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Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1 activation cascade: implications for HIF-1 gene therapy ¹

THOMAS HOFER^*, ISABELLE DESBAILLETS^*, GISELE HÖPFL^*, MAX GASSMANN^*,2 and ROLAND H. WENGER^*,

Institutes of
^* Physiology and
Veterinary Physiology, University of Zürich, CH-8057 Zürich, Switzerland; and
Institute of Physiology, Medical University of Lübeck, D-23538 Lübeck, Germany

²Correspondence: Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. E-mail: maxg{at}access.unizh.ch

SPECIFIC AIMS

The feasibility of hypoxia-inducible factor (HIF)-1 gene transfer is dependent on the (only partially characterized) role of the oxygen concentration in HIF-1 posttranslational modifications, nuclear translocation, heterodimerization with the ß-subunit, and target gene trans-activation. Thus, we analyzed the HIF-1 activation cascade in two newly established HeLa Tet-Off cell lines that inducibly overexpress high levels of HIF-1 under normoxic conditions both in vitro and in vivo.

PRINCIPAL FINDINGS

1. Inducible overexpression of HIF-1 in two HeLa Tet-off cell lines
Using the inducible Tet-off expression system, two HeLa cell lines were generated (termed HT42 and HT43) that stably overexpress the full-length human HIF-1 cDNA already under normoxic conditions. A coexpressed enhanced green fluorescent protein (EGFP) cDNA on the same construct served as a reporter for the induction efficiency of the expression system. Comparing hypoxic HIF-1 levels in repressed cells and normoxic HIF-1 levels in Tet-Off-induced cells, we found an at least 100-fold overexpression of HIF-1.

2. Kinetics of inducible HIF-1 overexpression
Time-dependent expression of Tet-Off-induced HIF-1 was analyzed under normoxic and hypoxic conditions. Over a period of 9 days, HT42 cells were split daily into doxycycline-free medium. On day 3, HIF-1 expression was fully induced and did not further increase. The HIF-1 signal was less intense in normoxic than in hypoxic cells, suggesting that partial amounts of overexpressed HIF-1 were degraded by the von Hippel-Lindau protein (pVHL)-ubiquitin-proteasome pathway. The HIF-1 levels became undetectable after 7 days of normoxic Tet-Off induction whereas under hypoxic conditions, HIF-1 was detectable until day 9. Therefore, all subsequent Tet-Off induction experiments were performed for 4 to 5 days.

3. Overexpression of HIF-1 is not sufficient to stabilize the p53 tumor suppressor protein
HIF-1 has been suggested to form a heterotrimeric complex with p53 and Mdm-2, thereby stabilizing the p53 protein. We therefore analyzed whether HIF-1 Tet-Off overexpression is able to influence p53 expression levels in HT42 cells. However, neither endogenous (24 h of hypoxia) nor overexpressed (5 days of normoxic Tet-Off induction) HIF-1 affected nuclear p53 levels. In contrast, treatment with actinomycin D readily induced p53, indicating that HT42 cells contain a fully responsive p53 activation pathway.

4. Overexpressed HIF-1 translocates to the nucleus under normoxic conditions
The subcellular localization of overexpressed HIF-1 was analyzed by immunofluorescence. In HT42 cells, Tet-Off overexpressed HIF-1 translocates into the nucleus under normoxic (Fig. 1 A) and hypoxic (Fig. 1B ) conditions, with hardly any detectable immunoreactivity remaining in the cytoplasm. In contrast, weak (repressed) and strong (Tet-Off-induced) EGFP fluorescence signals were distributed equally over the cells independent of the oxygen concentration.

View larger version (16K): [in this window] [in a new window]   Figure 1. Subcellular localization of EGFP and HIF-1 expression. Doxycycline-repressed and Tet-Off-induced HT42 cells were cultured under normoxic (A) or hypoxic (B) conditions for 24 h. EGFP fluorescence was detected using the FITC filter. HIF-1 was detected by indirect immunofluorescence using an anti-HIF-1 monoclonal antibody, followed by a rhodamine-coupled secondary goat anti-mouse antibody.

5. DNA binding activity of overexpressed HIF-1
Electrophoretic mobility shift assays revealed that normoxically overexpressed HIF-1 in HT42 and HT43 cells is able to recruit aryl hydrocarbon receptor nuclear translocator and form a functional DNA binding complex. Under hypoxic conditions, HIF-1 derived from both Tet-Off-induced and repressed cells showed HIF-1 DNA binding activity. Despite a 100-fold higher expression level of HIF-1 in Tet-Off-induced normoxic vs. repressed hypoxic cells, HIF-1 DNA binding activity was not further enhanced by Tet-Off induction in hypoxic cells.

6. Target gene activation by overexpressed HIF-1
RNA-blot analysis revealed that after Tet-Off induction and/or hypoxic exposure, HT42 and HT43 cells expressed higher levels of the HIF-1 target genes vascular endothelial growth factor (VEGF) and glucose transporter 1 (Glut-1) but not of the ribosomal protein L28 control mRNA. Tet-Off-induced cells grown under hypoxic conditions could not further enhance VEGF or Glut-1 mRNA levels. As expected, the HIF-1 mRNA levels were drastically elevated by Tet-Off induction but were not altered by hypoxic induction.

7. Trans-activation activity of overexpressed HIF-1
Reporter gene experiments were performed to analyze whether induction of endogenous HIF-1 target gene mRNA expression in Tet-Off-induced cells is due to the induction of HIF-1 trans-activation activity. Therefore, firefly luciferase plasmid constructs containing three HIF binding sites were transiently transfected into HT42 cells. Reporter gene expression increased 2.5-fold by Tet-Off induction in normoxic cells, 6-fold by hypoxic stimulation alone, and 11-fold by Tet-Off induction in hypoxic cells (Fig. 2 ). Thus, overexpressed HIF-1 is able to trans-activate reporter gene expression under normoxic conditions and hypoxia additively activates reporter gene expression.

View larger version (21K): [in this window] [in a new window]   Figure 2. HIF-1-dependent reporter gene expression in Tet-Off-induced cells. SV40 promoter-driven firefly luciferase reporter gene constructs containing three copies of either wild-type or mutant erythropoietin 3' enhancer-derived HBSs were transiently transfected into HT42 cells with or without the addition of the MEK-1 inhibitor PD98059 (50 µM). Doxycycline-repressed and Tet-Off-induced transfected cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions for 12 h and the luciferase activity was determined. A cotransfected renilla luciferase expression vector served as an internal control for transfection efficiency and extract preparation. All values were normalized to the normoxic firefly luciferase activities in repressed cells that were arbitrarily defined as 1.

8. The MEK-1 inhibitor PD98059 blocks trans-activation but not stabilization and DNA binding of overexpressed HIF-1
To analyze the involvement of MAP kinases in the activation of HIF-1, the selective MEK-1 inhibitor PD98059 was added to HT42 cell cultures. PD98059 completely abolished the elevation of reporter gene activity by Tet-Off and/or hypoxic induction. In contrast, neither the HIF-1 protein levels nor HIF-1 DNA binding activity were affected, indicating that the MEK/Erk MAPK pathway regulates trans-activation but not stabilization and DNA binding of HIF-1.

9. Exogenous HIF-1 overexpression in vivo
To demonstrate the feasibility of HIF-1 gene transfer under normoxic conditions in vivo, HT42 cells were transplanted into nude mice. The mice were kept under normoxic conditions; 25–28 days after injection, the cells were excised and analyzed for exogenous HIF-1 expression by immunoblotting. The mouse monoclonal antibody mgc3 does not recognize mouse HIF-1 by immunoblotting and allowed us to distinguish between HT42-dervied and mouse tissue-derived HIF-1. These results suggest that exogenous HIF-1 is overexpressed in HT42 cells under normoxic conditions in vivo as well as in vitro.

CONCLUSIONS

We established two cell lines capable of overexpressing HIF-1 protein at least 100-fold in an inducible manner under normoxic conditions. However, after 1 wk of Tet-Off induction, HIF-1 protein levels decreased again despite the ongoing absence of the repressor (doxycycline). One explanation might be the existence of a negative feedback loop. Indeed, a so far unidentified ‘HIF-1 proteasome targeting factor’ (HPTF) has been postulated that is distinct from pVHL, is transcriptionally induced by HIF-1, and subsequently leads to the degradation of HIF-1. Another possibility for a negative feedback loop might involve p53-induced degradation of HIF-1. According to this model, p53 induces Mdm-2-mediated HIF-1 ubiquitinylation and proteasomal degradation. On the other hand, p53, which is normally only present at low levels, is stabilized by the interaction with HIF-1. Thus, induction of HIF-1 should lead to higher levels of p53, which in turn degrade HIF-1 via Mdm-2 recruitment. However, despite the massive HIF-1 levels in Tet-Off-induced HT42 cells, no concomitant p53 induction could be observed.

Using a transiently transfected GFP-HIF-1 fusion construct, it has been reported that HIF-1 is localized predominantly in the cytoplasm of normoxically cultured cells and translocates to the nucleus after exposure to hypoxia or hypoxia-mimicking chemicals. However, our experiments demonstrate that even high levels of HIF-1 undergo nuclear translocation and accumulation without the need of further hypoxia-dependent signals. This contradictory finding might be related to the fact that we used wild-type HIF-1 instead of a GFP-HIF-1 fusion construct that might interfere with nuclear translocation and accumulation of HIF-1.

Despite the overexpression of HIF-1 in Tet-Off-induced HT42 cells, only a moderate enhancement of DNA binding and reporter gene activation could be observed. This might be explained by two different mechanisms: limiting amounts of critical cofactors (squelching) and/or insufficient (hypoxia-dependent) HIF-1 protein modifications. As for the first possibility, neither ectopic expression of ARNT nor the CREB binding protein (CBP) resulted in enhanced transactivation of reporter gene expression. The second possibility for the lack of full HIF-1 trans-activation activity might be insufficient posttranslational HIF-1 protein modification, most probably by phosphorylation. Indeed, using the selective MEK-1 inhibitor PD98059, the trans-activation activity of both normoxically overexpressed as well as hypoxic HIF-1 was completely abolished, suggesting that the activity of the p42/44 MAP kinases is the critical factor in the HIF-1 activation cascade that might thus be the rate-limiting step in Tet-Off induction of HIF-1. This view is supported by the finding that the Western blotting pattern of HIF-1 was clearly distinct when hypoxically induced and Tet-Off-induced cells were compared: a high proportion of HIF-1 in Tet-Off-induced cells was of lower molecular weight than the major band in hypoxic cells, indicating incomplete phosphorylation.

In summary, our results demonstrate the feasibility of HIF-1 gene transfer despite the fact that endogenous HIF-1 is unstable under normoxic conditions. The availability of cell lines stably overexpressing HIF-1 will aid in the identification of critical cofactors required for full HIF-1 activation that might be used for HIF-1-mediated therapeutic angiogenesis.

View larger version (51K): [in this window] [in a new window]   Figure 3. Schematic illustration of the HIF-1 activation cascade. A) In repressed cells, HIF-1 is either phosphorylated and accumulates in the nucleus under hypoxic conditions or is hydroxylated, ubiquitinylated, and proteasomally destroyed under normoxic conditions. B) In Tet-Off-induced cells, most HIF-1 accumulates in the nucleus in a transcriptionally inactive form. The oxygen concentration plays a marginal role.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0546fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0546fje

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