An endoplasmic reticulum transmembrane prolyl 4-hydroxylase is induced by hypoxia and acts on HIF-alpha

Koivunen P, Tiainen P, Hyvärinen J, Williams KE, Sormunen R, Klaus SJ, Kivirikko KI, Myllyharju J
Source: J Biol Chem
Publication Date: (2007)
Issue: 282(42): 30544-52
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Species: human
Tissue Origin: brain
Nucleofector™ I/II/2b

Kit V, D-23 siRNA Transfections and HIF-1a Protein Determination - Kelly cells (1.6 x 106) were transfected with 120 pmol of siRNA for HIF-P4H-1, 2 or 3 (Ambion) or P4H-TM (Dharmicon) or a non-targeting control siRNA (siCNTL, Dharmicon) with an Amaxa nucleoporator according to the manufacturers' instructions (Solution V, Program D-23) and seeded to 50% confluency. The cells were cultured in RPMI and 10% fetal bovine serum for 48 h, after which cell extracts were prepared and analyzed for HIF-1a protein levels using a HIF-1a Whole Cell Lysate Kit (MesoScale Discovery).


Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of HIF-alpha subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence resembles more closely those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide-substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in ER membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-alpha ODDD reporter construct levels and its siRNA increased HIF-1alpha protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1alpha ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.