FIH-1-Mint3 Axis Does Not Control HIF-1{alpha} Transcriptional Activity in Nucleus Pulposus Cells [Glycobiology and Extracellular Matrices]

May 27th, 2014 by Hirose, Y., Johnson, Z. I., Schoepflin, Z. R., Markova, D. Z., Chiba, K., Toyama, Y., Shapiro, I. M., Risbud, M. V.

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. While both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxia sensitive. Overexpression of FIH-1 was able to reduce HIF-1 function as seen by changes in activities of HRE-luciferase reporter and HIF-1α-CTAD and HIF-2α-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 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 Notch pathway changed in FIH-1 silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF1-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.