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Molecular and Cellular Biology, June 2007, p. 4133-4141, Vol. 27, No. 11
0270-7306/07/$08.00+0     doi:10.1128/MCB.01867-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Ternary Complex Factor Net Is Downregulated by Hypoxia and Regulates Hypoxia-Responsive Genes ^,

Christian Gross, Gilles Buchwalter, Hélène Dubois-Pot, Emilie Cler, Hong Zheng, and Bohdan Wasylyk^*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France

Received 3 October 2006/ Returned for modification 11 December 2006/ Accepted 23 March 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hypoxia and the Net ternary complex factor (TCF) regulate similar processes (angiogenesis, wound healing, and cellular migration) and genes (PAI-1, c-fos, erg-1, NOS-2, HO-1, and vascular endothelial growth factor genes), suggesting that they are involved in related pathways. We show here that hypoxia regulates Net differently from the other TCFs and that Net plays a role in the hypoxic response in vivo in mice and in cells. Hypoxia induces Net depletion from target promoters, nuclear export, ubiquitylation, and proteasomal degradation. Key mediators of the hypoxic response, the prolyl-4-hydroxylases containing domain proteins (PHDs), regulate Net. PHD downregulation in normoxia leads to Net degradation, and PHD overexpression delays Net downregulation by hypoxia. Net inhibition by RNA interference or mutation leads to altered regulation by hypoxia of the Net targets PAI-1, c-fos, and egr-1. We propose that hypoxia stimulates transcription of target promoters through removal of the repressor function of Net. Interestingly, the hematocrit response to a chemical inducer of hypoxia-like responses (cobalt chloride) is strongly altered in Net mutant mice. Our results show that the Net TCF is part of the biological response to hypoxia, adding a new component to an important pathological and physiological process.

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Hypoxia is a pleiotrophic condition encountered during embryonic development and organogenesis and in many pathological situations, like wound healing, inflammation, and tumorigenesis. In cancer, proliferating tumor cells outstrip the vascular supply, resulting in hypoxia, which induces adaptive changes (7, 22), including activation of the transcription factor hypoxia-inducible factor 1 (HIF-1) (20, 31) and angiogenesis (35). Inhibition of angiogenesis is a novel aspect of cancer therapy, but more precise manipulation of tumor vasculation may improve treatment strategies (27). Elucidation of the molecular mechanisms by which cells respond and adapt to hypoxia is of great interest.

We have identified a new component of the hypoxic response, the Net (Elk-3) transcription factor. Net, together with Elk-1 and Sap-1, forms the subfamily of ternary complex transcription factors (TCFs) that are notably known for their participation in the early response of quiescent cells to growth factor stimulation. Activation of the growth factor-Ras-mitogen-activated protein kinase (MAPK) pathway leads to phosphorylation and activation of Net and the other TCFs. Under basal conditions, Net is a strong transcriptional repressor (8, 15, 19, 30). Net negatively regulates a number of proteins, including c-fos (44) and egr-1 (2, 44), which are involved in the immediate early response; PAI-1, a serpin that regulates matrix remodeling (9); CTP:phosphocholine cytidylyltransferase (41), an enzyme involved in the phosphatidylcholine biosynthesis; and nitric oxide synthase 2 (10) and heme-oxygenase 1 (13), which are both implicated in the inflammatory response. Net is involved in various physiological processes. Net regulates cell migration through repression of PAI-1 (9). Mice that express mutant Net lacking its DNA binding domain exhibit respiratory distress, chylothorax (the accumulation of chyle in the thoracic cage) at autopsy (2), and delayed wound healing due to impaired angiogenesis (51).

The pathways that regulate the repressor function of Net remain to be identified. Several observations suggested that hypoxia could be one of these regulators. The physiological responses mediated by hypoxia and Net, including cell migration, inflammation, wound healing, and angiogenesis, are similar. Moreover, all the identified target genes of Net are known to be regulated by hypoxia (3, 11, 12, 21, 28, 34, 38, 39, 48). In this study, we show that the transcription repressor function of Net is specifically downregulated in hypoxia. Downregulation is mediated by the oxygen sensors, the prolyl-4-hydroxylases containing domain proteins (PHDs) (5), and involves Net degradation at the protein level. Net is required for the induction of gene expression by hypoxia and for the physiological response to a hypoxia mimic in mice. This study adds an additional component to the understanding of the hypoxic response and a potential new dimension to the control of tumor vasculature, providing hope for better treatments.

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Cell culture, transfections, and hypoxia treatment. The culture conditions were as follows: modified Eagle's medium (MEM), 10% fetal calf serum (FCS), and 40 µg/ml gentamicin for mouse skin endothelial (SEND) cells (a generous gift from Kari Alitalo); Dulbecco's MEM (DMEM)-Ham's F-12K medium (1:1), 10% FCS, and 40 µg/ml gentamicin for PC-3 cells; MEM (Eagle), 10% FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 40 µg/ml gentamicin for DU 145 cells; and DMEM, 10% FCS, and 40 µg/ml gentamicin for mouse embryonic fibroblasts, C11 cells, 444 cells, and COS-7 cells. COS-7 cells were transfected with Jet-Pei (Polyplus Transfection) according to the manufacturer's instructions. 444 and SEND cells were transfected with Lipofectamine (Invitrogen) as previously described (9, 44). The hypoxia conditions were 1% O₂ in a ThermoForma model 3110 incubator.

Immunoblotting and antibodies. For immunoblotting, see reference 9. The antibody uses and dilutions were as follows: rabbit anti-mouse Net no. 1996 for immunoprecipitation (IP; 7 µl per IP) and Western blotting (WB; 1/2,000), mouse anti-mouse phospho(Ser364)-Net no. 2F3 for WB (1/2,000), rabbit anti-mouse Elk-1 no. 512 for WB (1/2,000), rabbit anti-mouse Sap-1a no 644 for WB (1/2,000), mouse anti-mouse TATA binding protein (TBP; a gift from L. Tora) for WB (1/5,000), mouse anti--tubulin clone DM 1A for WB (1/10,000) (Sigma), mouse anti-mouse HIF-1 for WB (1/1,000) (Novus Biologicals), mouse anti-mouse hemagglutinin (HA; IGBMC core facilities) for WB (1/1,000), mouse anti-mouse ubiquitin (Ub) for WB (1/1,000) (Santa Cruz Biotechnologies), rabbit anti-mouse phospho-Erk1/Erk2 for WB (1/1,000) (P-ERK; Cell Signaling Technology), rabbit anti-p44/42 for WB (1/1,000) (Cell Signaling Technology), and rabbit anti-green fluorescent protein (anti-GFP) for WB (1/3,000) (Torrey Pines Biolabs, Inc.).

Chemicals. The chemicals were as follows: proteasome inhibitor MG132 (Calbiochem), cobalt chloride (CoCl₂; Alfa Aesar), MEK inhibitor U0126 (Promega), dimethyloxalylglycine (DMOG; Frontier Scientific), desferrioxamine (DFX) methanesulfonate (Sigma); ethyl 3,4-dihydroxybenzoate (Sigma), and cycloheximide (CHX; Calbiochem).

Plasmids. The plasmids were as follows: pTL2-Net, HA-tagged Ub (Ub-HA), pGFP-Net, PAI-1-luciferase reporters (9), pCMV-LacZ (IGBMC core facilities), and pCDNA3-FLAG-PHD1 (a generous gift from Frank S. Lee, University of Pennsylvania School of Medicine, Philadelphia, PA) (24).

Immunofluorescence. SEND cells were grown on coverslips, subjected to hypoxia for 8 h, fixed with 3.7% formaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 3% bovine serum albumin for 40 min. The cells were incubated with a rabbit anti-mouse Net no. 1996 (1/500) antibody for 2 h at 37°C and incubated for 1 h at 37°C with Texas Red-conjugated anti-rabbit antibody (Beckman Coulter). Finally, the cells were stained for 1 min at room temperature with Hoechst (Sigma), covered with coverslips using mounting solution (5% propylgallate in glycerol), and observed with a fluorescence microscope (Leica; magnification, x40).

RNA interference (RNAi). The small interfering RNAs (siRNAs) were as follows: mouse Net siRNA (Dharmacon, Inc.) (9); Human PHD1, PHD2, or PHD3 siRNA (siGENOME SMARTpool reagent; Dharmacon, Inc.); and GL2 luciferase control siRNA (9). SEND cells were transfected with siRNA duplexes (final concentration, 20 nM) by using Lipofectamine (Invitrogen, Carlsbad, CA) or Jet Pei (Polyplus Transfection) according to the manufacturer's guidelines.

RT-PCR. Total RNA was prepared using TRIzol reagent (Invitrogen). Reverse transcription (RT) was performed using a SuperScript II kit according to instructions from Invitrogen. The PCR steps were as follows: 5 min at 95°C and 30 cycles of 45 s at 95°C, 45 s at 60°C, 1 min at 72°C, and 5 min at 72°C. The PCR products were analyzed on a 2% agarose gel. The oligonucleotides were as follows: PHD1, 5'-CTGGGCAGCTATGTCATCAA and 5'-AAATGAGCAACCGGTCAAAG; PHD2, 5'-GAAAGCCATGGTTGCTTGTT and 5'-TTGGGTTCAATGTCAGCAAA; PHD3, 5'-AGATCGTAGGAACCCACACG and 5'-CAGATTTCAGAGCACGGTCA; 28S, see below.

Q-RT-PCR. Total RNA was prepared using TRIzol reagent (Invitrogen). Quantitative RT-PCR (Q-RT-PCR) was performed with the LightCycler system (Roche Diagnostics) and the SYBR green I (Roche Diagnostics) protocol. The reaction mixtures, containing 100 ng of RNA and 1x master mixture (0.5 µM primers, 4 mM MgCl₂, 200 µM deoxynucleoside triphosphates, 1x PCR buffer [Sigma], 1 U/µl Superscript reverse transcriptase [Invitrogen], Taq polymerase [Promega], anti-Taq antibody diluted 1/200 (Taqstart antibody; Clontech), 4.3% glycerol, 0.15 mg/ml bovine serum albumin, and 0.25x SYBR green I), were reverse transcribed for 10 min at 55°C, denatured for 30 s at 95°C, and cycled 40 times, each time for 2 s at 95°C, 10 s at 61°C, and 10 s at 72°C. Amplification specificity was verified by melting-curve analysis, and the data were quantified with LightCycler software. The absence of genomic DNA contamination was verified by repeating the procedure with the same samples without reverse transcriptase. The oligonucleotides were as follows: Net, first couple, 5'-GGCCGAACACACTTTTCCAG and 5'-GATTTCTGAGAGCTGGGGGA; Net, second couple, 5'-ACTAGCCCTGCTCTCTTCAT and 5'-GTTTGTTTCCCCACCACGCC; PAI-1, 5'-CTCCGAGAATCCCACACAG and 5'-ACTTTGAATCCCATAGCATC; c-fos, 5'-AAGGGAACGGAATAAGATGGC and 5'-CAACGCAGACTTCTCATCTTCAA; 28S, 5'-GGCGGCCAAGCGTTCATAGC and 5'-GCCAAGCACATACACCAAAT; egr-1, 5'-GCCGAGCGAACAACCCTA and 5'-TCCACCATCGCCTTCTCATT; VEGF, 5'-GGGGGTACCTGGACCTGGTGTTCTGCGT and 5'-CCACCATTTTGACAAACAGCACAATCACACCTTGCACGAAGAAG.

ChIP. Chromatin IP (ChIP) assays were performed as previously described (9) using a kit from Upstate Biotechnology. IPs were done with 10 µl of rabbit anti-mouse Net no. 1996 antibody or 10 µl of rabbit anti-clathrin heavy-chain H-300 (Santa Cruz Biotechnology). The coimmunoprecipitated DNA was quantified by semiquantitative PCR and quantitative PCR (denatured for 5 min at 95°C and cycled 35 times, each time for 5 s at 95°C, 30 s at 61°C, and 30 s at 72°C). Amplification specificity was verified by melting-curve analysis, and the data were quantified with LightCycler software.

Luciferase assays. SEND cells were transfected in triplicate with Jet Pei (Polyplus Transfection) according to the manufacturer's instructions in six-well clusters with 4 µg of DNA per well containing 1 µg of the PAI-1 reporters, 1 µg pCMV-LacZ, and 2 µg of pBSK. Forty-eight hours after transfection, the cells were subjected to hypoxia. Luciferase activities were measured with the Promega luciferase assay system (according to the manufacturer's protocol) and an EG&G Berthold luminometer and corrected for transfection efficiency by using ß-galactosidase activity as an internal control.

Co-IP. SEND cells treated with MG132 and subjected to hypoxia or transfected COS-7 cells were washed with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail (Roche Diagnostics). Immune complexes were formed by incubating the lysates overnight at 4°C with rabbit anti-mouse Net no. 1996, mixed for 1 h at 4°C with protein A/G Sepharose, washed three times with radioimmunoprecipitation buffer, resuspended in Laemmli buffer, boiled, separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk, incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, visualized with the Pierce enhanced chemiluminescence detection system, and exposed to film.

Animal studies. Age- and sex-matched, 17- to 30-week-old, 129Sv strain wild-type and Net mutant mice were used. Fourteen mice (7 per genotype) were injected intraperitoneally three times per week with 30 mg/kg CoCl₂, and blood samples were collected once a week during 4 weeks. Significance was evaluated by the Student t test.

Hematology and plasma PAI-1. Blood samples were obtained by retro-orbital sinus puncture. The hematological parameters were as follows. Thirty-microliter blood samples were collected in EDTA tripotassium salt microcuvettes (Sarstedt) and analyzed in a blood cell counter (AcT Diff Vet; Beckmann Coulter). For PAI-1, 9 volumes of blood was collected in 1 volume of 0.1 M trisodium citrate and immediately centrifuged at 3, 000 x g for 15 min. The plasma was transferred to a clean plastic tube, and total PAI-1 concentrations were determined by an enzyme-linked immunosorbent assay (MPAIKT-TOT; Innovative Research Inc.).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hypoxia specifically downregulates Net at the protein level. We studied Net regulation by hypoxia in endothelial cells, one of the cell types involved in the hypoxic response. Net protein levels decreased in SEND cells subjected to atmospheric hypoxia (1% O₂ in a hypoxic cell culture incubator), leading to complete loss of Net after 18 h (Fig. 1A). As expected, HIF-1 protein levels increased under the same conditions, peaking after 6 h of hypoxia. The levels of the two other TCFs, Elk-1 and Sap-1a, as well as the internal control TBP remained stable under the same conditions, showing that downregulation is specific for Net. Net degradation in response to hypoxia was not restricted to endothelial cells, since it was also observed in prostate cancer cells (PC3 and DU145), murine embryonic fibroblasts, and immortalized fibroblasts (C11) (Fig. 1B).

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FIG. 1. Specific downregulation of Net protein levels under hypoxic conditions. SEND (A), PC3, murine embryonic fibroblast (MEF), DU145, and C11 (B) cells were placed under hypoxic (Hypx) atmospheric conditions (1% O2), and the levels of HIF-1, Net, Elk-1, Sap-1a, and TBP were determined by WB analysis, as indicated.

In order to determine whether hypoxia affects Net expression at the RNA level, Net mRNA from hypoxia-treated SEND cells was quantitated by real-time Q-RT-PCR. There were relatively small variations in Net mRNA levels (Fig. 2A) (similar results were obtained with a second primer pair [data not shown]), which could not account for the dramatic decrease at the protein level. Under the same conditions, the RNA levels for several other genes (c-fos, PAI-1, vascular endothelial growth factor [VEGF], and egr-1 genes) increased (see below). To confirm that Net is regulated at the protein level, we studied the effect of hypoxia on the expression of Net from a heterologous promoter (simian virus 40 early region in pSG5) in the presence of an inhibitor of protein synthesis (CHX). The expression of the fusion protein GFP-Net in hypoxia was reduced compared to that in normoxia and compared to the expression of GFP alone (Fig. 2B and C). These results show that hypoxia downregulates Net at the protein level.

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FIG. 2. Net protein stability is decreased in hypoxia (Hypx). (A) mRNA from cells incubated in 1% O2 for 0 to 18 h was measured by Q-RT-PCR and normalized to 28S rRNA levels. (B, C) SEND cells were transfected with GFP or GFP-Net expression vectors, treated with CHX (50 µM) for 0 to 10 h as indicated, analyzed by WB for GFP and GFP-Net levels with a GFP antibody (B), and quantified with ChemiGenius XE (Syngene) (C).

PHDs are involved in Net degradation. PHDs are cellular sensors of oxygen that require oxygen, iron, ascorbate, and 2-oxoglutarate to hydroxylate HIF-1 in normoxia (5). PHDs are inhibited by iron chelators, such as DFX; iron-displacing transition metals, such as cobalt; or synthetic 2-oxoglutarate antagonists, such as DMOG. In cobalt chloride (CoCl₂)-treated cells, Net protein levels decreased in both a dose (Fig. 3A, panel a, 8 h of treatment)- and time (Fig. 3A, panel b, 250 µM of CoCl₂)-dependent manner, whereas HIF-1 levels increased (Fig. 3A, panel b). Other PHD inhibitors (DMOG, DFX, and ethyldihydroxybenzoate) also decreased Net and increased HIF-1 protein levels (see Fig. S1 in the supplemental material). However, these chemicals are not completely specific. To confirm that the PHDs have a role in Net regulation, we used siRNAs against the three isoforms of the human PHDs. In 444 cells (described previously [44]), PHDs 1, 2, and 3 were efficiently and specifically downregulated, as shown by RT-PCR (Fig. 3B, panel a). Downregulation of PHD2 increased HIF-1 levels, whereas downregulation of the two other isoforms had only slight effects (Fig. 3B, panel b), in agreement with another study (4). In contrast, downregulation of PHD2 as well as PHD3 had little effect on Net, whereas inhibition of PHD1 decreased Net expression (Fig. 3B, panel b). Overexpression of PHD1, which can counterbalance diminution of activity due to the lack of oxygen in hypoxia (32), delayed Net hypoxic downregulation (Fig. 3C, compare lanes 2, 3, and 4 with lanes 8, 9, and 10). These results implicate PHDs, and in particular PHD1, in Net regulation in hypoxia.

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FIG. 3. PHDs are involved in Net regulation. (A) SEND cells were treated with cobalt chloride (0 to 250 µM) for 8 h (a) and 250 µM cobalt chloride (b) for 0 to 12 h, and extracts were analyzed by WB. (B) 444 cells were transfected with anti-PHD1, -PHD2, or -PHD3 siRNAs; PHD RNA levels were estimated by RT-PCR (a); and HIF-1, Net, and -tubulin protein levels were analyzed by WB (b). (C) SEND cells were transfected with an expression vector for PHD1 and subjected to hypoxia (Hypx) (1% O2) for different times. The protein levels of Net and -tubulin (Tub) were analyzed by WB.

Hypoxia induces Net nuclear export. Many nuclear proteins that are degraded by the proteasome, such as p53, are concomitantly exported from the nucleus (50). Since Net has a nuclear export sequence (16), we studied the effects of hypoxia on its cellular localization. In SEND cells transfected with a GFP-Net expression vector, GFP-Net was nuclear in normoxia and nucleocytoplasmic after 8 h of hypoxia (Fig. 4A) . Similarly, endogenous Net, detected by immunocytochemistry with a Net antibody in nontransfected cells, was nuclear in normoxia and nucleocytoplasmic after 8 h of hypoxia (Fig. 4B). We also studied whether nuclear export and the JNK pathway are involved in hypoxia-induced Net degradation. Inhibition of nuclear export with leptomycin B (LMB) delayed hypoxia-induced Net degradation (Fig. 4C, compare lane 5 with lane 4 and lane 7 with lane 6), suggesting that nuclear export is involved, at least in part, in Net degradation. In contrast, inhibition of the JNK pathway with SP600125 did not affect Net levels (Fig. 4D), indicating that JNK signaling is not involved in hypoxia-induced Net degradation.

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FIG. 4. Nuclear export contributes to hypoxia downregulation of Net in SEND cells. (A) Cells were transfected with a GFP-Net expression vector, incubated 24 h later in normoxia or hypoxia (1% O2) for 8 h, stained with Hoechst, and visualized by fluorescence microscopy (magnification, x40). (B) Cells were incubated in normoxia (Normx) or hypoxia (Hypx) (1% O2) for 8 h, analyzed by immunocytochemistry with the Net antibody, stained with Hoechst, and visualized by fluorescence microscopy (magnification, x40). (C, D) Cells were incubated in hypoxia (1% O2) for 0 to 18 h and treated with inhibitors of nuclear export (2 ng/µl LMB) or JNK1-3 (10 µM SP600125), as indicated, and analyzed by WB with Net and TBP antibodies.

Hypoxia induces ubiquitylation and proteasomal degradation of Net. We used different protease inhibitors to elucidate the pathway of Net degradation. Whereas lysosomal and calpain inhibitors had no effect (data not shown), the proteasome inhibitor MG132 prevented Net protein degradation in hypoxia (Fig. 5A). The increase in Net levels (Fig. 5A, compare lanes 1 and 3) suggests that MG132 also inhibits Net turnover under normal conditions. Since proteasomes degrade ubiquitylated proteins (23), we studied whether Net can be ubiquitylated. COS-7 cells were transfected with Net and Ub-HA expression vectors and treated with MG132 as indicated, and extracts were analyzed by WB (Fig. 5B, panel a). Coexpression of Net with Ub-HA led to the appearance of a series of bands of increased molecular weights (Fig. 5B, panel a, lane 3) that was enhanced by the proteasome inhibitor (Fig. 5B, panel a, lane 4), consistent with them being ubiquitylated forms of Net. In order to confirm this hypothesis, Net immunoprecipitates were analyzed with HA antibodies by WB. The higher-molecular-weight forms were detected only with COS-7 cells expressing both Net and Ub-HA (Fig. 5B, panel b, compare lane 3 with lanes 1 and 2), suggesting that Net can be ubiquitylated in vivo. To determine whether endogenous Net ubiquitylation is enhanced in hypoxia, we submitted SEND cells to 12 h of either hypoxia or normoxia and analyzed Net immunoprecipitates by WB with an Ub antibody (Fig. 5C). Ubiquitylated higher-molecular-weight forms of Net were detected in Net immunoprecipitates after 12 h of treatment with MG132 (Fig. 5C, panel b, lanes 2 and 3), and the levels of ubiquitylated Net were higher in hypoxia than in normoxia (Fig. 5C, panel b, lanes 2 and 3). This confirms that hypoxia stimulates the ubiquitylation of Net, which presumably accelerates proteasomal degradation. Since many proteins are phosphorylated prior to ubiquitylation (18), we tested whether Net phosphorylation by the extracellular signal-regulated kinase (ERK) pathway is required for degradation. SEND cells were incubated with the potent MEK inhibitor U0126 in hypoxia (Fig. 5D). U0126 decreased ERK phosphorylation but did not prevent Net degradation, showing that ERK phosphorylation, under these conditions, is not necessary for the decrease of Net levels in response to hypoxia.

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FIG. 5. Hypoxia-induced Net ubiquitylation enhances Net proteasomal degradation. (A) SEND cells were incubated under hypoxic (Hypx) conditions (1% O2) with or without 35 µM MG132 for 0 to 12 h. (B) COS-7 cells were transfected with Net and Ub-HA expression vectors and treated with 35 µM MG132 for 6 h, as indicated. The extracts were analyzed directly (a) or after IP with Net antibodies (b) by WB. (C) SEND cells were incubated for 12 h in hypoxia (1% O2) and treated with 35 µM MG132, and extracts were analyzed directly (a) or after IP with a Net antibody (b) by WB. (D) SEND cells were treated with hypoxia (1% O2) and U0126 for 0 to 12 h and analyzed by WB.

Hypoxia induces loss of Net from target gene promoters. In order to determine whether hypoxia decreases the amounts of Net on the c-fos (44) and PAI-1 (9) promoters, which are known to bind Net in normoxia, we performed ChIP assays with SEND cells by using antibodies against Net as well as against clathrin, which does not bind to DNA (as a negative control). The products were analyzed by both semiquantitative (data not shown) and quantitative PCR. As expected, in normoxia, Net was detected on the serum response element-containing region (–338 to –153) of the c-fos promoter (Fig. 6A, panel a) and the proximal region (–715 to –367) of the PAI-1 promoter (Fig. 6A, panel b). The negative control did not give specific products (data not shown). After exposure to hypoxia, binding of Net was lost from both promoters (Fig. 6A, panels a and b). The loss of Net as a transcriptional repressor could account for the hypoxic induction of these genes.

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FIG. 6. Effect of hypoxia on binding of Net to the c-fos and PAI-1 promoters and regulation of PAI-1 promoter activity. (A) SEND cells incubated in hypoxia (1% O2) for the indicated times were analyzed by ChIP with Net and clathrin (control) antibodies. Input and coprecipitated (IP) DNA samples were analyzed by quantitative PCR, using primers for regions of the c-fos (–338 and –153) (a) and PAI-1 (–715 and –367) (b) promoters that contain Ets binding sites. The values were normalized to those for the inputs. (B) SEND cells were transfected with PAI-1 promoter luciferase reporters containing wild-type or mutated Net binding sites, and 48 h after transfection, the cells were incubated in hypoxia (1% O2) for 0 to 24 h. Luciferase activity was corrected for ß-galactosidase activity expressed from the pCMV-LacZ internal control.

Net is required for hypoxia induction of PAI-1 promoter activity. To study Net regulation of PAI-1 promoter activity in response to hypoxia, we used a luciferase reporter with mutations that inhibit Net binding (9) (Fig. 6B, panel a). SEND cells were transfected with either wild-type or mutated reporters, and after 48 h, the cells were incubated in hypoxia for up to 24 h. In normoxia, the activity of the mutated PAI-1 promoter was three times higher than that of the wild type, as expected for loss of transcriptional repression by Net (Fig. 6B, panel b, 0 h). However, after 18 h of hypoxia, both promoters exhibited similar activities, as expected for hypoxia-induced relief of repression by Net, but only for the wild-type promoter. It appears that hypoxic induction of the wild-type promoter is due to relief of repression by Net, among other factors, whereas the mutated promoter is induced only by the other factors, explaining the similar activities of the two promoters after 18 h of hypoxia. When luciferase activities were expressed relative to normoxic levels (0 h, induction), wild-type promoter activity increased about 11-fold after 18 h of hypoxia, whereas mutant promoter activity increased only about 4-fold (Fig. 6B, panel c). These results show that the Net binding sites are required in part for the induction of PAI-1 promoter activity by hypoxia and suggest that the loss of Net repression participates in this response.

Net is required for the hypoxic induction of target genes. To examine whether Net is required for the induction of PAI-1, c-fos, egr-1, and VEGF expression, we decreased Net levels with siRNA before incubation in hypoxia. Anti-Net siRNA abrogated Net protein expression (Fig. 7A, lane 3) in comparison with control siRNA (Fig. 7A, lane 2) or nontransfected (Fig. 7A, lane 1) cells. Hypoxia induced Net downregulation to similar extents in the nontransfected and control-transfected cells (Fig. 7A, lanes 4, 5, 7, and 8). mRNA levels were measured by Q-RT-PCR (Fig. 7B). In normoxia, at 0 h, Net is a transcriptional repressor of PAI-1, c-fos, and egr-1 and an activator of VEGF (Fig. 7B, panel a), as already shown by previous studies (2, 9, 44, 51). Hypoxia induced a gradual increase in PAI-1 expression that was reduced by prior downregulation of Net, from about six- to twofold, after 24 h (Fig. 7B, panel b). Hypoxia induced a more rapid increase in c-fos expression, which was about sixfold at the peak (6 h) and then gradually decreased. The increase was less pronounced and only about threefold in cells lacking Net (Fig. 7B, panel c). The apparently more complex effect of hypoxia on Egr-1 expression was strongly inhibited by downregulation of Net (Fig. 7B, panel d). Finally, the induction of VEGF was not significantly affected by prior downregulation of Net (Fig. 7B, panel e). These results show that Net contributes to hypoxia induction of PAI-1, c-fos, and egr-1 expression and is not required for induction of VEGF.

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FIG. 7. Net is required for hypoxic induction of target genes. SEND cells were transfected with siRNAs against Net and luciferase (negative control), and 48 h later, the cells were incubated in hypoxia (1% O2) for 0 to 24 h. Protein extracts were analyzed by WB (A) and mRNA levels by Q-RT-PCR, and the levels were normalized to 28S rRNA levels (B). (a) The mRNA levels at 0 h in normoxia for cells transfected with siRNA Net were normalized to the control siLuc values. (b to e) The mRNA levels at various times in hypoxia were normalized to the levels observed at 0 h before induction. NT, not transfected.

Net plays a role in the response to a hypoxia mimic in mice. We studied the effect of Net mutation in mice on PAI-1 expression induced by cobalt chloride, which is often used as a hypoxia mimic in small rodents (37, 47). CoCl₂ (30 mg/kg) enhanced PAI-1 levels in plasma during 4 weeks (Fig. 8A, panel a). Overall, averaging all the data revealed a slight but significant decrease in PAI-1 induction in Net mutant mice (Fig. 8A, panel b). We also analyzed several hematological parameters that are known to be induced by hypoxia. As expected, hemoglobin and hematocrit were induced by CoCl₂ (Fig. 8B, panels a and b). Interestingly, there were marked differences (P < 0.05) between wild-type and Net mutant mice in levels of both hemoglobin and hematocrit (Fig. 8B, panels a and b). Treatment with CoCl₂ induced both hemoglobin and hematocrit levels about 5 to 10% in wild-type mice and 20 to 25% in mutant mice (Fig. 8B, panel c). There were no significant differences in the numbers of platelets (Fig. 8B, panel d). These results show that Net is involved in regulation of hematological parameters in vivo.

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FIG. 8. Impaired response to a hypoxia inducer in Net mutant mice. (A) PAI-1 plasma levels. (a) Wild-type and Net mutant mice were injected intraperitoneally for 4 weeks with CoCl2 (30 mg/kg), plasma was collected every week, and PAI-1 levels were determined by an enzyme-linked immunosorbent assay. (b) Average PAI-1 levels for all the treated mice shown in panel a. *, P < 0.05 (Student t test). (B) Hematological parameters. Wild-type and Net mutant mice were injected intraperitoneally for 1 week with saline or CoCl2 (45 mg/kg). Blood samples were collected before and after treatment. Hematological parameters, including those for hemoglobin (a), hematocrit (b), and platelets (d), were analyzed. (c) Inductions (%) of hemoglobin (Hb) and hematocrit (Ht) were calculated. *, P < 0.05 (Student t test).

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In this study, we have shown that hypoxia downregulates the TCF Net in several cell types without affecting the two other members of the subfamily (Elk-1 and Sap-1a). Hypoxia enhances Net ubiquitylation, nuclear export, and subsequent proteasomal degradation. Net degradation in hypoxia is inhibited by overexpression of PHD1, and degradation in normoxia is induced by PHD1 downregulation, mimicking the loss of activity that occurs in hypoxia. These results indicate that the cellular oxygen sensor PHD1 is involved in Net regulation. Hypoxia induces loss of Net from the PAI-1 and c-fos gene promoters, and the Net binding sites of the PAI-1 promoter are necessary for hypoxic induction of promoter activity. Prior inhibition of Net by RNAi decreases hypoxic induction of PAI-1, c-fos, and egr-1. These results suggest that stimulation of transcription of these genes by hypoxia results in part from relief of transcription repression by Net. Our results also implicate Net in physiological responses to hypoxia in vivo, since Net mutant mice display altered responses to the hypoxia inducer cobalt chloride in terms of both plasma PAI-1 and hematocrit levels. In summary, this study implicates the TCF Net in the transcriptional and biological responses to hypoxia.

The transcriptional repressor Net can be switched to an activator upon phosphorylation. Previous studies have shown that posttranslational modifications, especially phosphorylation, play a key role in the cellular response to hypoxia (29). The transcription factor TAL1/SCL is ubiquitylated in hypoxia after phosphorylation by MAPK (42). The transcriptional coactivator CREB is ubiquitylated after hypoxic repression of protein phosphatase 1 (43). HIF-1 phosphorylation by p44/p42 MAPKs enhances its transcriptional activity (6, 36). Net is known to be phosphorylated through the ERK pathway (19). Interestingly, a specific inhibitor of the ERK pathway did not prevent Net degradation, suggesting that the ERK phosphorylation of Net is not required for hypoxia-induced degradation. Moreover, under our conditions, we did not detect hypoxia-induced changes in phosphorylation of Net on one of the important MAPK phosphorylation sites (serine 364). However, we cannot exclude that phosphorylation of Net could participate in the response to hypoxia, for example, at time points or sites that we have not tested.

The PHDs regulate HIF-1 protein levels by hydroxylation of specific proline residues (25, 26). We show here that the PHDs also regulate Net protein levels. Inhibition of PHDs by different chemicals as well as by RNAi leads to downregulation of Net in normoxia. Furthermore, overexpression of PHD1 in hypoxia delays Net downregulation. These results suggest that decreased PHD activity in hypoxia, resulting from decreased cellular oxygen concentrations, is involved in Net downregulation. Under our experimental conditions, only PHD1 seems to regulate Net. Cellular localization of the PHDs may contribute to this apparent specificity. Net is lost from target promoters in the nucleus, suggesting that PHD regulation may occur in the nucleus. PHD1 is exclusively nuclear (32), PHD3 is distributed homogenously in the cytoplasm, and PHD2 is mainly in the cytoplasm but can shuttle between the cytoplasm and the nucleus. HIF-1 appears to be mainly regulated by PHD2 (4), although some studies suggest that all three isoforms can hydroxylate HIF-1 with different efficiencies. Similarly, all three PHDs could regulate Net, but with different relative activities, depending on the cell type and the relative abundances of the PHDs (1). We are currently testing the possibility that Net is hydroxylated directly by the PHDs, using various approaches, including mass spectrometry.

Several observations raise the possibility that relief of Net repression by hypoxia may also involve its corepressor CtBP1, which interacts with the CID domain of Net (15). CtBP is upregulated in lung cancer cells in hypoxia (49), and high concentrations of NADH in hypoxia inhibit the interaction of hCtBP2 with Hdm2, resulting in relief of transcriptional inhibition of p53 (33). We did not detect changes in CtBP1 levels in hypoxia (data not shown). Furthermore, ChIP experiments indicate that Net is depleted from target promoters in response to hypoxia, which could be sufficient for relief of repression. However, we cannot exclude the possibility that the effects of hypoxia on CtBP1 could also modulate Net repression under certain conditions. Several studies have shown that hypoxia induces sumoylation and changes the balance between the sumoylation and ubiquitylation of several factors (14, 40). Net can be ubiquitylated (this study) and sumoylated, and sumoylation increases repression by Net (45). We did not detect any obvious changes in Net mobility in response to hypoxia that might correspond to sumoylation (data not shown). However, the possibility that hypoxia induces alterations in both the extents and the balance of Ub and SUMO modifications of Net needs to be investigated further. Interestingly, sumoylation can induce LMB-sensitive nuclear export of the Ets family transcription factor Tel (46). Nuclear export appears to be involved in Net degradation, since an inhibitor of nuclear export decreases but does not prevent degradation. Nuclear export could enhance Net degradation by involving both nuclear and cytoplasmic proteasomes. Moreover, nuclear export could contribute, along with degradation, to the loss of Net promoter binding.

We report here that Net is implicated in the hypoxic induction of c-fos (44), PAI-1 (9), and egr-1 (2). Our findings agree with a previous study that demonstrates that hypoxic induction of the c-fos promoter requires the TCF binding site and the serum response element (34). PAI-1 is known to be induced in hypoxia through HIF-1 (17, 28). We show here that PAI-1 hypoxic induction is strongly impaired by mutation of the Net binding sites in the PAI-1 promoter and Net inhibition by RNAi. Net may similarly mediate induction of egr-1 through the hypoxia-responsive Ets binding sites (48). Interestingly, another Net target gene, the nitric oxide synthase 2 gene (10), also responds to hypoxia (38), suggesting that Net also has a role in this process. HIF-1 is classically considered to be the key transcription mediator of the hypoxic response, raising the question of how Net fits into this already well-characterized pathway. It will be interesting to compare the global contributions of HIF-1 and Net to the transcriptional response to hypoxia, in order to help define their common and distinct physiological roles.

We provide evidence that Net plays a role in the hypoxic response in mice, suggesting that Net has perhaps many target genes involved in the physiological response to hypoxia. Net appears to be involved in hypoxic induction of PAI-1 in vivo, since Net mutant mice display lower plasma PAI-1 levels after CoCl₂ treatment. PAI-1 levels are similar in mutant and wild-type mice in normoxia, suggesting that PAI-1 is regulated by other mechanisms under these conditions. Hypoxic induction of PAI-1 in Net mutant mice is not dramatically impaired, suggesting that there are other compensatory mechanisms in mice in addition to the cellular models that we have studied. Interestingly, we found that Net contributes to the induction of several hematological parameters in mice, such as the hematocrit, raising the possibility that Net has a more global role in the hypoxic response. This observation opens new avenues for exploring the physiological and pathological mechanisms of the hypoxic response in vivo.

We have previously shown that the inhibition of Net in a tumor model decreases tumor mass and angiogenesis and increases hypoxia levels in tumors (51). These observations concord with our current study, which shows that inhibition of Net impairs hypoxic induction of genes involved in adaptive responses to hypoxia. During tumorigenesis, the hypoxic regulation of the TCF Net could be part of the pathways that are involved in tumor progression, such as the angiogenic response to hypoxia.

 
We thank Frank S. Lee for providing the PHD expression vectors, William Magnant (IGBMC) for help in collecting blood samples, Marie-France Champy (MCI) for the blood analyses, Christine Wasylyk for providing mice, the Wasylyk laboratory members and Patricia Marchal for support and encouragement, Marion Poulard for her contribution in the beginning of this project, and the IGBMC core facilities.

C.G. received fellowships from the Ministère de la Recherche et Technologie, the Association pour la Recherche contre le Cancer and the CGE-DKF2 Regional Council of Alsace. G. Buchwalter received a fellowship from AICR (05-390). The Wasylyk laboratory is supported by the Ligue Nationale Française contre le Cancer (Equipe labelisée), the Ligue Régionale (Bas-Rhin) contre le Cancer and the Ligue Régionale (Haut-Rhin) contre le Cancer, the Association pour la Recherche contre le Cancer, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the EU (FP5 Procure project QLK6-2000-00159 and FP6 Prima project no 504587), INCA Cancéropole Grande Est (Axe IV), and AICR (05-390).

 
* Corresponding author. Mailing address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 10142, Illkirch cedex 67404, France. Phone: 33 3 88 65 34 11. Fax: 33 3 88 65 32 01. E-mail: boh{at}igbmc.u-strasbg.fr

Published ahead of print on 2 April 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

These authors contributed equally to this work.

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