Hypoxia-inducible factor-1 and neuroglobin expression

Abstract

Neuroglobin (Ngb) is a hypoxia-inducible protein with cytoprotective effects in animal models of stroke, Alzheimer's disease, and related disorders, but the molecular mechanisms involved in its induction are unknown. We tested the hypothesis that hypoxia-inducible factor-1 (HIF-1) regulates Ngb levels, using shRNA-mediated knockdown and lentiviral vector-mediated overexpression of the HIF-1α subunit, in cultured neural (HN33) cells. HIF-1α knockdown decreased and HIF-1α overexpression increased Ngb levels, consistent with a connection between HIF-1 and Ngb induction. These findings may have implications for understanding the hypoxia-response repertoire of neural cells and devising therapeutic strategies for neurologic disorders.

Figure 1. shRNA-mediated knockdown of HIF-1α decreases Ngb protein levels

(A) Normalized densitometric analysis and (B) western blots of nuclear HIF-1α protein in HN33 cells infected with control (left) or HIF-1α shRNA-expressing (right) lentivirus (*, p<0.02). Band at far right is from cells treated for 4 h with the prolyl hydroxylase domain-containing protein inhibitor, 2,2-dipyridyl (50 μM). (C) Normalized densitometric analysis and (D) western blots of cytoplasmic Ngb protein in HN33 cells infected with control (left) or HIF-1α shRNA-expressing (right) lentivirus (*, p<0.01).

Figure 2. CMV-mediated HIF-1α overexpression increases Ngb protein levels

(A) Normalized densitometric analysis and (B) western blots of nuclear HIF-1α protein in HN33 cells infected with CMV-GFP control (left) or CMV-HIF-1α (right) vector (*, p<0.001). Band at far right is from cells treated for 4 h with the prolyl hydroxylase domain-containing protein inhibitor, 2,2-dipyridyl (50 μM). (C) Normalized densitometric analysis and (D) western blots of cytoplasmic Ngb protein in HN33 cells infected with CMV-GFP control (left) or CMV-HIF-1α (right) vector (*, p<0.001).

Figure 3. ChIP analysis of predicted HIF-1α binding sites upstream of the Ngb ATG start site

Anti-HIF-1α was used to immunoprecipitate HIF-1α-crosslinked DNA sequences amplified with primers for candidate HIF-1α binding sites located 7736 (mNgb1), 6725 (mNgb2), 5012 (mNgb3), and 1993 (mNgb4) base pairs upstream of the neuroglobin ATG start site. All except mNgb4 yielded bands. mEpo served as a positive control for the ability to detect HIF-1α binding sites, hVEGF as a negative control for DNA amplification, and goat IgG (lanes labeled “-”) as a negative control for immunoprecipitation.