VEGF production by primary human renal proximal tubular cells: requirement of HIF-1, PI3-kinase and MAPKK-1 signaling

Cell Physiol Biochem. 2005;15(1-4):99-108. doi: 10.1159/000083642.

Abstract

Renal proximal tubular epithelial cells (PTEC) respond to hypoxia exposure or interleukin-1beta (IL-1beta) treatment with increased vascular endothelial growth factor (VEGF) production. With respect to O2 deprivation, the hypoxia-inducible factor 1alpha/ beta (HIF-1) is the most important transcription factor driving VEGF mRNA expression. HIF-1 is also activated by IL-1beta and may thus be involved in the stimulation of VEGF production by this cytokine. However, the molecular mechanisms of HIF-1 dependent VEGF synthesis are poorly understood. Herein, human PTEC in primary culture were challenged by hypoxic incubation and/or IL-1beta treatment in absence or presence of specific phosphatidylinositol 3-kinase (PI3K) or mitogen activated protein kinase kinase-1 (MAPKK-1) inhibitors for assay of VEGF protein, VEGF mRNA and detection of HIF-1alpha by Western Blotting, EMSA and fluorescence microscopy. In addition, the activities of PI3K and MAPKK-1 were studied following hypoxia and IL-1beta treatment of the cultures. The study shows that PI3K but not MAPKK-1 inhibition resulted in the loss of hypoxic and IL-1beta induced HIF-1alpha accumulation, whereas VEGF synthesis was reduced by either intervention. Thus, PI3K signaling is required for HIF-1alpha accumulation and VEGF synthesis, whereas MAPKK-1 signaling is required for VEGF synthesis only. Furthermore, hypoxia alone was sufficient to activate PI3K in PTEC in contrast to MAPKK-1, whose activity was lowered in hypoxia.

MeSH terms

  • Blotting, Western
  • Cell Hypoxia / drug effects
  • Cells, Cultured
  • Chromones / pharmacology
  • DNA-Binding Proteins / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Fluorescent Antibody Technique
  • Humans
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / enzymology
  • Kidney Tubules, Proximal / metabolism*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Morpholines / pharmacology
  • Nuclear Proteins / metabolism*
  • Oxygen / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction* / drug effects
  • Transcription Factors / metabolism*
  • Vascular Endothelial Growth Factors / biosynthesis*
  • Vascular Endothelial Growth Factors / genetics

Substances

  • Chromones
  • DNA-Binding Proteins
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Morpholines
  • Nuclear Proteins
  • Phosphoinositide-3 Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Transcription Factors
  • Vascular Endothelial Growth Factors
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinase Kinases
  • Oxygen