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.
Copyright 2005 S. Karger AG, Basel.
MeSH terms
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Blotting, Western
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Cell Hypoxia / drug effects
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Cells, Cultured
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Chromones / pharmacology
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DNA-Binding Proteins / metabolism*
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Dose-Response Relationship, Drug
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Enzyme Activation / drug effects
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Fluorescent Antibody Technique
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Humans
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Kidney Tubules, Proximal / cytology
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Kidney Tubules, Proximal / enzymology
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Kidney Tubules, Proximal / metabolism*
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / metabolism
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Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinase Kinases / metabolism*
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Morpholines / pharmacology
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Nuclear Proteins / metabolism*
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Oxygen / metabolism
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Phosphatidylinositol 3-Kinases / metabolism*
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Phosphoinositide-3 Kinase Inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Reverse Transcriptase Polymerase Chain Reaction
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Signal Transduction* / drug effects
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Transcription Factors / metabolism*
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Vascular Endothelial Growth Factors / biosynthesis*
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Vascular Endothelial Growth Factors / genetics
Substances
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Chromones
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DNA-Binding Proteins
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HIF1A protein, human
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Morpholines
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Nuclear Proteins
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Phosphoinositide-3 Kinase Inhibitors
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Proto-Oncogene Proteins
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Transcription Factors
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Vascular Endothelial Growth Factors
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinase Kinases
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Oxygen