Abstract
We report a Notch signal-induced pathway that leads to transcriptional activation of HIF1-alpha gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-alpha and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-alpha expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.
Highlights
Notch signaling is highly conserved through evolution and is crucial for cell death, proliferation, differentiation, and development
To evaluate the role of Notch signaling on HIF1-α expression, we analyzed transcriptional expression of HIF1-α after the induction of Notch signaling in HeLa/rtTAA/tetracycline response element (TRE)-N1-IC cells
Notch signaling significantly increased luciferase reporter activity driven by hypoxia response elements (HRE) in HeLa/ rtTAA/TRE-N1-IC cells under hypoxic condition (Fig. 2D)
Summary
Notch signaling is highly conserved through evolution and is crucial for cell death, proliferation, differentiation, and development. HES family proteins are transcriptional repressors that act as Notch effectors by negatively regulating the expression of downstream target genes [2]. HIF1-α is hydroxylated at specific proline residues by prolyl hydroxylases that act as oxygen sensors leading to the rapid ubiquitination followed by proteosomal degradation [6, 7]. HIF1-α expression is elevated as a result of reduced ubiquitination and decreased proteosomal degradation. HIF1-α binds, in conjugation with HIF1-β, to specific hypoxia response elements (HRE) in the promoters and enhancers of HIF1 target genes. Among the best characterized HIF1-α downstream target genes are genes involved in angiogenesis, erythropoiesis, and glycolysis, namely differentiated embryo-chondrocyte expressed gene 1 (DEC1), vascular endothelial growth factor (VEGF) gene, erythropoietin (EPO) gene, and phosphoglycerate kinase 1 (PGK1) gene [8,9,10,11]
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