Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1

Int J Cancer. 2005 Jun 10;115(2):202-13. doi: 10.1002/ijc.20871.

Abstract

Interleukin-6 (IL-6) expression is strongly correlated with the degree of human glioma malignancy and necessary for tumor formation in a mouse model of spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant progression of these brain tumors is still unclear. We have scrutinized the mechanism of transcriptional activation of vascular endothelial growth factor (VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We demonstrate here that IL-6 drives transcriptional upregulation of VEGF in astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green fluorescent protein (GFP) double transgenic mice. We further show that IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells is dependent on signal transducer and activator of transcription 3 (STAT3). By progressive 5'-deletion analysis we defined the minimal VEGF promoter region for IL-6-responsiveness to nucleotides -88/-50. Surprisingly, this promoter region is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the -88/-50 element upon IL-6 stimulation. Interestingly, preincubation with STAT3 antibody prevented the binding of Sp1 and Sp3 to the -88/-50 element, indicating that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation. Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation. The functional relevance of the STAT3/Sp1 association was corroborated by transient transfection experiments, which showed that overexpression of constitutively active STAT3 increased the minimal VEGF promoter activity. Taken together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas and describes a novel transcriptional activation mechanism for STAT3 in the context of a STAT3 binding element (SBE)-free promoter.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / drug effects*
  • Astrocytes / metabolism
  • COS Cells
  • Chlorocebus aethiops
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism*
  • Electrophoretic Mobility Shift Assay
  • GC Rich Sequence / genetics
  • Gene Expression Regulation*
  • Glial Fibrillary Acidic Protein / genetics
  • Glial Fibrillary Acidic Protein / metabolism
  • Glioblastoma / metabolism*
  • Glioblastoma / pathology
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • Interleukin-6 / pharmacology*
  • Mice
  • Mice, Transgenic
  • NIH 3T3 Cells
  • Neovascularization, Physiologic
  • Promoter Regions, Genetic*
  • STAT3 Transcription Factor
  • Sequence Deletion
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*
  • Sp3 Transcription Factor
  • Trans-Activators / antagonists & inhibitors
  • Trans-Activators / genetics
  • Trans-Activators / immunology
  • Trans-Activators / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcriptional Activation
  • Vascular Endothelial Growth Factor A / genetics*
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • DNA-Binding Proteins
  • Glial Fibrillary Acidic Protein
  • Interleukin-6
  • SP3 protein, human
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Sp1 Transcription Factor
  • Sp3 protein, mouse
  • Stat3 protein, mouse
  • Trans-Activators
  • Transcription Factors
  • Vascular Endothelial Growth Factor A
  • Green Fluorescent Proteins
  • Sp3 Transcription Factor