Molecular and Cellular Biochemistry

, Volume 393, Issue 1–2, pp 199–207 | Cite as

ZNF580 mediates eNOS expression and endothelial cell migration/proliferation via the TGF-β1/ALK5/Smad2 pathway

  • Yuyu Luo
  • Ying Zhao
  • Xiaodong Li
  • Juan Zhao
  • Wencheng Zhang


ZNF580 is a novel C2H2 zinc-finger nuclear transcription factor with potential involvement in the transforming growth factor-β1 (TGF-β1) signal transduction pathway. Emerging evidence suggests that TGF-β1 can regulate endothelial nitric oxide synthase (eNOS) expression in endothelial cells. This study aimed to determine if ZNF580 mediated eNOS expression and participated in endothelial cell migration and proliferation via the TGF-β1/Smad2/ZNF580/eNOS signaling pathway. Overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of eNOS expression. TGF-β1 downregulated both ZNF580 and eNOS at the mRNA and protein levels in concentration- and time-dependent manners. ZNF580 and eNOS downregulation induced by TGF-β1 was blocked by the specific TGF-β1 type I receptor ALK5 inhibitor, SB431542. Overexpression of ZNF580 attenuated TGF-β1-induced inhibition of EAhy926 cell growth and mobility, and vice versa. These results suggest that ZNF580 mediates eNOS expression and endothelial cell migration/proliferation via the TGF-β1/ALK5/Smad2 pathway, and thus plays a crucial role in vascular endothelial cells.


ZNF580 Endothelial nitric oxide synthase Transforming growth factor-β1 Endothelial cell 



Transforming growth factor-β


Fetal bovine serum


Glyceraldehyde-3-phosphate dehydrogenase


RNA interference


Green fluorescent protein


Endothelial nitric oxide synthase


Polymerase chain reaction





We are grateful to Dr. Thomas Michel (Brigham and Women’s Hospital, Boston, MA, USA) for the generous gifts of the plasmids pGL3-basic and human pGL3-eNOS-Luc. This project was supported by the National Natural Science Foundation of China Grants (No. 81170106, No. 81200223) and Science Foundation of Logistics University CPAF (No. WHB201207).

Conflict of interest

No competing financial interests exist.


  1. 1.
    Zhang WC, Chen BS, Zeng WW, Wu G (2003) Cloning and tissue expression of a novel gene down-regulated by low density lipoprotein. Basic Med Sci Clin 23:279–282Google Scholar
  2. 2.
    Zhang WC, Chen BS, Zeng WW, Wu G, Xue H (2001) Cloning and structural analysis of a novel gene down-regulated by low density lipoprotein. Nat Med J China 81:435–436Google Scholar
  3. 3.
    Urrutia R (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol 4:231PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Iuchi S (2001) Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58:625–635PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang WC, Sun HY, Luo YY, Cui ML (2008) Construction of eukaryotic expression vector for ZNF580 and EGFP fusion protein and its expression and localization in HEK293 cells. Acta Acad Med CPAF 17:161–165Google Scholar
  6. 6.
    Zhang WC, Sun HY, Meng XY (2008) Subcellular localization of ZNF580-EGFP fusion protein. Chin J Pathophysiol 24:1590–1594Google Scholar
  7. 7.
    Sun HY, Wei SP, Xu RC, Xu PX, Zhang WC (2010) Sphingosine- 1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: novel insights into angiogenesis. Biochem Biophys Res Commun 395:361–366PubMedCrossRefGoogle Scholar
  8. 8.
    Luo YY, Hu WL, Xu RC, Hou B, Zhang LJ, Zhang WC (2011) ZNF580, a novel C2H2 zinc-finger transcription factor, interacts with the TGF-b signal molecule Smad2. Cell Biol Int 35:1153–1157PubMedCrossRefGoogle Scholar
  9. 9.
    Massagué J (2008) TGF in cancer. Cell 134:215–230PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Pardali E, ten Dijke P (2009) Transforming growth factor-beta signaling and tumor angiogenesis. Front Biosci 14:4848–4861CrossRefGoogle Scholar
  11. 11.
    Keiji M, Masahiko S, Takane H, Toshio F, Kohei M (2002) Two major Smad pathways in TGF-b superfamily signaling. Genes Cells 7:1191–1204CrossRefGoogle Scholar
  12. 12.
    Inoue N, Venema RC, Sayegh Hassan S, Ohara Y, Murphy TJ, Harrison DG (1995) Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-ß1. Arterioscler Thromb Vasc Biol 15:1255–1261PubMedCrossRefGoogle Scholar
  13. 13.
    Saura M, Zaragoza C, Cao W, Bao C, Rodrı´guez-Puyol M, Rodrı´guez-Puyol D et al (2002) Smad2 mediates transforming growth factor-beta induction of endothelial nitric oxide synthase expression. Circ Res 91:806–813PubMedCrossRefGoogle Scholar
  14. 14.
    Yamauchi K, Nishimura Y, Shigematsu S, Takeuchi Y, Nakamura J, Aizawa T, Hashizume K (2004) Vascular endothelial cell growth factor attenuates actions of transforming growth factor-β in human endothelial cells. J Biol Chem 279:55104–55108PubMedCrossRefGoogle Scholar
  15. 15.
    Dong Hu, Wan Yong (2011) Regulation of Krüppel-like factor 4 by the anaphase promoting complex pathway is involved in TGF-β signaling. J Biol Chem 286:6890–6901CrossRefGoogle Scholar
  16. 16.
    Ozawa A, Lick AN, Lindberg I (2011) Processing of proaugurin is required to suppress proliferation of tumor cell lines. Mol Endocrinol 25:776–784PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Castañares C, Redondo-Horcajo M, Magán-Marchal N, ten Dijke P, Lamas S, Rodríguez-Pascual F (2007) Signaling by ALK5 mediates TGF -β-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci 120:1256–1266PubMedCrossRefGoogle Scholar
  18. 18.
    Maruyama S, Shibata R, Kikuchi R, Izumiya Y, Rokutanda Y, Araki S (2012) Fat-derived factor omentin stimulates endothelial cell function and ischemia-induced revascularization via endothelial nitric oxide synthase-dependent mechanism. J Biol Chem 287:408–417PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Froese N, Kattih B, Breitbart A, Grund A, Geffers R, Molkentin JD, Kispert A, Wollert KC, Drexler H, Heineke J (2011) GATA6 promotes angiogenic function and survival in endothelial cells by suppression of autocrine transforming growth factor activin receptor-like kinase 5 signaling. J Biol Chem 286:5680–5690PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Li H, Wallerath T, Förstermann U (2002) Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide 7:132–147PubMedCrossRefGoogle Scholar
  21. 21.
    Tai SC, Robb GB, Marsden PA (2004) Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel. Arterioscler Thromb Vasc Biol 24:405–412PubMedCrossRefGoogle Scholar
  22. 22.
    Paduch R, Kandefer-Szerszeń M (2009) Transforming growth factor-beta1 (TGF-beta1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1beta (IL-1beta) secretion in human colon adenocarcinoma cells. In Vitro Cell Dev Biol Anim 45:543–550PubMedCrossRefGoogle Scholar
  23. 23.
    Chen H, Li D, Saldeen T, Mehta JL (2001) TGF-beta(1) modulates NOS expression and phosphorylation of Akt/PKB in rat myocytes exposed to hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 281:H1035–H1039PubMedGoogle Scholar
  24. 24.
    Pan CC, Bloodworth JC, Mythreye K, Lee NY (2012) Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation. Biochem Biophys Res Commun 424:620–623PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Manganini M, Maier JA (2000) Transforming growth factor beta2 inhibition of hepatocyte growth factor-induced endothelial proliferation and migration. Oncogene 19:124–133PubMedCrossRefGoogle Scholar
  26. 26.
    Oberringer M, Meins C, Bubel M, Pohlemann T (2008) In vitro wounding: effects of hypoxia and transforming growth factor beta1 on proliferation, migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model. J Mol Histol 39:37–47PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Yuyu Luo
    • 1
    • 2
  • Ying Zhao
    • 1
  • Xiaodong Li
    • 3
  • Juan Zhao
    • 1
  • Wencheng Zhang
    • 1
    • 2
  1. 1.Department of Physiology and PathophysiologyLogistics University of Chinese People’s Armed Police ForceTianjinChina
  2. 2.Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury Institute of Cardiovascular DiseaseTianjinChina
  3. 3.Department of OrthopaedicsTianjin Third Central HospitalTianjinChina

Personalised recommendations