Abstract
Oxygen sensing transcription factor HIF-1 is activated due to accumulation of regulatory subunit HIF-1α by posttranslational stability mechanism during hypoxia or by several other stimuli even in normoxia. HIF-1α is also regulated by NF-kB mediated transcription mechanism. Reactive oxygen species (ROS) act as an important regulator of HIF-1 either by affecting prolyl hydroxylase activity, the critical determinant of HIF-1α stabilization or by activating NF-kB to promote HIF-1α transcription. Insulin is known to activate HIF-1 by a ROS dependent mechanism but the molecular mechanism of HIF-1α regulation is not known so far. Here we show that insulin regulates HIF-1α by a novel transcriptional mechanism by a ROS-sensitive activation of Sp1 in 3T3-L1 preadipocyte. Insulin shows little effect on HIF-1α protein stability, but increases HIF-1α promoter activity. Mutation analyses, electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirm the role of Sp1 in HIF-1α transcription. We further demonstrate that insulin-induced ROS generation initiates signaling pathway involving phosphatidylinositol 3-kinase and protein kinase C for Sp1 mediated HIF-1α transcription. In summary, we reveal that insulin regulates HIF-1α by a novel transcriptional mechanism involving Sp1.
Highlights
The oxygen sensing transcription factor hypoxia-inducible factor-1 (HIF-1) is a heterodimer of regulatory subunit HIF-1a and constitutive subunit HIF-1b [1]
In this study we reveal that insulin regulates HIF-1a by a novel transcriptional mechanism involving Sp1 in 3T3-L1 preadipocytes
We reported that NADPH-oxidase mediated Reactive oxygen species (ROS) generation and subsequent phosphatidylinositol 3-kinase (PI3K) activation was needed for HIF-1 activation [19]
Summary
The oxygen sensing transcription factor hypoxia-inducible factor-1 (HIF-1) is a heterodimer of regulatory subunit HIF-1a and constitutive subunit HIF-1b [1]. HIF-1 binds to the hypoxia response elements (HREs) of target genes implicated in energy metabolism, angiogenesis, apoptosis, and iron homeostasis [4,5,6]. HIF-1a is usually unstable due to hydroxylation of two proline residues; Pro402 and Pro564 that promotes ubiquitination and subsequent proteasomal degradation [7,8,9,10]. Three different HIF prolyl-hydroxylases termed PHD1, PHD2, and PHD3 are able to hydroxylate HIF-1a using oxygen and 2-oxoglutarate as substrates and iron as well as ascorbate as essential cofactors [2,3]. Hypoxic conditions lead to HIF-1a stabilization due to inhibition of prolyl hydroxylses and subsequent decrease in HIF-1a ubiquitination and degradation
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