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Hypoxia rapidly activates HIF-3 mRNA expression ¹

Institute of Experimental and Clinical Pharmacology and Toxicology, University of Luebeck, D-23538 Luebeck, Germany

²Correspondence: Institute of Experimental and Clinical Pharmacology and Toxicology, University Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany. E-mail: heidbred{at}medinf.mu-luebeck.de

SPECIFIC AIMS

Although the pivotal role of the transcriptional complex hypoxia-inducible factor 1 (HIF-1) for protection against hypoxia is well documented, the expression patterns and functional properties of HIF-2 and HIF-3 remain to be elucidated. The present study addressed early in vivo changes of expression of HIF-1, HIF-1ß, HIF-2,, and HIF-3 during moderate hypoxic conditions and their correlation with the expression of the HIF target genes erythropoietin (EPO) and glucose transporter 1 (GLUT1).

PRINCIPAL FINDINGS

1. HIF-1, HIF-1ß, HIF-2, and HIF-3 are expressed during normal O₂ tensions in an organ-dependent manner
By quantitative real-time RT-PCR and immunoblotting, physiological levels of expression of HIF-1, HIF-1ß, HIF-2, and HIF-3 were determined in cerebral cortex, hippocampus, lung, heart, liver, and kidney. Distinct mRNA levels of all HIF subunits were detectable in these tissues during normoxia whereby the amounts of mRNA appeared to differ in an organ-specific fashion (Fig. 1 A–D). The cerebral cortex, hippocampus, and lung featured highest mRNA levels regarding all HIF subunits. In lung tissue, abundant amounts of all HIF mRNAs were present. In contrast, myocardial tissue and the liver contained minor amounts of all HIF subunits mRNAs. Referring to the absolute numbers of HIF-1, HIF-1ß, and HIF-2, all examined organs yielded HIF-3-specific mRNA amounts less than the other subunits.

View larger version (23K): [in this window] [in a new window]   Figure 1. Analysis of HIF-1 (A), HIF-1ß (B), HIF-2 (C), and HIF-3 (D) mRNA levels in cortex, hippocampus (hippo), lung, heart, liver, and kidney of male Wistar rats during normoxic conditions (baseline expression), obtained by real-time RT-PCR. Data are presented as means ± SE in a logarithmic scale.

The mRNA expression patterns were paralleled by results of immunoblots, where even in normoxia a clear presence of all HIF proteins in the brain and the lung became visible. In heart, liver, and kidney, HIF-1, HIF-1ß, HIF-2, and HIF-3 proteins were not detectable (data not shown).

2. Moderate hypoxia is capable of activation of the HIF target genes EPO and GLUT1
Healthy male Wistar rats were exposed to half of normal O₂ tensions (65 mmHg) for intervals of 0.5 or 2 h. In contrast to earlier studies that applied severe and/or long-lasting hypoxia either in vivo or in cell culture experiments, we simulated a more physiological in vivo situation using O₂ tensions that corresponded to values observable in pulmonary diseases. GLUT1 and EPO were chosen as established target genes of HIF to reflect the extent of activation during this moderate hypoxia. EPO mRNA was not detectable during normoxic conditions apart from the kidney, where low mRNA amounts were observed. As expected, a pronounced increase of EPO mRNA occurred in the kidney after 2 h of hypoxia. In contrast, GLUT1 mRNA was already traceable in normoxic animals. High amounts of GLUT1 mRNA were present in cerebral cortex, hippocampus, and lung. When hypoxia was terminated after 2 h, a rise of GLUT1 mRNA was observable in all organs and was significant in cerebral cortex, hippocampus, liver, and kidney.

3. Moderate hypoxia causes a rapid increase of HIF-3 mRNA transcripts
Regarding mRNA levels of HIF-1, HIF-1ß, and HIF-2, no significant changes of mRNA amounts were observed during hypoxia (Fig. 2 A–C). HIF-3 presented a completely different expression pattern. Levels of HIF-3 mRNA rose significantly with the duration of systemic hypoxia (Fig. 2 D). This increase was not restricted to specific organs; rather, it occurred in all tissues examined with exception of the liver. Here, HIF-3 mRNA levels showed a trend to increase (150%) that was not significant. In contrast, marked up-regulation occurred in the cortex (430%), hippocampus (630%), lung (460%), kidney (720%), and myocardial tissue (690%).

View larger version (30K): [in this window] [in a new window]   Figure 2. Representative levels of HIF-1 (A), HIF-1ß (B), HIF-2 (C), and HIF-3 (D) mRNAs during normoxic and hypoxic conditions in the cerebral cortex. The left panel presents corresponding numbers of mRNA copies during normoxia (n), 0.5 h and 2 h of moderate systemic hypoxia obtained by quantitative real time RT-PCR. Data presented as means ± SE; *P < 0.05 vs. control, #P < 0.05 vs. 0.5 h. Right panel shows amplified cDNA fragments during normoxia (n) and moderate systemic hypoxia (0.5 h, 2 h) visualized by agarose gel electrophoresis. M denotes a 100 bp DNA ladder, with a brighter band at 500 bp.

CONCLUSIONS AND SIGNIFICANCE

The results of this study clearly demonstrate the presence of HIF-1, HIF-1ß, HIF-2, and HIF-3 during normoxic conditions in all tissues examined, indicating a constitutive expression. This is in line with the suggestion that HIF-1 and HIF-1ß are kept at a certain level of expression that is necessary to maintain target gene transcription for physiological basal energy homeostasis; based on our results, this function may also apply to HIF-2 and HIF-3. The amounts of the various HIF mRNAs depended on the type of tissue. High steady-state mRNA levels of all HIF subunits were determined in brain tissue and the lung, implicating an organ-specific priority that is high in these organs and lower in heart and liver, proposing that in latter organs other or additional mechanisms of protection against hypoxic damage are active. The high priority of brain and lung in the oxygen supply hierarchy is supported by the fact that patterns of GLUT-1 mRNA distribution were similar to that obtained for the various HIF subunits. The highest baseline amounts of mRNA encoding for GLUT1 were again detected in cortex, hippocampus, and lung. GLUT-1 mRNA increased after 30 min of moderate hypoxia and reached significant peaks in the cortex, hippocampus, and liver at the end of 2 h of hypoxia, indicating an efficient activation of the HIF system.

During normoxia, EPO mRNA was traceable only in the kidney and remained undetectable in all other organs during hypoxia. In the kidney, the increase of EPO mRNA after 2 h hypoxia again indicated a sufficient activation by moderate hypoxia. This pattern of activation may point to a sequential activation of the numerous target genes of the HIF system during hypoxia depending on duration and severity of hypoxia.

HIF-1, HIF-1ß, and HIF-2 mRNA levels were not affected by moderate hypoxia, presumably because the hypoxic protocol we used was not severe enough or the interval too short to induce changes of mRNA content of these subunits. In contrast, a significant increase of mRNA encoding HIF-3 came about not only in the brain, but in all other peripheral tissues. The moderate decrease in O₂ tension as used in our model was therefore sufficient to induce HIF-3 mRNA synthesis. At this time, a role of HIF-3 in cellular response to tissue hypoxia has not yet been established in detail. The present immunoblotting results indicate that HIF-3 protein amounts are increasing during hypoxia, denoting a role for this subunit in hypoxia. Despite the structural similarities of HIF-3 to other -class subunits, HIF-3 clearly is regulated in a different manner. It may be possible that this subunit constitutes a more rapidly reacting component of the response to tissue hypoxia or that HIF-3 responds in a more sensitive way than HIF-1 or HIF-2 (see Fig. 3 ). HIF-3 may also be capable of inducing target genes like EPO and GLUT-1, especially in early phases of tissue adaption to low O₂ tensions. The precise regulation of HIF-3 in hypoxia and its potential importance in pathophysiological conditions other than hypoxia have to be determined in further studies.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram of potential implications of the HIF-1, HIF-2, and HIF-3 in various states of hypoxic conditions. Moderate hypoxia induced mRNA transcription of HIF-3 whereas HIF-1 and HIF-2 mRNA level remained unchanged. As a very sensitive and rapidly reacting component of the HIF system, HIF-3 may therefore contribute to protection during early intervals of hypoxia and/or moderate hypoxia. In contrast, HIF-1 and HIF-2 may confer protection against severe and/or prolonged hypoxia.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0963fje; doi: 10.1096/fj.02-0963fje

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