1 Department of Pediatric Oncology/Hematology, Charité Campus Virchow-Klinikum, Medical University of Berlin, 13353 Berlin, Germany
2 Department of Neonatology, Charité Campus Virchow-Klinikum, Medical University of Berlin, 13353 Berlin, Germany
3 Department of Clinical Molecular Biology, Kyoto University, Kyoto 606-8507, Japan
* Author for correspondence (e-mail: christoph.buehrer{at}charite.de)
Accepted 1 December 2003
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Summary |
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Key words: RBM3, CIRP, Hypoxia, HIF-1
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Introduction |
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The list of genes regulated in response to hypoxia by HIF-1 is ever increasing, and most research in hypoxia has concentrated on the HIF-1 pathway. The central role of HIF-1 is emphasized by its ubiquitous expression, hampering efforts to study alternative pathways regulating gene transcription in response to hypoxia. Serendipitously, we identified a human B-cell acute lymphoblastic leukemia cell line that failed to respond to hypoxia by expression of VEGF, a known HIF-1 target (Forsythe et al., 1996). Further investigations revealed this cell line to harbour a homozygous deletion for HIF-1
. Subsequent gene expression profiling showed that none of the HIF-1 inducible genes was upregulated under hypoxia whereas expression of two related RNA-binding proteins, RBM3 (Derry et al., 1995
) and CIRP (Nishiyama et al., 1997
), significantly increased in response to hypoxia.
Here we demonstrate that mRNA and protein levels of the two RNA-binding proteins, RBM3 and CIRP, increase in response to hypoxia by a mechanism not involving HIF-1. We further show that the oxygen threshold required for RBM3 and CIRP induction differs from that of HIF-1-regulated gene expression. Finally, we report that this novel mechanism of hypoxic adaptation is inhibited by the respiratory chain inhibitors NaN3 and cyanide but is also fully functional in the absence of mitochondria.
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Materials and Methods |
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Reagents
The following pharmacological inhibitors were purchased from Calbiochem-Novabiochem (Bad Soden, Germany): carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2-thenoyltrifluoroacetone 4,4'-trifluoro-1-(2-thienyl)-1,3 butanedione (TTFA), nordihydroguairaretic acid (NDGA) and ebselen; or from Sigma-Aldrich: NaN3, rotenone, antimycin, oligomycin, N-acetyl cystein (NAC), dithiothreitol (DTT), ascorbic acid, pyrrolidine dithiocarbamate (PDTC) and H2O2. Stock solution of inhibitors were prepared by dissolving them in ethanol, dimethylsulfoxide or water, as recommended by the manufacturers.
DNA analysis
Preparation of genomic DNA and PCR were performed as described previously (Taube et al., 1997). The following forward (F) and reverse (R) oligonucleotides (TIB Molbiol, Berlin, Germany) were used for detection of the HIF-1
gene (HIF1A):
Markers for HIF1A flanking chromosomal regions were obtained from the Ensemble Human Genome browser (http://www.ensembl.org/Homo_sapiens/contigview). Markers located proximal to the HIF1A gene are:
Markers located distal from the HIF1A gene are:
ß-globin was used as control:
Mitochondrial DNA was quantitated by real-time PCR according to a previously reported method (Chiu et al., 2003), ß-globin was used as control.
Gene expression profiling
Total RNA was extracted using TriReagent (Sigma-Aldrich) and subsequently purified employing the QiagenRNeasy kit (Qiagen, Hilden, Germany). RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). cDNA synthesis was performed from 9 µl (13.5 µg) of total RNA using a T-7 linked oligo-dT primer, and cRNA was then synthesized with biotinylated UTP and CTP; a detailed description is given elsewhere (Dürig et al., 2003). Fragmentation of cRNA, hybridization to Human Genome U133A oligonucleotide arrays (Affymetrix, Santa Clara, CA), washing and staining as well as scanning of the arrays in a GeneArray scanner (Agilent, Palo Alto, CA) were performed as recommended in the Affymetrix Gene Expression Analysis Technical Manual (Ludger Klein-Hitpass, Institute of Cell Biology, Medical Faculty, University of Essen, Germany). Signal intensities (MAS5 signal) and detection calls for statistical analysis were determined using the GeneChip 5.0 software (Affymetrix). A scaling across all probe sets of all four arrays to an average intensity of 1000 units was included to compensate for variations in the amount and quality of the cRNA samples and other experimental variables. Results show the mean signal intensities determined for the genes overexpressed in hypoxic versus normoxic cells, where a difference is significant at P values less than 0.001.
Ribonuclease protection assay (RPA) and reverse-transcriptase-PCR (RT-PCR)
RPA for HIF1A and HIF-2 (EPAS1) were kindly performed by M. Wiesener (Department of Nephrology and Medical Intensive Care, Charité Medical Center, Berlin) as described (Maxwell et al., 1993
), with parallel hybridization using 30 µg for HIF1A, 30 µg for EPAS1, and 1 µg for RNU6 (U6 small nuclear RNA). 32P-labeled riboprobes were generated using SP6 or T7 RNA polymerase. The templates used yielded protected fragments as follows: 221 bp for EPAS1 (nucleotides 2542 to 2762, accession no. U81984), 255 bp for HIF1A (nucleotides 764 to 1018, U22431), and 106 bp for RNU6 (nucleotides 1 to 107, X01366). After resolution on 8% polyacrylamide gels, quantification was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Signals for HIF1A mRNA and EPAS1 mRNA were normalized to a value of 100 for EPAS1 in Hep3B cells, allowing for the different number of labeled nucleotides in the two protected fragments. For RT-PCR, total RNA was extracted using the QiagenRNeasy kit (Qiagen), transcribed into cDNA by use of Superscript II Reverse Transcriptase (Invitrogen) and random hexamers (Invitrogen) according to the manufacturer's recommendations. The cDNA was amplified by real-time PCR on the LightCycler as previously described (Wellmann et al., 2001
) using the following forward (F) and reverse (R) oligonucleotides (TIB Molbiol, Berlin, Germany):
SYBR Green I was used as a fluorescent dye (Applied Biosystems, Foster City, CA, USA). Quantitation of mRNA expression was carried out by relating the PCR crossing point obtained from probe samples (automatically determined by the LightCycler software 3.3 in the second derivative maximum mode) to the appropriate plasmide calibration curve. Data were normalized against B2M RNA levels and, for experiments with Hepa-1 cells, against RPL13a RNA levels and are given as fold changes compared with the control experiments.
Nuclear run-on assay
Nuclei of HeLa cells were isolated, and run-on transcription experiments were performed by a method modified from Greenberg et al. (Greenberg and Bender, 1997). Briefly, 1x107 cells were collected by scraping and washed. After lysis for 12 minutes in ice-cold nuclear extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (v/v) Nonidet p-40) nuclei were isolated by centrifugation through 10% sucrose in nuclear extraction buffer. Pelleted nuclei were immediately forwarded to run-on transcription. During isolation, nuclear morphology was monitored by phase contrast microscopy. Cell membrane permeability was assessed by Tryptan Blue exclusion. For nuclear run-on analysis, 50 µl of nuclei suspension were incubated for 40 minutes at 30°C in a total of 0.1 ml reaction mixture containing 20 mM Tris-HCl, pH 8.0, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM each of CTP, ATP, UTP, GTP (Promega, Mannheim, Germany) and 20% glycerol. RNase inhibitor (Invitrogen) was included (20 U/vial) to prevent RNA degradation. The reaction was terminated by adding DNase I (10 U/vial) and proteinase K (200 µg/ml) (both purchased from Qiagen). Immediately before transcription a sample of each condition was removed. Total RNA before and after transcription was isolated, and RBM3, CIRP and B2M mRNA were quantitated using real-time RT-PCR, as described above. The extent of RBM3 and CIRP mRNA transcription was determined by subtracting the amount of RBM3 and CIRP mRNA standardized to B2M mRNA prior to transcription from the amounts post transcription. Data are given as the ratio of copies of target gene mRNA to B2M mRNA copies.
Protein extraction and immunoblot analysis
Cell protein extracts were quantified as described previously (Wiesener et al., 1998). For immunoblotting, proteins were separated by SDS-PAGE, blotted on Hybond-P PVDF (polyvinylidine fluoride) membranes (Amersham Biosciences, Freiburg, Germany), and stained with monoclonal anti-HIF-1
antibody (Transduction Laboratories, Lexington, KY), polyclonal anti-RBM3 antibody (Danno et al., 2000
) or polyclonal anti-CIRP antibody (Nishiyama et al., 1997
) as detailed previously. Bound antibodies were detected using secondary antibodies conjugated with horseradish peroxidase (for HIF-1
from DAKO, Ely, UK, and for RBM3 and CIRP from DPC Biermann, Bad Nauheim, Germany) and enhanced chemiluminescence systems (ECL, from Amersham Biosciences).
Determination of cell viability
Apoptotic and necrotic cells were quantitated by use of the phosphatidyl serine detection kit (IQProducts, Groningen, Netherlands). Briefly, cells were sequentially incubated at 4°C for 20 minutes with fluorescein isothiocyanate (FITC)-conjugated Annexin V and for 5 minutes with propidium iodide as suggested by the manufacturer. Stained cells (104 per sample) were analyzed on a FACSCalibur flow cytometer with standard CellQuest software (BD Biosciences, Palo Alto, CA).
Measurement of intracellular ATP
Cellular ATP content was quantitated by luminometry of the luciferin firefly luciferase reaction using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the procedure recommended by the manufacturer.
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Results |
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HIF-1-deficient Z-33 cells increase expression of several genes in response to hypoxia
Z-33 cells were exposed to room air or 1% O2 for 8 hours, and the relative difference in expression of mRNA was analyzed by the U133A human cDNA expression array. Out of more than 15,000 genes analyzed, 9 genes fulfilled both criteria, genes overexpressed in hypoxic versus normoxic cells with P values <0.001 and absolute expression level over 800 units in hypoxia of Z-33 (Table 1A). Although expression of house keeping genes, such as ß-actin (ACTB), ß2-microglobulin (B2M) and RPL13a, was virtually identical for hypoxia and normoxia in both Z-33 and REH (Table 1C), HIF1A was present in REH but not in Z-33, whereas the HIF1A homologue, EPAS1, was dimly expressed in Z-33 and absent in REH cells (Table 1B, Fig. 1). ARNT expression showed little variation in response to hypoxia in both Z-33 and REH (Table 1C). In contrast, HIF1A-dependent genes, such as WT1 (Wagner et al., 2003), RTP801 (Shoshani et al., 2002
), BNIP3 (Sowter et al., 2001
), prolyl 4-hydroxylase
(P4HA1) (Takahashi et al., 2000
), phosphofructo-2-kinase fructose-2,6-biphosphophatase-3 (PFKFB3) (Minchenko et al., 2002
), glucose transporter 3 (GLUT3) (O'Rourke et al., 1996
), DEC-1/Stra13 (Miyazaki et al., 2002
), and VEGF (Forsythe et al., 1996
), were significantly overexpressed in response to hypoxia in HIF1A-competent REH cells but not in Z-33 cells.
The RNA-binding proteins RBM3 and CIRP are induced in response to hypoxia independently of HIF-1
The two related proteins RBM3 and CIRP were among the five most strongly expressed genes displaying increased mRNA transcription in response to hypoxia in both HIF1A-deficient Z-33 and HIF1A-competent REH cells (Table 1A). Therefore, we investigated RBM3 and CIRP regulation at the protein level. Western blot analysis showed strongly increased RBM3 expression in both leukemic cells lines, whereas CIRP increased moderately (Fig. 3A). Hypoxia increased RBM3 and CIRP mRNA (Fig. 3B) as well as protein expression (Fig. 3C) in Hepa-1 wildtype and ARNT-deficient Hepa-1 c4 mutant cells.
The dynamic range of oxygen tension differs between HIF-1-dependent and HIF-1
-independent genes
Increased gene expresssion in response to hypoxia was further characterized in the two adherently growing human cancer cell lines, HeLa and Hep3B. Increased RBM3 and CIRP expression was equally induced by 8% and 1% oxygen but not by the iron chelator DFO (Fig. 4). In contrast, expression of VEGF, a known target of HIF, required 1% oxygen but did not increase when oxygen was lowered to only 8%. DFO was equally potent as 1% oxygen to induce VEGF transcription. Similarly, the transition metal chelator 2,2'-dipyridyl or cobalt chloride induced VEGF but not RBM3 or CIRP (data not shown). Decreasing the incubator temperature to 32°C induced RBM3 and CIRP expression similarly to hypoxia but had no effect on VEGF expression (Fig. 4). As a corollary, protein levels of RBM3 and CIRP increased in HeLa cells after 24 hours of hypothermia (32°C) or hypoxia (1% oxygen) but were unaffected by DFO (Fig. 5).
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The increase of RBM3 and CIRP is sustained during prolonged hypoxia and requires de novo mRNA synthesis
Kinetic analysis of RBM3 and CIRP protein content in HeLa cells showed that both proteins increased continuously during 24 hours of hypoxia (Fig. 6A). The increase of RBM3 and CIRP mRNA was abrogated in the presence of actinomycin-D (Fig. 6B). To demonstrate that the rise of RBM3 and CIRP mRNA in response to hypoxia was mediated by increased transcription, nuclear in vitro run-on assays were performed. Nuclei were isolated from control and hypoxic HeLa cells, and the RBM3/B2M and CIRP/B2M mRNA ratios were determined from samples taken before and after 40 minutes of in vitro transcription. De novo synthesis of RBM3 and CIRP mRNA normalized to B2M mRNA increased by more than threefold in response to hypoxia (Fig. 6C).
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Reduced oxygen tension, not energy depletion, leads to RBM3 and CIRP expression
To investigate whether ATP depletion following hypoxia plays a role in inducing RBM3 and CIRP, we depleted cellular energy stores by reducing the glucose concentration in the medium and blocking the oxidative phosphorylation at various mitochondrial sites. Culturing cells in glucose-free medium for 24 hours decreased intracellular ATP concentrations to 37% of baseline while increasing RBM3 and CIRP expression 1.5-2-fold (data not shown). This effect was additive to that of hypoxia and paralleled changes observed for VEGF expression. However, NaN3 inhibited hypoxia-induced RBM3 and CIRP expression in a dose-dependent fashion. RBM3 expression after hypoxia was reduced by 50% at 5 mM NaN3 and was down to normoxia baseline levels at 10 mM NaN3 for 24 hours. Hypoxia-mediated CIRP induction was even more sensitive to NaN3, with expression reduced by 50% or down to normoxia baseline levels at 1 mM and 5 mM NaN3, respectively (Fig. 7A). In sharp contrast, NaN3 at 1-10 mM was strongly synergistic with hypoxia in inducing VEGF. Under normoxia, NaN3 up to 10 mM by itself only slightly reduced RBM3 and CIRP expression, whereas VEGF remained unchanged. The pan-respiratory chain uncoupler CCCP (15 µM) reduced ATP concentrations to 36% of baseline while simultaneously reducing hypoxia-mediated RBM3 and CIRP induction, inducing VEGF expression under normoxia and superinducing VEGF in response to hypoxia (Fig. 7A). Concentrations of NaN3 or CCCP that reduced overall cell viability by less than 50% (Fig. 7A,B) were able to elicit a robust increase in VEGF mRNA, similarly to hypoxia, while abolishing (NaN3) or diminishing (CCCP) hypoxia-mediated RBM3 and CIRP mRNA expression. A moderate reduction of hypoxia-mediated RBM3 and CIRP expression was observed with the mitochondrial inhibitors targeting complex I (rotenone, 1-5 µg/ml), complex II (TTFA, 20-100 µM), complex III (antimycin, 0.2-1 µg/ml), or oligomycin (10-50 µM), which downstream of complex IV blocks the H+ transporting ATP synthase and the Na+/K+ transporting ATPase (data not shown).
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Hypoxia-induced RBM3 and CIRP expression does not involve changes of cellular redox status
Several drugs known to affect the cellular redox status were tested for their ability to influence RBM3 and CIRP expression. The antioxidants NAC (1-10 mM), DTT (0.5 mM), ascorbic acid (1-5 mM), PDTC (50-100 µM), NDGA (1-10 µM), or ebselen (10-100 µM) had only minimal effects on RBM3 or CIRP expression under normoxic or hypoxic conditions (data not shown). H2O2 (1-10 mM) did not affect RBM3 or CIRP expression under normoxic conditions while increasing VEGF two-to threefold. Hypoxia-mediated RBM3 or CIRP expression was slightly reduced by H2O2 whereas hypoxia-mediated VEGF induction was enhanced (data not shown).
Induction of RBM3 and CIRP in response to hypoxia also occurs in the absence of mitochondria
As the mitochondrial complex IV inhibitors NaN3 and CCCP abolished RBM3 and CIRP induction in response to hypoxia, we assessed the involvement of mitochondria in this hypoxia-response pathway. When HeLa cells were grown in the presence of EB at either 50 or 100 ng/ml, in medium enriched for glucose, pyruvate and uridine, for 6 days, mitochondrial DNA concentrations dropped to 15.3% or 3.9%, respectively, while no change was noted for baseline and hypoxia-induced RBM3 and CIRP transcripts (Fig. 8). In addition, baseline and hypoxia-induced RBM3 and CIRP transcripts did not differ between 143B TK- (mitochondria+) and rho0 (mitochondria-) osteosarcoma cells.
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Discussion |
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CIRP, also known as A18 hnRNP (Sheikh et al., 1997), and RBM3 belong to the hnRNP subgroup of RNA-binding proteins. While CIRP is expressed ubiquitously, RBM3 tissue distribution appears to be more restricted (Danno et al., 1997
). Most notably, both RBM3 and CIRP belong to the small number of genes that show upregulation in response during mild hypothermia (32°C) but not by heat stress (Sonna et al., 2002
). In addition, CIRP induction and translocation from the nucleus to the cytoplasm has also been observed after ultraviolet irradiation (Yang and Carrier, 2001
).
RBM3 and CIRP are poorly characterized proteins that may participate in transcriptional and post-transcriptional events of gene expression. Target genes of CIRP identified after exposure to UV radiation include replication protein A and thioredoxin (Yang and Carrier, 2001). Possibly, CIRP plays a protective role against various stressors by stabilizing specific transcripts involved in cell survival. The RBM3 mRNA 5' leader sequence contains a number of specialized sequences that allow initiation of translation independently of the methylated G nucleotide 5'-cap that is typically used by cells to tag an mRNA molecule for initiation of protein synthesis (Chappell et al., 2001
). RBM3 has been identified in five genes to be highly involved in suppression of cell death in various cell lines (Kita et al., 2002
). In addition, RBM3 was found to be involved in maintaining cytokine-dependent proliferation in the human premyeloid cell line, TF-1 (Baghdoyan et al., 2000
). Thus, the increased intracellular levels of RBM3 and CIRP in response to hypoxia may contribute to the physiological changes enabling cell integrity and survival under conditions of reduced oxygen supply.
The mechanism involved in increasing RBM3 and CIRP mRNA and protein synthesis in response to hypoxia appears to involve enhanced transcription of the genes, as shown by inhibition by actinomycin-D and direct nuclear in vitro run-on assays. Glucose deprivation was able to induce RBM3 and CIRP, but only to levels approximating less than 50% of those seen with hypoxia. Low oxygen tension, but not energy depletion, appears to be the critical event resulting in RBM3 and CIRP induction, as inhibition of the respiratory chain was not able to induce RBM3 and CIRP. On the contrary, CCCP, which depletes cellular energy stores by uncoupling electron transfer of mitochondrial complex I, II, III and IV, diminished the induction of RBM3 and CIRP seen after hypoxia or hypothermia. RBM3 and CIRP induction was totally blocked by NaN3, which more specifically targets complex IV (cytochrome C oxidase) by binding to its heme moiety (Palmer, 1993). In contrast, hypoxia-induced RBM3 and CIRP induction was only moderately reduced by drugs targeting the complex I, II or III of the mitochondrial respiratory chain. However, reduction of mitochondrial DNA to 10% or 3% by culture with ethidium bromide did not alter the hypoxic induction of RBM3 and CIRP, and the hypoxic inducibility of RBM3 and CIRP was comparable in osteosarcoma cells devoid of mitochondria and wild-type osteosarcoma cells, arguing against a role for mitochondria in mediating the RBM3/CIRP response to hypoxia. As NaN3 and CCCP display strong affinity for heme (Palmer, 1993
), we propose that the O2 sensor governing hypoxic expression of RBM3 and CIRP involves a heme-containing protein (Rodgers, 1999
).
Requirements for RBM3 and CIRP induction by hypoxia differ in several points from those described for HIF-1-dependent mechanisms. First, iron chelators or divalent transition metal ions replacing ferrous iron from the HIF-prolyl hydroxylase were ineffective in inducing RBM3 or CIRP, while inducing HIF-1 or HIF-1-regulated genes such as VEGF. Second, the threshold of oxygen tension evoking gene transcription was found to be equivalent to 8% oxygen for CIRP and RBM3, as opposed to 1% oxygen for HIF-1. Third, RBM3 and CIRP induction by hypoxia was abolished by CCCP or NaN3 whereas both mitochondrial inhibitors strongly superinduced hypoxia-mediated VEGF induction.
HIF-1-independent induction of gene expression has also been described for inhibitor of apoptosis protein 2 (IAP-2) (Dong et al., 2001). IAP-2 expression is not induced by iron chelators or divalent transition metal ions, glucose deprivation, or pharmacological inhibition of mitochondrial respiration. However, IAP-2 induction was observed only after virtually complete lack of oxygen. Whereas RBM3 and CIRP, as well as HIF-1-regulated genes, required several hours of hypoxia, IAP-2 expression occurred within the first hour of oxygen depletion. Thus we propose that there are at least three different mechanisms enabling mammalian cells to respond to hypoxia in a graded fashion. Moderate hypoxia induces transcription of RBM3 and CIRP, more pronounced hypoxia leads to the stabilization of HIF-1 that subsequently upregulates the transcription of a variety number of genes such as VEGF or erythropoietin, and severe anoxia induces IAP-2.
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Acknowledgments |
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