BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition

Carcinogenesis. 2013 Nov;34(11):2558-67. doi: 10.1093/carcin/bgt242. Epub 2013 Jul 8.

Abstract

Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Blotting, Western
  • Cell Proliferation
  • Cells, Cultured
  • Fluorescent Antibody Technique
  • Gene Expression Regulation, Neoplastic
  • Heterografts
  • Human Umbilical Vein Endothelial Cells / cytology
  • Human Umbilical Vein Endothelial Cells / metabolism*
  • Humans
  • Hypoxia*
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • Immunoenzyme Techniques
  • Immunoprecipitation
  • Lung Neoplasms / blood supply
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / secondary*
  • Melanoma / blood supply
  • Melanoma / metabolism
  • Melanoma / pathology*
  • Mice
  • Mice, Nude
  • Neovascularization, Pathologic / metabolism
  • Neovascularization, Pathologic / pathology*
  • Protein Interaction Domains and Motifs
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism*
  • Wound Healing

Substances

  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Messenger
  • Vascular Endothelial Growth Factor A