FIH-1-Mint3 axis does not control HIF-1 transcriptional activity in nucleus pulposus cells

J Biol Chem. 2014 Jul 25;289(30):20594-605. doi: 10.1074/jbc.M114.565101.

Abstract

The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxiasensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1-C-TAD and HIF-2-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Hypoxia / genetics
  • Cell Line
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Dental Pulp / cytology
  • Dental Pulp / metabolism*
  • Humans
  • Hypoxia-Inducible Factor 1 / genetics
  • Hypoxia-Inducible Factor 1 / metabolism*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Proteins / genetics
  • Proteins / metabolism*
  • Rats
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Signal Transduction*
  • Transcription, Genetic*

Substances

  • APBA3 protein, human
  • Adaptor Proteins, Signal Transducing
  • Apba3 protein, rat
  • Carrier Proteins
  • Hypoxia-Inducible Factor 1
  • Proteins
  • Receptors, Notch
  • Repressor Proteins
  • Mixed Function Oxygenases
  • HIF1AN protein, human