eNOS-NO-induced small blood vessel relaxation requires EHD2-dependent caveolae stabilization

Autoři: Claudia Matthaeus aff001;  Xiaoming Lian aff002;  Séverine Kunz aff003;  Martin Lehmann aff004;  Cheng Zhong aff002;  Carola Bernert aff001;  Ines Lahmann aff005;  Dominik N. Müller aff006;  Maik Gollasch aff002;  Oliver Daumke aff001
Působiště autorů: Crystallography, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany aff001;  Charité—Universitätsmedizin Berlin, Experimental and Clinical Research Center (ECRC), Campus Buch, Berlin, Germany aff002;  Electron Microscopy Core Facility, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany aff003;  Department of Molecular Pharmacology & Cell Biology and Imaging Core Facility, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany aff004;  Signal Transduction/Developmental Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany aff005;  Experimental & Clinical Research Center, a cooperation between Charité Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine, Berlin, Germany aff006;  Charité—Universitätsmedizin Berlin, Medical Clinic for Nephrology and Internal Intensive Care, Campus Virchow, Berlin, Germany aff007;  Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany aff008
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223620


Endothelial nitric oxide synthase (eNOS)-related vessel relaxation is a highly coordinated process that regulates blood flow and pressure and is dependent on caveolae. Here, we investigated the role of caveolar plasma membrane stabilization by the dynamin-related ATPase EHD2 on eNOS-nitric oxide (NO)-dependent vessel relaxation. Loss of EHD2 in small arteries led to increased numbers of caveolae that were detached from the plasma membrane. Concomitantly, impaired relaxation of mesenteric arteries and reduced running wheel activity were observed in EHD2 knockout mice. EHD2 deletion or knockdown led to decreased production of nitric oxide (NO) although eNOS expression levels were not changed. Super-resolution imaging revealed that eNOS was redistributed from the plasma membrane to internalized detached caveolae in EHD2-lacking tissue or cells. Following an ATP stimulus, reduced cytosolic Ca2+ peaks were recorded in human umbilical vein endothelial cells (HUVECs) lacking EHD2. Our data suggest that EHD2-controlled caveolar dynamics orchestrates the activity and regulation of eNOS/NO and Ca2+ channel localization at the plasma membrane.

Klíčová slova:

Arteries – Cell membranes – Cell staining – Membrane proteins – Mouse models – Small interfering RNAs – Coated pits – Mesenteric arteries


1. Sinha B, Köster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell. 2011;144: 402–413. doi: 10.1016/j.cell.2010.12.031 21295700

2. Cheng JPX, Nichols BJ. Caveolae: One Function or Many? Trends Cell Biol. 2016;26: 177–189. doi: 10.1016/j.tcb.2015.10.010 26653791

3. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol. 2007;8: 185–94. doi: 10.1038/nrm2122 17318224

4. Cheng JPX, Mendoza-Topaz C, Howard G, Chadwick J, Shvets E, Cowburn AS, et al. Caveolae protect endothelial cells from membrane rupture during increased cardiac output. J Cell Biol. 2015;211: 53–61. doi: 10.1083/jcb.201504042 26459598

5. Razani B, Combs TP, Wang XB, Frank PG, Park DS, Russell RG, et al. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. J Biol Chem. 2002;277: 8635–8647. doi: 10.1074/jbc.M110970200 11739396

6. Pohl J, Ring A, Ehehalt R, Schulze-Bergkamen H, Schad A, Verkade P, et al. Long-Chain Fatty Acid Uptake into Adipocytes Depends on Lipid Raft Function. Biochemistry. 2004;43: 4179–4187. doi: 10.1021/bi035743m 15065861

7. Ring A, Le Lay S, Pohl J, Verkade P, Stremmel W. Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts. Biochim Biophys Acta—Mol Cell Biol Lipids. 2006;1761: 416–423. doi: 10.1016/j.bbalip.2006.03.016 16702023

8. Liu L, Brown D, McKee M, LeBrasseur NK, Yang D, Albrecht KH, et al. Deletion of Cavin/PTRF Causes Global Loss of Caveolae, Dyslipidemia, and Glucose Intolerance. Cell Metab. 2008;8: 310–317. doi: 10.1016/j.cmet.2008.07.008 18840361

9. Stoeber M, Stoeck IK, Hänni C, Bleck CKE, Balistreri G, Helenius A. Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin. EMBO J. 2012;31: 2350–2364. doi: 10.1038/emboj.2012.98 22505029

10. Le PU. Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J Cell Sci. 2003;116: 1059–1071. doi: 10.1242/jcs.00327 12584249

11. Pelkmans L, Bürli T, Zerial M, Helenius A. Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell. 2004;118: 767–780. doi: 10.1016/j.cell.2004.09.003 15369675

12. Rath G, Dessy C, Feron O. Caveolae, caveolin and control of vascular tone: nitric oxide (NO) and endothelium derived hyperpolarizing factor (EDHF) regulation. J Physiol Pharmacol. 2009;60 Suppl 4: 105–109.

13. Kraehling JR, Hao Z, Lee MY, Vinyard DJ, Velazquez H, Liu X, et al. Uncoupling Caveolae from Intracellular Signaling in Vivo. Circ Res. 2016;118: 48–55. doi: 10.1161/CIRCRESAHA.115.307767 26602865

14. Tran J, Magenau A, Rodriguez M, Rentero C, Royo T, Enrich C, et al. Activation of endothelial nitric oxide (eNOS) occurs through different membrane domains in endothelial cells. PLoS One. 2016;11: 1–20. doi: 10.1371/journal.pone.0151556 26977592

15. Pani B, Singh BB. Lipid rafts/caveolae as microdomains of calcium signaling. Cell Calcium. 2009;45: 625–633. doi: 10.1016/j.ceca.2009.02.009 19324409

16. Sowa G., Caveolae caveolins, cavins, and endothelial cell function: New insights. Front Physiol. 2012;2 JAN: 1–13. doi: 10.3389/fphys.2011.00120 22232608

17. Lockwich TP, Liu X, Singh BB, Jadlowiec J, Weiland S, Ambudkar IS. Assembly of Trp1 in a Signaling Complex Associated with Caveolin-Scaffolding Lipid Raft Domains *. J Bioenerg Biomembr. 2000;275: 11934–11942.

18. Murata T, Lin MI, Stan R V., Bauer PM, Yu J, Sessa WC. Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells. J Biol Chem. 2007;282: 16631–16643. doi: 10.1074/jbc.M607948200 17416589

19. Brazer SW, Singh BB, Liu X, Swaim W, Ambudkar IS. Caveolin-1 Contributes to Assembly of Store-operated Ca2+ Influx Channels by Regulating Plasma Membrane Localization of TRPC1 *. J Biol Chem. 2003;278: 27208–27215. doi: 10.1074/jbc.M301118200 12732636

20. Isshiki M, Ando J, Korenaga R, Kogo H, Fujimoto T, Fujita T, et al. Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges. Proc Natl Acad Sci USA. 1998;95: 5009–5014. doi: 10.1073/pnas.95.9.5009 9560219

21. Parton RG, Del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol. 2013;14: 98–112. doi: 10.1038/nrm3512 23340574

22. Parton RG, Hanzal-Bayer M, Hancock JF. Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci. 2006;119: 787–796. doi: 10.1242/jcs.02853 16495479

23. Parton RG, Tillu VA, Collins BM. Caveolae. Curr Biol. 2018;28: R402–R405. doi: 10.1016/j.cub.2017.11.075 29689223

24. Senju Y, Itoh Y, Takano K, Hamada S, Suetsugu S. Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting. J Cell Sci. 2011;124: 2032–2040. doi: 10.1242/jcs.086264 21610094

25. Hansen CG, Howard G, Nichols BJ. Pacsin 2 is recruited to caveolae and functions in caveolar biogenesis. J Cell Sci. 2011;124: 2777–2785. doi: 10.1242/jcs.084319 21807942

26. Senju Y, Rosenbaum E, Shah C, Hamada-Nakahara S, Itoh Y, Yamamoto K, et al. Phosphorylation of PACSIN2 by protein kinase C triggers the removal of caveolae from the plasma membrane. J Cell Sci. 2015;128: 2766–2780. doi: 10.1242/jcs.167775 26092940

27. Seemann E, Sun M, Krueger S, Tröger J, Hou W, Haag N, et al. Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination. Elife. 2017;6: 1–37. doi: 10.7554/eLife.29854 29202928

28. Ludwig A, Howard G, Mendoza-Topaz C, Deerinck T, Mackey M, Sandin S, et al. Molecular Composition and Ultrastructure of the Caveolar Coat Complex. PLoS Biol. 2013;11. doi: 10.1371/journal.pbio.1001640 24013648

29. Morén B, Shah C, Howes MT, Schieber NL, McMahon HT, Parton RG, et al. EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization. Mol Biol Cell. 2012;23: 1316–29. doi: 10.1091/mbc.E11-09-0787 22323287

30. Simone LC, Naslavsky N, Caplan S. Scratching the surface: Actin’ and other roles for the C-T terminal Eps15 homology domain protein, EHD2. Histol Histopathol. 2014;29: 285–292. doi: 10.14670/HH-29.285 24347515

31. Daumke O, Lundmark R, Vallis Y, Martens S, Butler PJG, McMahon HT. Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling. Nature. 2007;449: 923–7. doi: 10.1038/nature06173 17914359

32. Shah C, Hegde BG, Morén B, Behrmann E, Mielke T, Moenke G, et al. Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2. Structure. 2014;22: 409–420. doi: 10.1016/j.str.2013.12.015 24508342

33. Matthaeus C, Lahmann I, Kunz S, Jonas W, Melo A, Lehmann M, et al. EHD2‐mediated restriction of caveolar dynamics regulates cellular lipid uptake 2. bioRxiv. 2019; 1–46. http://dx.doi.org/10.1101/511709.

34. Melo AA, Hegde BG, Shah C, Larsson E, Isas JM, Kunz S, et al. Structural insights into the activation mechanism of dynamin-like EHD ATPases. Proc Natl Acad Sci. 2017;114: 5629–5634. doi: 10.1073/pnas.1614075114 28228524

35. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33: 829–837. doi: 10.1093/eurheartj/ehr304 21890489

36. Sessa WC. eNOS at a glance. J Cell Sci. 2004;117: 2427–2429. doi: 10.1242/jcs.01165 15159447

37. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Anderson RGW, et al. Acylation Targets Endothelial Nitric-oxide Synthase to Plasmalemmal Caveolae *. 1996;271: 6518–6522.

38. Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, et al. UltraRapid Communication Estrogen Receptor ␣ and Endothelial Nitric Oxide Synthase Are Organized Into a Functional Signaling Module in Caveolae. Circ Res. 2000;87: 44–52.

39. Ju H, Zou R, Venema VJ, Venema RC. Direct Interaction of Endothelial Nitric-oxide Synthase and Caveolin-1 Inhibits Synthase Activity. J Biol Chem. 1997;272: 18522–18525. doi: 10.1074/jbc.272.30.18522 9228013

40. Gratton JP, Fontana J, O’Connor DS, García-Cardeña G, McCabe TJ, Sessa WC. Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro: Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1. J Biol Chem. 2000;275: 22268–22272. doi: 10.1074/jbc.M001644200 10781589

41. Rafikov R, Fonseca F V, Kumar S, Pardo D, Darragh C, Elms S, et al. eNOS activation and NO function: Structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity. J Endocrinol. 2011;210: 271–284. doi: 10.1530/JOE-11-0083 21642378

42. Miller SG, Kennedy MB. Regulation of Brain Type II Ca2+/Calmodulin- Dependent Protein Kinase by Autophosphorylation: A Ca2+-Triggered Molecular Switch. Cell. 1986;44: 861–870. doi: 10.1016/0092-8674(86)90008-5 3006921

43. Lou LL, Lloyd SJ, Schulman H. Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci USA. 1986;83: 9497–9501. doi: 10.1073/pnas.83.24.9497 3467320

44. Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res. 2002;43: 532–541. doi: 10.1016/s0008-6363(99)00094-2

45. Hercule HC, Schunck WH, Gross V, Seringer J, Leung FP, Weldon SM, et al. Interaction between P450 eicosanoids and nitric oxide in the control of arterial tone in mice. Arterioscler Thromb Vasc Biol. 2009;29: 54–60. doi: 10.1161/ATVBAHA.108.171298 18927469

46. Majed BH, Khalil RA. Molecular mechanisms regulating the vascular prostacyclin pathways and their adaptation during pregnancy and in the newborn. Pharmacol Rev. 2012;64: 540–582. doi: 10.1124/pr.111.004770 22679221

47. Holton M, Mohamed TMA, Oceandy D, Wang W, Lamas S, Emerson M, et al. Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells. 2010; 440–448. doi: 10.1093/cvr/cvq077 20211863

48. Mota MM, Mesquita TRR, Da Silva TLTB, Fontes MT, Lauton Santos S, Dos Santos Aggum Capettini L, et al. Endothelium adjustments to acute resistance exercise are intensity-dependent in healthy animals. Life Sci. 2015;142: 86–91. doi: 10.1016/j.lfs.2015.10.007 26455551

49. Wilkinson DD. In situ Hybridization. A Practical Approach. Genet Res. 1993;61: 234. doi: 10.1017/S0016672300031402

50. Carvajal J a, Germain a M, Huidobro-Toro JP, Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol. 2000;184: 409–420. doi: 10.1002/1097-4652(200009)184:3<409::AID-JCP16>3.0.CO;2-K 10911373

51. Lin S, Fagan KA, Li K, Shaul PW, Cooper DMF, Rodman DM. Sustained Endothelial Nitric-oxide Synthase Activation Requires Capacitative Ca 2 ؉ Entry *. J Biol Chem. 2000;275: 17979–17985. doi: 10.1074/jbc.275.24.17979 10849433

52. Nilius B, Droogmans GUY. Ion Channels and Their Functional Role in Vascular Endothelium. Physiol Rev. 2001;81: 1416–1459.

53. Gifford SM, Grummer MA, Pierre SA, Austin JL, Zheng J, Bird IM. Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production. J Endocrinol. 2004;182: 485–499. doi: 10.1677/joe.0.1820485 15350190

54. Mount PF, Kemp BE, Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. J Mol Cell Cardiol. 2007;42: 271–279. doi: 10.1016/j.yjmcc.2006.05.023 16839566

55. Naidoo A, Naidoo K, Yende-zuma N, Gengiah TN. Endothelial Caveolar Subcellular Domain Regulation of Endothelial Nitric Oxide Synthase. Clin Exp Pharmacol Physiol. 2013;40: 753–764. doi: 10.1111/1440-1681.12136 23745825

56. Goligorsky M, Li H, Brodsky S, Chen J. Relationships between caveolae and eNOS: everything in proximity and the proximity of everything. Am J Physiol Ren Physiol. 2002;283: F1–F10. doi: 10.1002/mus.10181

57. Sowa G, Pypaert M, Sessa WC. Distinction between signaling mechanisms in lipid rafts vs. caveolae. 2001;

58. Heijnen HFG, Waaijenborg S, Crapo JD, Bowler RP, Akkerman JN, Slot JW. Colocalization of eNOS and the Catalytic Subunit of PKA in Endothelial Cell Junctions: A Clue for Regulated NO Production The Journal of Histochemistry & Cytochemistry. J Histochem Cytochem. 2004;52: 1277–1285. doi: 10.1177/002215540405201004 15385574

59. Reiner M, Bloch W, Addicks K. Functional interaction of caveolin-1 and eNOS in myocardial capillary endothelium revealed by immunoelectron microscopy. J Histochem Cytochem. 2001;49: 1605–1609. doi: 10.1177/002215540104901214 11724908

60. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, et al. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science (80-). 2001;293: 2449–2452. doi: 10.1126/science.1062688 11498544

61. Tran Q, Ohashi K, Watanabe H. Calcium signalling in endothelial cells. 2000;48: 13–22. doi: 10.1016/s0008-6363(00)00172-3 11033104

62. Calles-Escandon J, Cipolla M. Diabetes and endothelial dysfunction: A clinical perspective. Endocr Rev. 2001;22: 36–52. doi: 10.1210/edrv.22.1.0417 11159815

63. Tabit C, Chung W, Hamburg N, Vita J. Endothelial dysfunction in diabetes mellitus: Molecular mechanisms and clinical implications. Rev Endocr Metab Disord. 2010;11: 61–74. doi: 10.1007/s11154-010-9134-4 20186491

64. Caballero AE. Endothelial dysfunction in obesity and insulin resistance: A road to diabetes and heart disease. Obes Res. 2003;11: 1278–1289. doi: 10.1038/oby.2003.174 14627747

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