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,12,1,
* Liver Laboratories, University of Birmingham, Birmingham, UK;
Institutes of Physiology, University of Zürich, Zürich, Switzerland; and
Medical University Lübeck, Lübeck, Germany
2Correspondence: Liver Laboratories, Clinical Research Block, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. E-mail: d.m.stroka{at}bham.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and a constitutively expressed ß subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1 is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1 expression varies among different organs. In liver and kidney, HIF-1 reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1 is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1ß is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1. Overall, HIF-1 expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.—Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A. H., Bauer, C., Gassmann, M., Candinas, D. HIF-1 is expressed in normoxic tissue and displays an organ specific regulation under systemic hypoxia.
Key Words: hypoxia-inducible factor 1 • ARNT • tissue hypoxia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and HIF-1ß, also known as ARNT (aryl hydrocarbon receptor nuclear translocator) (1)
, which helps to restore oxygen homeostasis at a cellular, local, and systemic level. HIF-1 functions by regulating many of the genes involved in angiogenesis, erythropoiesis, glycolysis, iron metabolism, and cell survival (reviewed in ref 2
). In addition to its role in oxygen homeostasis, HIF-1 has been implicated as a critical factor in the pathogenesis of tumor vascularization, myocardial ischemia, and stroke by having a regulatory role in localized tissue hypoxia prevailing under these conditions.
HIF-1 protein is regulated mainly through the oxygen-dependent proteolysis of its subunit. Under normoxic conditions, HIF-1 is degraded by the ubiquitin-proteasome pathway by binding of the von Hippel-Lindau tumor suppressor protein (pVHL) to the oxygen-dependent degradation domain (ODD) (3
4
5
6
7
8
9)
. It was recently shown that the interaction between pVHL and HIF-1 is regulated through hydroxylation of a proline residue that requires molecular oxygen and Fe2+ (10
, 11)
. Under hypoxic conditions, HIF-1 protein is stabilized and initiates a multistep pathway of activation that includes nuclear translocation, dimerization with its partner HIF-1ß, recruitment of transcriptional coactivators, and subsequent binding to hypoxia response elements of target genes (reviewed in ref 12
).
Although HIF-1 is regulated mainly by oxygen tension, other factors also modulate HIF-1 expression and consequent function. For example, nitric oxide (NO) regulates HIF-1 accumulation (13)
. The cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-) (14)
stimulate DNA binding of HIF-1. The transcriptional activity of HIF-1 can also be enhanced by the activation of p44/42 MAP kinase (15
, 16)
and by trophic stimuli such as serum, insulin, and insulin-like growth factors (IGF-1, IGF-2) (17
, 18)
. Genetic alterations such as overexpression of the v-src oncogene (19)
or inactivation of the tumor suppressor genes p53 (20)
, pVHL (4)
and PTEN (21)
overexpress HIF-1 and enhance transcriptional activity of downstream genes.
In addition to the in vitro studies elucidating the regulation of HIF-1 protein, in vivo studies have determined specific aspects of HIF-1 expression in inflammation, ischemia, anemia, and various cancers (22
23
24
25
26
27)
as well as during development (28)
, all of which may involve oxygen-independent pathways of HIF-1 regulation. Conversely, the temporal and spatial pattern of HIF-1 protein expression in vivo in response to oxygen tension as a single specific physiological stimulus in a healthy organism has not been examined in detail. Therefore, we exposed wild-type C57BL/6 mice to various fractions of inspiratory O2 (FIO2) and determined the kinetics and oxygen-dependent induction of the two subunits, HIF-1 and HIF-1ß in various mouse tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry
Frozen tissues were cut into 6 µm serial sections, dried on a 50°C hot plate for 2 min, and fixed in 4% formaldehyde in PBS (pH 7.4) for 10 min. All antibodies were diluted in 0.05 mol/l Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 and 10% normal sheep serum (NSS). Sections were incubated with our chicken polyclonal anti-HIF-1 antibody (1:50) (29)
or 10% NSS (negative control) overnight at 4°C. A peroxidase-conjugated rabbit anti-chicken IgY antibody (1:100; Pierce, Rockford, IL) was added for 45 min at RT, followed by a peroxidase-conjugated goat anti-rabbit IgG antibody (1:100; Dako, Carpenteria, CA) for another 45 min. Reactions were visualized with DAB (Sigma, St. Louis, MO) containing 0.01% sodium azide. Sections were counterstained with hematoxylin, dehydrated, and mounted in DPX medium.
Protein extraction and immunoblot analysis
Nuclear proteins from tissue were extracted according to a previously described protocol (30)
. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA); equal amounts of nuclear extracts were separated by 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane. Transfer and equal loading of proteins were confirmed by Ponceau S staining. For immunoblotting, the membranes were incubated with either the chicken polyclonal anti-HIF-1 antibody (1:100) (29)
, a rabbit polyclonal anti-ARNT antibody (1:500; Affinity BioReagents, Neshanic Station, NJ), or a rabbit polyclonal anti-Sp1 antibody (1:10,000; Santa Cruz, Santa Cruz, CA) and with the appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence and quantitated using a Gel Doc 2000 scanner with Quantity One software (Bio-Rad).
RNA extraction and Northern blot analysis
Steady-state RNA levels were assayed by Northern analysis from total RNA prepared by the guanidinium thiocyanate method (31)
. RNA was separated on a 1.3% agarose gel containing 0.66 mol/l formaldehyde and transferred onto a nylon membrane (Hybond, Escondido, CA). Mouse full-length HIF-1 , human L28, and mouse VEGF (32)
cDNA fragments were labeled with [-32P]dCTP using a standard random primed Klenow polymerase reaction (Boehringer, Mannheim, Germany). Membranes were hybridized overnight at 42°C in 50% formamide, 10% dextran sulfate, 5x SSPE, 2x Denhardt’s reagent, and 0.1% SDS and washed at 50°C for 1 h with 0.2x SSC/0.1% SDS. Membranes were either exposed to film (X-Omat AR, Kodak, Rochester, NY) with an intensifying screen at -80°C or to PhosphorImaging screens (Molecular Dynamics, Sunnyvale, CA) to quantitate signals with ImageQuant software.
EPO measurements
EPO serum levels were measured by a radioimmunoassay using 125I-EPO (Amersham, Arlington Heights, IL), recombinant human EPO (100 U/µg; Boehringer), and polyclonal antibodies raised against EPO (Boehringer) as described (33)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and HIF-1ß expression. As the FIO2 was decreased from 21% (normoxic) to 6% (hypoxic) in the brain, we observed a gradual increase in HIF-1 protein (Fig. 1
A). HIF-1 protein was already detectable at normoxic conditions (21% O2). Its expression increased when the FIO2 was reduced to 18% and reached maximum intensity at 6%. In comparison, renal and hepatic HIF-1 protein became detectable only in mice exposed to a FIO2 of 6% (Fig. 1B
, 1C
). In consideration of the extreme conditions at 6% O2, mimicking an altitude of 9100 m above sea level, responses to O2 levels below 6% were not tested.
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As expected, the subunit of HIF-1 accumulated in the nucleus in an oxygen-dependent manner. Although the ß subunit of HIF-1 is constitutively present, the nuclear accumulation in response to hypoxia appears to differ between organs. By reincubating the same membranes with an anti-HIF-1ß antibody, we observed in the brain and kidney that HIF-1ß remained unchanged with decreasing FIO2 (Fig. 1A
, 1B
). In the liver, however, HIF-1ß was not detected in normoxic nuclear extracts and was detected only when the FIO2 was decreased to 12%. The strongest expression of hepatic HIF-1ß protein was at a FIO2 of 6%, concomitant with HIF-1 protein expression (Fig. 1C
). An antibody against the transcription factor Sp1 was used to ensure that equal amounts of nuclear proteins were compared.
HIF-1 protein was also detected by immunoblotting in hypoxic heart, spleen, and skeletal muscle (Fig. 1D
) but not in the lung (data not shown). Intriguingly, a strong HIF-1 signal was present in normoxic skeletal muscle (Fig. 1D
). Considering that HIF-1 was detected in the brain and skeletal muscle of normoxic mice, we questioned whether it was present in the other organs at levels below the detection limit of the immunoblot assay. Therefore, we decided to detect and localize HIF-1 protein in normoxic and hypoxic mouse tissue by immunohistochemistry.
Localization of HIF-1 protein in mouse tissue
Various tissues from normoxic mice or from mice exposed to a FIO2 of 6% were analyzed by immunohistochemistry. HIF-1 protein was clearly present in normoxic mouse brain, kidney, liver, and heart (Fig. 2
A–D). HIF-1 protein was detected within each tissue in distinct cell types, and its expression was further increased in response to hypoxia. In the brain, we detected strong HIF-1 staining of the neurons of the cerebral cortex and granular layer of the dentate gyrus and the hippocampus proper (Fig. 3
A, B). In the cerebellum, the Purkinje cells stained positive whereas the neurons of the adjacent granular layer were negative (see Fig. 5
). Throughout the brain, all cells in the white matter, as well as the ependymal and endothelial cells, were negative (Fig. 3A, C, D
). In the kidney, there was nuclear staining of distal convoluted tubules (Fig. 3E
). The glomerular endothelial, epithelial, and mesangial cells (Fig. 3F
) and vascular endothelial and smooth muscle cells were negative (not shown). In the liver, there was widespread nuclear staining of hepatocytes with no pattern of zonal distribution. The endothelial cells of the central and portal veins as well as the sinusoids were negative. There is faint nuclear and cytoplasmic staining of cells in the sinusoidal regions, which may be Kupffer cells (Fig. 3G
). In the heart, the nuclei of myocytes (Fig. 3H
), arterial endothelial, and smooth muscle cells stained positive for HIF-1 (not shown).
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Kinetics of HIF-1 protein expression in vivo
We observed by immunoblotting and immunohistochemical analysis that 1 h of systemic hypoxia was sufficient to increase HIF-1 expression in all tissues examined. In the next step, we used the brain, kidney, and liver to compare the kinetics of HIF-1 expression in the different tissues and to determine the time HIF-1 protein reaches its maximum expression level. After 1 h of acclimatization, mice were exposed to a FIO2 of 6% for 0 to 12 h (brain) or 0 to 6 h (kidney and liver) (Fig. 4
). In the brain, there was an increase of HIF-1 protein after 1 h of hypoxia, with maximum levels observed after 4 to 5 h (Fig. 4A
). These signals decreased after 9 to 12 h to normoxic levels. Compared with the brain, the highest expression of HIF-1 in the kidney and liver was at 1 to 2 h of hypoxia, and the signal declined to undetectable levels after 3 to 4 h (Fig. 4B
, 4C
). Corroborating the observations in Fig. 1
, HIF-1ß nuclear protein levels in the brain and kidney remained unchanged throughout the time course independent of the presence of HIF-1 protein (Fig. 4A
, 4B
). In the liver, nuclear HIF-1ß protein levels were again concomitant with HIF-1 protein expression (Fig. 4C
).
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Paralleling the results obtained by immunoblotting (Fig. 4A
), the kinetics of HIF-1 protein expression could also be observed by immunohistochemistry (Fig. 5
). In the Purkinje cells, a faint HIF-1 immunostaining was observed in normoxic mice. HIF-1 protein began to accumulate in both the nucleus and cytoplasm after 1 h at 6% O2 and increased after 6 h, with strong nuclear and cytoplasmic staining. After 12 h of continuous hypoxia, the intensity of HIF-1 staining decreased to basal levels (Fig. 5A-D
).
To determine whether the decrease of HIF-1 protein expression during hypoxia was regulated at a transcriptional level, steady-state mRNA levels of HIF-1 in brain, kidney, and liver were assayed by Northern blotting (Fig. 6
). However, there was no significant change in HIF-1 mRNA expression of mice exposed for up to 12 h of continuous hypoxia compared with control animals at any of the times tested. HIF-1 mRNA was constitutively expressed with relative levels in the brain and kidney greater than in the liver (Fig. 6A-C
).
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The function of HIF-1 was tested by measuring serum levels of the HIF-1 target gene erythropoietin (EPO). As expected, the EPO serum level was fourfold higher in mice exposed for 6 h to a FIO2 of 6% (133.3±36.8 u/l; n=5) vs. normoxic mice (32.1±4.4 u/l; n=5).
HIF-1 protein degradation on reoxygenation in vivo
In vitro, nuclear HIF-1 protein levels are rapidly decreased within 4–8 min upon reoxygenation. To determine the degradation rate of HIF-1 on reoxygenation in vivo, mice were exposed to a FIO2 of 6% for 75 min and returned to room air. In the brain, HIF-1 protein was half its maximum level after 15 min and decreased to normoxic levels by 60 min whereas HIF-1ß protein levels remained unchanged (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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protein is not expressed in normal human tissue, with the exception of the bone marrow (26
, 27)
. We demonstrated by immunohistochemistry in normal mouse brain, kidney, liver, and heart (as well as skeletal muscle shown by immunoblotting) that HIF-1 protein is present in the nucleus under normoxic conditions. Oxygen concentrations in normal tissue range from 2 to 5%, in vitro, these concentrations produce a dramatic response with respect to HIF-1 activation (34
, 35)
. Nuclear HIF-1 protein was detected in distinct cell types in vivo and its expression was further increased in response to hypoxia. Physiological oxygen tension in tissue probably sustains a low level of HIF-1 protein to maintain a form of tissue homeostasis. It has been suggested that HIF-1 may be present in normal tissue for a basal induction of genes that are necessary to provide the cellular energy requirements. This was supported by the observation that the normoxic basal transcriptional level of HIF target genes is diminished upon targeted disruption of either the HIF-1 or HIF-1ß gene in embryonic stem cells (36
37
38)
. Our finding that HIF-1 protein is present in normal tissue strongly supports this suggestion.
HIF-1 was detected in specific cell types only and was notably more abundant in larger cells of each tissue (e.g., neurons and hepatocytes). It is possible that HIF-1 is present in other cell types, but expressed at undetectable levels as a result of cell size regulation. Cell size, as determined by its RNA:DNA ratio, is determined by the overall degree of transcriptional activity and is an important factor in the tissue distribution of transcription factors (39)
. It has been shown in mice by in situ hybridization that there was enhanced expression of HIF-1 mRNA in the hippocampus proper and the dentate gyrus compared with other areas of the brain (22
23
24)
. Correspondingly, we observed strong expression of HIF-1 protein in the neurons of these two structures. Alternatively, cells negative for HIF-1 may preferentially be expressing other family members, such as HIF-2 (40
41
42
43)
or HIF-3 (44)
.
We observed by immunohistochemistry in hypoxic tissues an increase in both nuclear and cytoplasmic HIF-1 levels. The strong cytoplasmic presence of HIF-1 appears to be a result of enhanced protein stabilization and accumulation before to nuclear translocation. Likewise, cytoplasmic staining of HIF-1 was detected in various adenocarcinomas (45)
, hypoxic differentiated U937 cells (26)
, and normoxic rat hepatocytes (46)
.
HIF-1 expression in vivo is tightly regulated by O2 tension as demonstrated by the rapid decay rate on reoxygenation. In the brain, a slight reduction of oxygen was sufficient to increase HIF-1 protein accumulation. However, to detect an increase of HIF-1 protein in the kidney, liver, heart, and spleen, it was necessary to expose mice to a severe hypoxic condition. Indeed, expression of other family members may be increased since, when comparing the hypoxic induction of HIF-1 to HIF-2 in HeLa cells, HIF-2 was induced by slightly less severe hypoxia (47)
. In our model, HIF-1 protein was never detected in the lung. As previously reported, HIF-1 was identified in ferret lungs ventilated with 1.3% O2, but was undetectable at 7% O2 (48)
. This agrees with our findings that even at the extreme condition of 6% O2, the pulmonary cells were probably still too ‘oxygenated’ to elicit hypoxia-induced up-regulation of HIF-1.
Otherwise, the response to the acute and severe hypoxic stimulus was immediate in all tested tissues, with an induction of HIF-1 protein detected within 1 h of exposure. In the brain, the maximum levels of HIF-1 are reached after 4–5 h of hypoxia, followed by a decrease to basal level between 9–12 h. In the kidney and the liver, HIF-1 expression is highest between 1 and 2 h of hypoxia, then decreases to undetectable levels by 3–4 h. The in vivo response we observed differs from that demonstrated in vitro (35)
, in which the response of HIF-1 is also instantaneous and is sustained up to 18 h. In contrast to homogeneous culture conditions, we assume that, in vivo, systemic and local responses such as regional changes in blood flow and differences in cellular oxygen consumptions may affect the degree of cellular hypoxia and thus the response of individual cell types. Our model uses a severe hypoxic stimulus. Recent studies have shown that tissue-specific changes in protein synthesis are integral for survival under hypoxic/anoxic stress and that a metabolic down-regulation occurs solely within the translational aspect of protein metabolism (49
, 50)
. In our model, there were no significant changes in the steady-state mRNA level in response to hypoxia at all times tested. Therefore, the decrease of HIF-1 protein we observed is probably not regulated at the transcriptional level.
In vivo studies have reported an increase of HIF-1 mRNA in ischemic conditions (23
, 24
, 51)
. However, a significant increase in mRNA was found only after 20 h (24)
. This late induction implies that factors other than hypoxia are necessary for the increase in HIF-1 mRNA. Two putative HIF-1 binding sites were identified in the promoter of the HIF-1 gene (52)
. It was assumed that these putative sites were not functional because HIF-1 mRNA levels in cell lines and in mice were not up-regulated by hypoxia. However, this result does not exclude that HIF-1 binding to its own promoter is just part of a functional DNA complex that, in cooperation with other transcription factors accompanying inflammation and ischemia, is necessary to up-regulate HIF-1 mRNA. Our results suggest that hypoxia alone is not sufficient to increase the steady-state mRNA levels during ischemic conditions.
Our studies demonstrate that in the absence of additional stimuli, hypoxia leads to a timely and spatially distinct pattern of HIF-1 protein expression in vivo. Our findings that HIF-1 is expressed in normoxic organs also indicate that it may have a physiological role in tissue homeostasis.
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ACKNOWLEDGMENTS |
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immunohistochemistry, D. Chilov, J. Kohl, and C. Schirlo for helpful discussions, M. Leuener for critical reading of the manuscript, and P. Spielmann and F. Parpan for excellent technical assistance. This work was supported by the Swiss National Science Foundation (grant #315090897), the Stiftung für Wissenschaftliche Forschung an der Universität Zürich, and the Endowment Fund of the United Birmingham Hospitals. |
FOOTNOTES |
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Received for publication June 13, 2001. Accepted for publication July 12, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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] denotes the point after 1 h of adaptation time (see Materials and Methods). HIF-1
