170183.  https://doi.org/10.1098/rstb.2017.0183 Google Scholar
  26. Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62(6):670–684.  https://doi.org/10.1007/s00018-004-4464-6 Google Scholar
  27. Moraes MS, Costa PE, Batista WL, Paschoalin T, Curcio MF, Borges RE, Taha MO, Fonseca FV, Stern A, Monteiro HP (2014) Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis. Arch Biochem Biophys 558:14–27.  https://doi.org/10.1016/j.abb.2014.06.011 Google Scholar
  28. Murshid A, Theriault J, Gong J, Calderwood SK (2011) Investigating receptors for extracellular heat shock proteins. Methods Mol Biol 787:289–302.  https://doi.org/10.1007/978-1-61779-295-3_22 Google Scholar
  29. Nigro P, Abe J, Berk BC (2011) Flow shear stress and atherosclerosis: a matter of site specificity. Antioxid Redox Signal 15(5):1405–1414.  https://doi.org/10.1089/ars.2010.3679 Google Scholar
  30. Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1(6):2650–2660.  https://doi.org/10.1038/nprot.2006.427. Google Scholar
  31. Pockley AG (2002) Heat shock proteins, inflammation, and cardiovascular disease. Circulation 105(8):1012–1017Google Scholar
  32. Pockley AG, Shepherd J, Corton JM (1998) Detection of heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol Investig 27(6):367–377Google Scholar
  33. Powers ET, Balch WE (2013) Diversity in the origins of proteostasis networks--a driver for protein function in evolution. Nat Rev Mol Cell Biol 14(4):237–248.  https://doi.org/10.1038/nrm3542 Google Scholar
  34. Radons J (2016) The human HSP70 family of chaperones: where do we stand? Cell Stress Chaperones 21(3):379–404.  https://doi.org/10.1007/s12192-016-0676-6 Google Scholar
  35. Schlecht R, Erbse AH, Bukau B, Mayer MP (2011) Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat Struct Mol Biol 18(3):345–351.  https://doi.org/10.1038/nsmb.2006 Google Scholar
  36. Shiota M, Kusakabe H, Izumi Y, Hikita Y, Nakao T, Funae Y, Miura K, Iwao H (2010) Heat shock cognate protein 70 is essential for Akt signaling in endothelial function. Arterioscler Thromb Vasc Biol 30(3):491–497.  https://doi.org/10.1161/ATVBAHA.109.193631 Google Scholar
  37. Shrestha L, Patel HJ, Chiosis G (2016) Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease. Cell Chem Biol 23(1):158–172.  https://doi.org/10.1016/j.chembiol.2015.12.006 Google Scholar
  38. Smith CL, Shah CM, Kamaludin N, Gordge MP (2015) Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C. Thromb Res 135(4):748–753.  https://doi.org/10.1016/j.thromres.2015.01.034 Google Scholar
  39. Soares Moretti AI, Martins Laurindo FR (2016) Protein disulfide isomerases: redox connections in and out of the endoplasmic reticulum. Arch Biochem Biophys 617:106–119.  https://doi.org/10.1016/j.abb.2016.11.007 Google Scholar
  40. Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3(12):995–1000.  https://doi.org/10.1038/nmeth947 Google Scholar
  41. Sperry JL, Deming CB, Bian C, Walinsky PL, Kass DA, Kolodgie FD, Virmani R, Kim AY, Rade JJ (2003) Wall tension is a potent negative regulator of in vivo thrombomodulin expression. Circ Res 92(1):41–47Google Scholar
  42. Takemoto H, Yoshimori T, Yamamoto A, Miyata Y, Yahara I, Inoue K, Tashiro Y (1992) Heavy chain binding protein (BiP/GRP78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase. Arch Biochem Biophys 296(1):129–136Google Scholar
  43. Tanaka LY, Araújo HA, Hironaka GK, Araujo TL, Takimura CK, Rodriguez AI et al (2016) Peri/epicellular protein disulfide isomerase sustains vascular lumen caliber through an anticonstrictive remodeling effect. Hypertension 67(3):613–622.  https://doi.org/10.1161/HYPERTENSIONAHA.115.06177 Google Scholar
  44. Thériault JR, Mambula SS, Sawamura T, Stevenson MA, Calderwood SK (2005) Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cells. FEBS Lett 579(9):1951–1960.  https://doi.org/10.1016/j.febslet.2005.02.046 Google Scholar
  45. Uchiyama T, Atsuta H, Utsugi T, Oguri M, Hasegawa A, Nakamura T, Nakai A, Nakata M, Maruyama I, Tomura H, Okajima F, Tomono S, Kawazu S, Nagai R, Kurabayashi M (2007) HSF1 and constitutively active HSF1 improve vascular endothelial function (heat shock proteins improve vascular endothelial function). Atherosclerosis 190(2):321–329.  https://doi.org/10.1016/j.atherosclerosis.2006.03.037 Google Scholar
  46. Vanhoutte PM, Zhao Y, Xu A, Leung SW (2016) Thirty years of saying NO: sources, fate, actions, and misfortunes of the endothelium-derived vasodilator mediator. Circ Res 119(2):375–396.  https://doi.org/10.1161/CIRCRESAHA.116.306531 Google Scholar
  47. Wang XL, Fu A, Raghavakaimal S, Lee HC (2007) Proteomic analysis of vascular endothelial cells in response to laminar shear stress. Proteomics 7(4):588–596.  https://doi.org/10.1002/pmic.200600568 Google Scholar
  48. White SJ, Hayes EM, Lehoux S, Jeremy JY, Horrevoets AJ, Newby AC (2011) Characterization of the differential response of endothelial cells exposed to normal and elevated laminar shear stress. J Cell Physiol 226(11):2841–2848.  https://doi.org/10.1002/jcp.22629 Google Scholar
  49. Wiest DL, Bhandoola A, Punt J, Kreibich G, McKean D, Singer A (1997) Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones. Proc Natl Acad Sci U S A 94(5):1884–1889Google Scholar
  50. Willems SH, Tape CJ, Stanley PL, Taylor NA, Mills IG, Neal DE, McCafferty J, Murphy G (2010) Thiol isomerases negatively regulate the cellular shedding activity of ADAM17. Biochem J 428(3):439–450.  https://doi.org/10.1042/BJ20100179 Google Scholar
  51. Xu Q (2002) Role of heat shock proteins in atherosclerosis. Arterioscler Thromb Vasc Biol 22(10):1547–1559Google Scholar
  52. Yurdagul A, Finney AC, Woolard MD, Orr AW (2016) The arterial microenvironment: the where and why of atherosclerosis. Biochem J 473(10):1281–1295.  https://doi.org/10.1042/BJ20150844 Google Scholar
  53. Zhang X, Xu Z, Zhou L, Chen Y, He M, Cheng L, Hu FB, Tanguay RM, Wu T (2010) Plasma levels of Hsp70 and anti-Hsp70 antibody predict risk of acute coronary syndrome. Cell Stress Chaperones 15(5):675–686.  https://doi.org/10.1007/s12192-010-0180-3 Google Scholar
  54. Zhang X, Tanguay RM, He M, Deng Q, Miao X, Zhou L et al (2011) Variants of HSPA1A in combination with plasma Hsp70 and anti-Hsp70 antibody levels associated with higher risk of acute coronary syndrome. Cardiology 119(1):57–64.  https://doi.org/10.1159/000329917. Google Scholar
  55. Zhu J, Quyyumi AA, Wu H, Csako G, Rott D, Zalles-Ganley A, Ogunmakinwa J, Halcox J, Epstein SE (2003) Increased serum levels of heat shock protein 70 are associated with low risk of coronary artery disease. Arterioscler Thromb Vasc Biol 23(6):1055–1059.  https://doi.org/10.1161/01.ATV.0000074899.60898.FD Google Scholar

Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  1. 1.Vascular Biology Laboratory, Heart Institute (InCor)University of São Paulo School of MedicineSão PauloBrazil
  2. 2.Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor)University of São Paulo School of MedicineSão PauloBrazil

Personalised recommendations