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. 2003 Jan;23(1):359-69.
doi: 10.1128/MCB.23.1.359-369.2003.

Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia

Nobuhito Goda  1 , Heather E Ryan, Bahram Khadivi, Wayne McNulty, Robert C Rickert, Randall S Johnson
Affiliations

Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia

Nobuhito Goda et al. Mol Cell Biol. 2003 Jan.

Abstract

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1alpha gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1alpha influenced p53, we also created double-knockout (HIF-1alpha null, p53 null) strains and cells. In both cell types, loss of HIF-1alpha abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1alpha genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1alpha causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1alpha-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1alpha dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.

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Figures

FIG. 1.
FIG. 1.
Deletion of the HIF-1α allele in B cells in vivo and in vitro. (A) Tissue-specific deletion of HIF-1α caused by CD19cre expression. Southern blot analysis of DNA from different organs of HIF-1α null (HIFDFCD19cre) mice was performed. DNA was digested with EcoRI and PstI and probed with a fragment of HIF-1α intron 1 as described elsewhere (27). The positions of DNA with the HIF-1α floxed allele (wt) and DNA in which the HIF-1α allele had been deleted (Δ) are shown to the right of the blot. (B) Increased HIF-1α-deficient B cells in response to mitogen. B cells isolated from HIF-1α null (HIFDFCD19cre) mice were stimulated with anti-IgM antibody (Ab) (10 μg/ml) plus IL-4 (0.5 ng/ml) for 48 h under either normoxia or hypoxia. After the stimulation, DNA was extracted and analyzed as described above for panel A. The data shown are representative results from three different preparations. The values immediately above and below the blot are the percentages of wild-type B cells and HIF-1α null cells, respectively. Note that HIF-1α null cells expanded preferentially in response to mitogen under both normoxia and hypoxia compared to wild-type B cells in culture. (C) Quantification of the proliferative advantage of HIF-1α null B cells in culture. The histogram is a graphic representation of information from panel B. Error bars show standard errors.
FIG. 2.
FIG. 2.
Cell cycle response to hypoxia in HIF-1α null mEFs and B cells. (A) Hypoxic arrest in mEFs. Primary mEF cells from p53 wild-type (p53+/+) and p53 knockout (p53−/−) mice with floxed alleles at the HIF-1α locus were infected with adenovirus expressing either Cre recombinase or β-galactosidase (as a control for adenovirus expression). The cells were incubated first in the presence of either 0.5 or 20% oxygen for 24 h and then with BrdU for 30 min and then subjected to cell cycle analysis. The x axis shows red propidium iodide (PI) fluorescence; the y axis shows green FITC fluorescence associated with anti-BrdU antibody. The percentage of cells positive for BrdU (S-phase cells) is indicated. Note that 0.5% hypoxia induced G1 arrest in wild-type (HIF-1α+/+ p53+/+) mEF cells. A deletion of HIF-1α allowed progress beyond the G1 checkpoint during hypoxia. Primary p53−/− mEF cells demonstrated pronounced hypoxia-induced cell cycle arrest. Inactivation of the HIF-1α gene causes a loss of growth arrest in p53−/− cells similar to that seen in p53+/+ cells. (B) Hypoxic arrest in splenic B cells. B cells isolated from either wild-type (CD19cre) or HIF-1α null (HIFDFCD19cre) mice were incubated with 0.5% (hypoxia) or 20% oxygen (normoxia) for 48 h in the presence of anti-IgM antibody (10 μg/ml) plus IL-4 (0.5 ng/ml) and then were subjected to cell cycle analysis as described above for panel A. The hypoxic responses in p53−/− B cells were also analyzed. The percentage of cells in S phase is indicated. Note that 0.5% hypoxia inhibited G1/S transition in wild-type B cells, while circa 50% of B cells with the HIF-1α gene deleted entered S phase under hypoxia. A deletion of p53 gene enhanced the hypoxia-induced HIF-1α-dependent G1 arrest. However, double-knockout cells do not respond in any fashion to hypoxia. (C and D) Graphic representation of the data shown in panels A and B, respectively. The percentile change in the number of cells in S phase during hypoxia relative to that during normoxia is shown. Error bars show standard errors.
FIG. 2.
FIG. 2.
Cell cycle response to hypoxia in HIF-1α null mEFs and B cells. (A) Hypoxic arrest in mEFs. Primary mEF cells from p53 wild-type (p53+/+) and p53 knockout (p53−/−) mice with floxed alleles at the HIF-1α locus were infected with adenovirus expressing either Cre recombinase or β-galactosidase (as a control for adenovirus expression). The cells were incubated first in the presence of either 0.5 or 20% oxygen for 24 h and then with BrdU for 30 min and then subjected to cell cycle analysis. The x axis shows red propidium iodide (PI) fluorescence; the y axis shows green FITC fluorescence associated with anti-BrdU antibody. The percentage of cells positive for BrdU (S-phase cells) is indicated. Note that 0.5% hypoxia induced G1 arrest in wild-type (HIF-1α+/+ p53+/+) mEF cells. A deletion of HIF-1α allowed progress beyond the G1 checkpoint during hypoxia. Primary p53−/− mEF cells demonstrated pronounced hypoxia-induced cell cycle arrest. Inactivation of the HIF-1α gene causes a loss of growth arrest in p53−/− cells similar to that seen in p53+/+ cells. (B) Hypoxic arrest in splenic B cells. B cells isolated from either wild-type (CD19cre) or HIF-1α null (HIFDFCD19cre) mice were incubated with 0.5% (hypoxia) or 20% oxygen (normoxia) for 48 h in the presence of anti-IgM antibody (10 μg/ml) plus IL-4 (0.5 ng/ml) and then were subjected to cell cycle analysis as described above for panel A. The hypoxic responses in p53−/− B cells were also analyzed. The percentage of cells in S phase is indicated. Note that 0.5% hypoxia inhibited G1/S transition in wild-type B cells, while circa 50% of B cells with the HIF-1α gene deleted entered S phase under hypoxia. A deletion of p53 gene enhanced the hypoxia-induced HIF-1α-dependent G1 arrest. However, double-knockout cells do not respond in any fashion to hypoxia. (C and D) Graphic representation of the data shown in panels A and B, respectively. The percentile change in the number of cells in S phase during hypoxia relative to that during normoxia is shown. Error bars show standard errors.
FIG. 3.
FIG. 3.
Enhanced expansion of HIF-1α null B cells in response to mitogen. (A) Thymidine incorporation upon mitogenic stimulation of isolated B cells under either normoxia or hypoxia. B cells isolated from either HIF-1α null (HIFDFCD19cre) or wild-type (WT) (CD19cre) mice were stimulated with anti-IgM antibody (10 μg/ml) plus IL-4 (1, 0.5, or 0.1 ng/ml) under either normoxia or hypoxia for 48 h. The incubation with [3H]thymidine was performed for the last 12 h. The results shown are the average values for two CD19cre and HIFDFCD19cre mice. Note that B cells in which the HIF-1α gene had been deleted (HIF-1α null B cells) incorporated more thymidine than wild-type cells under either normoxia or hypoxia in response to mitogenic stimulation. (B) Proliferation curves of isolated splenic B cells under normoxia and hypoxia. HIF-1α null B cells show increased growth rates during normoxic and hypoxic culture. Isolated B cells were incubated with anti-IgM antibody (10 μg/ml) plus IL-4 (0.5 ng/ml) under either normoxia or hypoxia for 3 days. Viable cells were counted after staining with trypan blue. The results shown are the average values for two wild-type (WT) (CD19cre) and HIF-1α null (HIFDFCD19cre) mice.
FIG. 4.
FIG. 4.
Hypoxic effects on cell cycle regulatory proteins in primary B cells. (A) Rb hyperphosphorylation in hypoxic HIF-1α null B cells. Isolated B cells were stimulated with anti-IgM antibody (10 μg/ml) plus IL-4 (0.5 ng/ml) under either normoxia or hypoxia and centrifuged, and pellets were collected at the indicated time points after stimulation. Western blotting analysis was performed with antibodies against Rb. As can be seen, at 48 h, hyperphosphorylation occurs in HIF-1α null B cells in hypoxia, but in wild-type cells there is virtually no detectable hyperphosphorylated Rb. (B) Following extended incubation in hypoxic conditions, p27 expression is elevated only in wild-type B cells, not in HIF-1α null B cells. (C) Hypoxia induces cyclin E kinase activity in HIF-1α null B cells after 48 h. Isolated B cells were stimulated, and after stimulation, samples were immunoprecipitated (IP) with the indicated antibodies. Kinase assays were performed as described in Materials and Methods. As can be seen, loss of HIF-1α activity is accompanied by increased cyclin E- and CDK2-associated kinase activity following prolonged hypoxia. (D) Increased cyclin E expression in HIF-1α null B cells under hypoxia. Hypoxia inhibits the mitogen-stimulated accumulation of cyclin E protein in wild-type cells but not in HIF-1α null cells. Cyclin A and CDK2 expression in both cell types is not affected by HIF-1 status.
FIG. 5.
FIG. 5.
Hypoxia-induced p21 and p27Kip1 transcriptional activation is dependent on HIF-1α. Real-time RT-PCR was performed to determine the relative amounts of the target transcription messages in B cells. The data are the means ± standard deviations (n = 3) of the ratio of message expressed under hypoxia compared to that under normoxia after normaliz against the rRNA as an internal control. Note that PGK expression was induced significantly by hypoxic treatment in wild-type cells, but not in HIF-1α null cells. In B cells from wild-type mice, the expression of both p21 and p27 was increased following 48 h of hypoxia, whereas induction in HIF-1α-deficient B cells was completely abolished. ANOVA tests of significance: null cells differ from the wild type at P <0.05 for all three genes.
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