Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production

Abstract

Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIFalpha subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules.

Fig. 1.

ODD-Luc fusion protein retains luciferase activity and binds to pVHL. (A) Autoradiogram of the indicated ³⁵S-labeled proteins after in vitro translation and SDS/polyacrylamide gel electrophoresis. (B) Luciferase activity (LU, light units) corresponding to 50% (by volume) of the in vitro translation products studied in A. Data shown are representative of two independent experiments. (C) Autoradiogram of the indicated ³⁵S-labeled proteins made by in vitro translation in rabbit reticulocyte lysate (Retic) or wheat germ extract (WG). Proteins were incubated with glutathione Sepharose preloaded with GST-pVHL, elongin B, and elongin C. Specifically bound proteins were released by boiling in SDS-containing sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (PD, pulldown). For comparison, 10% of the protein used for the binding studies was load directly and resolved in the same gel (I, input).

Fig. 2.

ODD-Luc is induced by hypoxia and hypoxia mimetics. (A) Firefly luciferase activity, corrected for renilla luciferase, of WT8 P → A cells [pVHL(+)] and 786-O [pVHL(–)] renal carcinoma cells transfected to produce wild-type firefly luciferase or ODD-Luc. Where indicated, 500 μM deferoxamine (D) or 100 μM cobalt chloride (C) was added to media 24 h before analysis. Luciferase data were normalized to D for each cell line-reporter pair. (B) Normalized luciferase values for HeLa VHL+/+ cervical carcinoma cells transfected to produce wild-type firefly luciferase or ODD-Luc. Where indicated, cells were exposed to 500 μM deferoxamine (D) or 0.2% oxygen (H) for 24 h before analysis. Error bars indicate one standard error.

Fig. 3.

Imaging hypoxia by using ODD-Luc mice. (A) Bioluminescent images (anterior view) of ROSA26 Luc/+, ODD-Luc/+, and +/+ FVB mice. (B) Bioluminescent images (posterior view) of the same two ROSA26 ODD-Luc/+ mice breathing 21% or 8% oxygen. For the latter, the ambient oxygen was reduced from 21% to 8% over 1 h and maintained at 8% for an additional 4 h. Color bar indicates photons/(cm²·sec·steradian) with minimum and maximum threshold values.

Fig. 4.

Inhibition of HIF prolyl hydroxylase by small molecules in cell-based assays. (A) Fold increase in normalized luciferase values of HeLa cells transfected to produce ODD-Luc and exposed to compound A, FG-4383, or deferoxamine (DFO) at the indicated concentrations relative to untreated cells. Error bars indicate one standard error. (B) Immunoblot analysis of HeLa cells treated as in A.

Fig. 5.

Pharmacodynamic monitoring of HIF prolyl hydroxylase inhibitor. (A) Bioluminescent images of ROSA26 ODD-Luc/+ mice 6 h after administration of vehicle, Compound A (100 mg/kg), or FG-4383 (100 mg/kg) by oral gavage. (B) Bioluminescent images of ROSA26 ODD-Luc/+ mice before and 6 h after administration of FG-4383 by oral gavage at indicated doses. Color bar indicates photons/(cm²·sec·steradian) with minimum and maximum threshold values. (C) Immunoblot analysis of tissue extracts prepared from wild-type (WT) and ROSA26 ODD-Luc/+ (ODD) mice 6 h after treatment with 100 mg/kg FG-4383.