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.
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.
Loeffler
Sébastien
S
Division of Neuropathology, Institute of Pathology, University of Bern, Bern, Switzerland.
Fayard
Bérengère
B
Weis
Joachim
J
Weissenberger
Jakob
J
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D020862:GC Rich Sequence / Q000235:genetics; D005786:Gene Expression Regulation; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000494:pharmacology*; D051379:Mice; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D018919:Neovascularization, Physiologic; D011401:Promoter Regions, Genetic; D050796:STAT3 Transcription Factor; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; D015850:Interleukin-6; C497121:SP3 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D051705:Sp3 Transcription Factor