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. 2000 Aug 1;97(16):9082-7.
doi: 10.1073/pnas.97.16.9082.

Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia

R K Bruick  1
Affiliations

Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia

R K Bruick. Proc Natl Acad Sci U S A. .

Abstract

The ability to sense and respond to changes in oxygen availability is critical for many developmental, physiological, and pathological processes, including angiogenesis, control of blood pressure, and cerebral and myocardial ischemia. Hypoxia-inducible factor-1alpha (HIF-1alpha) is a basic-helix-loop-helix (bHLH)containing member of the PER-ARNT-SIM (PAS) family of transcription factors that plays a central role in the response to hypoxia. HIF-1alpha, and its relatives HIF-2alpha/endothelial PAS domain protein (EPAS) and HIF-3alpha, are induced in response to hypoxia and serve to coordinately activate the expression of target genes whose products facilitate cell survival under conditions of oxygen deprivation. When cells are exposed to chronic hypoxia, the protective response can fail, resulting in apoptosis. This study shows that transcription of the gene encoding Nip3, a proapoptotic member of the Bcl-2 family of cell death factors, is strongly induced in response to hypoxia. The Nip3 promoter contains a functional HIF-1-responsive element (HRE) and is potently activated by both hypoxia and forced expression of HIF-1alpha. Exposure of cultured cells to chronic hypoxia results in the accumulation of a protein recognized by antibodies raised against Nip3. This study demonstrates a direct link between HIF-1alpha and a proapoptotic member of the Bcl-2 family and offers a reasonable physiological function for members of the Bcl-2 subfamily, including Nip3 and its close relative Nix. These observations indicate that Nip3 may play a dedicated role in the pathological progression of hypoxia-mediated apoptosis, as observed after ischemic injury.

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Figures

Figure 1
Figure 1
Nip3 mRNA levels increase in CHO-K1 cells in response to hypoxia. CHO-K1 cells were incubated under normoxic (20% O2) or hypoxic (1% O2) conditions or in the presence of 100 μM CoCl2, 100 μM deferoxamine mesylate (DFO), 5 mM N-(2-mercaptopropionyl)glycine (NMPG), or 10 μM Cbz-Leu-Leu-Leu-norvalinal (CBZ-LLnL) under normoxic conditions. Radiolabeled probes specific for Nip3, Glut-1, or 28S rRNA were used to visualize mRNAs from total RNA by Northern blot analysis.
Figure 2
Figure 2
Nip3 protein levels accumulate after prolonged exposure to hypoxia. (A) Total cell lysates from CHO-K1 cells incubated under normoxic (20% O2) or hypoxic (0.5% O2) conditions for 0–6 days were examined by Western blot analysis, using antiserum raised against recombinant Nip3 protein. The endogenous Nip3 protein has an observed molecular mass of approximately 60 kDa (13, 14). (B) CHO-K1 cells were maintained under normoxic (20% O2) or hypoxic (0.5% O2) conditions for 1–6 days. Cells were harvested and incubated in the presence of 0.2% trypan blue in PBS for 30 min at room temperature. Viable cells exclude trypan blue and are not stained. Values represent the average ± SD of three samples.
Figure 3
Figure 3
Nip3 mRNA levels are induced in response to hypoxia in several different cell lines. Cells were incubated under normoxic (20% O2) or hypoxic (0.5% O2) conditions for 15 h. Radiolabeled probes specific for Nip3, Glut-1, or 28S rRNA were used to visualize mRNAs from total RNA by Northern analysis.
Figure 4
Figure 4
Nip3 and Nix are the only members of the Bcl-2 family of apoptotic factors induced in response to hypoxia. CHO-K1 cells were incubated under normoxic (20% O2) or hypoxic (0.5% O2) conditions for 10 h. Radiolabeled probes specific for the indicated genes were used to visualize mRNAs from poly(A)+ mRNA by Northern analysis.
Figure 5
Figure 5
The Nip3 promoter is responsive to hypoxia and to HIF-1α. (A) The promoter sequence of Nip3 from CHO-K1 cells contains two consensus HREs (highlighted in gray). HRE1 is shown in boldface. The translation start codon of the Nip3 polypeptide at position +1 is in boldface. The 5′ end of the mRNA as determined by 5′ rapid amplification of cDNA ends is indicated by an arrow and is located 25 nucleotides downstream of a putative TATA box (underlined). HRE1-Mut changed the putative HRE beginning at position −234 from 5′-CACGTG-3′ to 5′-CACCAC-3′. HRE2-Mut changed the putative HRE beginning at position −160 from 5′-CGCGTG-3′ to 5′-CGACTG-3′. (B) Luciferase reporter constructs containing no promoter (pGL3-Basic), a minimal TATA box containing promoter (E1B-Luc), the Nip3 promoter, or the Nip3 promoter containing a mutation in the first (HRE#1-Mut) or second (HRE#2-Mut) putative HRE was transfected into 293 cells along with pcDNA3 (vector) or HIF-1α. Cells transfected with vector were incubated for 19 h in an atmosphere containing 20% O2 (normoxia) or for 5 h in an atmosphere containing 20% O2 followed by 14 h in an atmosphere containing 0.5% O2 (hypoxia). Cells transfected with HIF-1α were incubated for 19 h under normoxic (20% O2) conditions. The values represent the average luciferase activity of six samples; bars indicate standard error.
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