Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)

Abstract

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.

FIG. 1.

(a) Representative hTERT mRNA expression in placenta. Normal villi and placenta were analyzed by RT-PCR for hTERT and glyceraldehyde-3-phosphate dehydrogenase expression. (b) Relative mRNA level of hTERT by real-time RT-PCR. (c) Representative hTERT protein expression in placentas analyzed by Western blotting for HIF-1α, hTERT, and actin as follows: 6-, 8-, and 9-week normal villi (lanes 1 to 3, respectively); 15-, 39-, and 40-week normal placentas (lanes 4 to 6, respectively); and 32-, 35-, and 38-week preeclamptic placentas (lanes 7 to 9, respectively).

FIG. 2.

Hypoxia induces telomerase and hTERT expression. (a) JEG-3 cells were cultured at 20% O₂ or 1% O₂ for 24 h. Whole-cell extracts were prepared and subjected to TRAP assays. (b) JEG-3 cells were cultured at 20% O₂ or 1% O₂ for 1, 3, 6, 12, and 24 h. Total RNA was prepared and subjected to real-time RT-PCR analysis. (c) JEG-3 and JAR cells were cultured at 20% O₂ or 1% O₂ for 24 h. Whole-cell extracts (40 μg) were prepared and subjected to Western blot analysis.

FIG. 3.

Transactivation of hTERT promoter activity by hypoxia exposure and HIF-1 overexpression. (a) Schematic representations of the HIF-1 response elements and the pGL181-luc, pGL181mt1-luc, pGL181mt2-luc, and pGL181mt-luc reporters are shown. (b) JEG-3 cells were cotransfected with the pGL181-luc, pGL181mt1-luc, pGL181mt2-luc, and pGL181mt-luc (0.3 μg each) constructs and 1.0 μg (each) of the HIF-1 expression constructs (HIF-1α and ARNT) or the empty vector pcDNA3 under hypoxic conditions (1% O₂); activity is reported as relative luminescence units (RLU). Error bars indicate standard deviations in triplicate assays.

FIG. 4.

Disruption of hTERT expression by an AS oligonucleotide of HIF-1α. (a) JEG-3 and JAR cells were transfected with an AS or SE oligonucleotide relative to the HIF-1α cDNA sequence by using Lipofectamine 2000. Cells were then exposed to hypoxic conditions (1% O₂). A cell lysate was prepared after 24 h and analyzed by Western blotting for protein level by using antibodies to HIF-1α, hTERT, and actin. (b) JEG-3 cells were transfected with the HIF-1α AS or SE oligonucleotide by using Lipofectamine 2000. Cells were then exposed to hypoxic conditions (1% O₂). Whole-cell extracts were prepared and subjected to a TRAP assay.

FIG. 5.

Analysis of HIF-1 interaction with the putative HREs in the hTERT promoter. (a and b) ³²P end-labeled TERT/HRE1 or ³²P end-labeled TERT/HRE2 oligonucleotide was used as a probe. For the competition assays, a 50-fold or 200-fold molar excess of the HRE was used. For the supershift assay, anti-HIF-1α antibody (0.5 μg) was added to the binding reaction. The thick arrow indicates HIF-1/DNA complexes, and the thin arrows indicate supershifted bands. (c) The HIF-1 consensus oligonucleotide was end-labeled with ³²P. For the competition assays, a 50-fold or 200-fold molar excess of the TERT/HRE1 and of the TERT/HRE2 oligonucleotides were added to the binding reaction, respectively. For the supershift assay, anti-HIF-1α antibody (0.5 μg) was added to the binding reaction. The thick arrow indicates HIF-1/DNA complexes, and the thin arrows indicate supershifted bands. NS, nonspecific.

FIG. 6.

Analysis of HIF-1 interaction with the mutated HREs in the hTERT promoter. (a and b) ³²P end-labeled TERT/HREmt1 or ³²P end-labeled TERT/HREmt2 oligonucleotide was used as a probe. For the competition assays, a 200-fold molar excess of the HRE was used. For the supershift assay, anti-HIF-1α antibody (0.5 μg) was added to the binding reaction. (c) The HIF-1 consensus oligonucleotide was end labeled with ³²P. For the competition assays, 200-fold molar excesses of TERT/HRE1, TERT/HRE2, TERT/HREmt1, TERT/HREmt2, and HRE were added to the binding reactions. For the supershift assay, anti-HIF-1α antibody (0.5 μg) was added to the binding reaction. The thick arrow indicates HIF-1/DNA complexes, and thin arrows indicate supershifted bands. NS, nonspecific.