title: FIH-1-Mint3 axis does not control HIF-1 transcriptional activity in nucleus pulposus cells.
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.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Hirose
authors.forename: Zariel I
authors.initials: ZI
authors.lastname: Johnson
authors.forename: Zachary R
authors.initials: ZR
authors.lastname: Schoepflin
authors.forename: Dessislava Z
authors.initials: DZ
authors.lastname: Markova
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Chiba
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Toyama
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003782:Dental Pulp / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: C117536:APBA3 protein, human; D048868:Adaptor Proteins, Signal Transducing; C485532:Apba3 protein, rat; D002352:Carrier Proteins; D051793:Hypoxia-Inducible Factor 1; D011506:Proteins; D051880:Receptors, Notch; D012097:Repressor Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human