INTRODUCTION

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Reduced tissue oxygen tension levels (hypoxia) play a major role in the regulation of many physiological and pathological processes (for reviews, see references 8 and 55). Adaptive physiological responses to hypoxia include induced expression of genes encoding erythropoietin and vascular endothelial growth factor and activation of genes involved in glucose transport and metabolism (reviewed in references 8 and 55). Moreover, hypoxia is a critical determinant of the pathogenesis of many disorders, including tumor angiogenesis, ischemic heart disease, and stroke. Under hypoxic conditions, the diverse target genes described above are all transcriptionally up-regulated by the transcription factor hypoxia-inducible factor 1 (HIF-1).

HIF-1 is a heterodimeric complex of two basic helix-loop-helix (bHLH)/Per-Arnt-Sim domain (PAS) proteins, HIF-1 and Arnt (54). The HIF-1 protein is rapidly degraded in normoxic cells by the ubiquitin-proteasome pathway but is stabilized under hypoxic conditions (20, 21, 25, 27, 44) and shows hypoxia-induced import into the nucleus (26), thus allowing heterodimerization with the nuclear factor Arnt (12). The active HIF-1 heterodimer binds DNA at conserved hypoxia response elements (HREs) to activate transcription of target genes (55). The mechanism of hypoxia-dependent activation of HIF-1 function is not yet understood. We and others have recently demonstrated that the mechanism of signal transduction by HIF-1 is a multistep process since nuclear translocation of the protein per se is not sufficient for transcriptional activation by HIF-1 (26). In addition, within the nucleus HIF-1 requires hypoxia-dependent activation to enable it to recruit the CREB-binding protein (CBP)/p300 coactivator protein (26), which has previously been shown to play a role in activation of the erythropoietin and vascular endothelial growth factor genes in response to hypoxia (5).

CBP and p300 are ubiquitous, evolutionarily conserved, nuclear phosphoproteins that function, at least in part, by linking several different signal-responsive transcriptional activators to the basal transcription apparatus (28, 29, 32). It has been shown that CBP/p300 can form a complex with SRC-1, a protein originally identified as a coactivator of steroid hormone receptors (15, 28, 42, 60). SRC-1 belongs to a family of 160-kDa proteins that interact with several members of the nuclear receptor family in a hormone-dependent manner and thereby enhance transcriptional activation (50). To date, several structurally and functionally related proteins have been identified, including transcription intermidiary factor 2 (TIF2) (52) (also known as GRIP1 [17]), p/CIP (49) (also known as ACTR [10]), RAC3 (33), AIB1 (2), and TRAM-1 (48). The finding that the p160 coactivators can bind to other type of coactivators, such as CBP/p300 and p/CAF (10, 28, 60), suggests that p160 proteins play an important role in bridging the interactions of receptor activation functions with the basal transcription machinery to stimulate transactivation. Moreover, both the CBP/p300 and SRC-1/p160 classes of coactivators possess intrinsic histone acetyltransferase (HAT) activity which may contribute to locally remodel chromatin structures for better accessibility of the transcription machinery to DNA (6, 10, 41, 47).

The mechanism underlying hypoxia-dependent transcriptional activation by HIF-1 remains unclear. Understanding how functional activity of HIF-1 is triggered by low oxygen tension and how the activated protein is able to mediate transcriptional activation is necessary to elucidate the mechanism of signal transduction by HIF-1. In this study, we demonstrate that two p160 proteins, SRC-1 and TIF2, interact with HIF-1 to enhance its hypoxia-dependent transactivation function. We also show that SRC-1 and CBP/p300 synergistically enhance HIF-1-mediated transcriptional regulation under hypoxic conditions. Moreover, the redox-dependent enzyme Ref-1 appears to be critical in linking the effects of the two coactivator proteins CBP/p300 and SRC-1 to HIF-1.

	MATERIALS AND METHODS

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Plasmids and fusion proteins. Plasmids containing full-length HIF-1 (pCMV4/HIF-1), full-length Arnt (pCMV4/Arnt), and the GAL4-driven luciferase reporter gene have been previously described (14, 26). The Rc/RSV-mCBP-HA construct expressing full-length mouse CBP was obtained from R. H. Goodman (Vollum Institute, Portland, Oreg.), and plasmids pCMV-p300-HA and pCMV-p3001254-1376 were a kind gift from D. M. Livingston (Dana-Farber Cancer Institute and Harvard Medical School, Boston, Mass.). Plasmids containing human full-length SRC-1 and SRC-1PAS cDNAs in pSG5 vector (42) were kindly provided by B. W. O'Malley (Baylor College of Medicine, Houston, Tex.). pSG5/SRC-1 M1234 (16) was obtained from M. G. Parker (Imperial Cancer Research Fund, London, United Kingdom). pCMV5/Ref-1 was produced by inserting the 1.45-kb EcoRI fragment of pBS/Ref-1 (57) (kindly provided by T. Curran, St. Jude's Children Hospital, Memphis, Tenn.) into the EcoRI site of pCMV5. pSG5/TIF2 was a kind gift from P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). pT81/HRE-luc contains three tandem copies of the erythropoietin HRE in front of the herpes simplex thymidine kinase promoter and the luciferase gene (P. Coumailleau, P. Carrero, and L. Poellinger, unpublished data).

Cell culture. COS7 cells (from the American Type Culture Collection) were routinely maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 100 IU of penicillin per ml, 100 µg of streptomycin sulfate per ml, 2 mM L-glutamine, and 1× minimal essential medium containing nonessential amino acids. The human embryonic kidney cell line (293 cells) was cultured in 1:1 mixture of DMEM and F-12 medium supplemented with 10 and 5% FCS, respectively, penicillin-streptomycin sulfate, and nonessential amino acids as described above. All media and growth factors were purchased from Life Technologies, Inc.

Transfection and transient expression assays. One day before transfection, cells were seeded in six-well plates, and the medium was changed to OPTI-MEM medium lacking phenol red (Life Technologies) before transfection. A DNA mixture containing 0.5 µg of luciferase reporter, 0.2 µg of a combination of expression vectors for HIF-1 and Arnt, 0.75 to 1.5 µg of expression vectors for different coactivators, and 0.2 µg of internal control plasmid pRSV-AF together with carrier DNA pCMV4 was prepared and mixed with Lipofectamine (Life Technologies) according to the manufacturer's recommendations. DNA precipitates were allowed to form at room temperature for 30 min and added to the culture. After 6 h of transfection, the medium was replaced with DMEM-10% FCS. Hypoxic induction was achieved by incubation of the cells with 1% O₂, or 21% O₂ for normoxia. After 36 h, aliquots of medium were collected and analyzed for alkaline phosphatase activity, and cells were harvested and extracts were analyzed for luciferase activity. Alkaline phosphatase activity was used to correct for differences in transfection efficiency. All transfections were done in duplicate; results are presented as mean ± standard error (SE).

Assays of GST protein-protein interaction. The analysis of interactions between SRC-1 and HIF-1 bound to glutathione-Sepharose was carried out by the method of Chatton et al. (9), with minor modifications. Approximately 250 µg of whole-cell extracts from COS7 cells transiently expressing GST-HIF-1 was incubated with [³⁵S]methionine-labeled in vitro-translated SRC-1 protein in 250 µl of a low-stringency buffer (50 mM Tris-HCl [pH 7.8], 0.1% NP-40, 250 mM NaCl). The complexes were immobilized on 60 µl of a 50% suspension of glutathione-Sepharose beads (Pharmacia Biotech). After washing, the radiolabeled proteins were eluted, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and detected by fluorography.

	RESULTS

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TIF2 enhances HIF-1 activity and interacts with HIF-1 in a hypoxia-dependent manner in mammalian cells. To gain a better understanding of the mechanism of transcriptional activation by HIF-1, we tested the effect of transient expression of the transcriptional coactivator TIF2 on the functional activity of HIF-1. TIF2 belongs to the p160 family of coactivators which have been shown to be required for nuclear receptor-mediated transcriptional activation (17, 48, 52). Transient transfection experiments were initially performed with COS7 cells. As shown in Fig. 1A, the reporter gene activity was not significantly altered by hypoxic treatment (1% O₂) in the absence of coexpressed proteins, most probably due to the low levels of endogenous HIF-1 activity in COS7 cells (unpublished observations). Upon transient coexpression of HIF-1 and Arnt, pT81/HRE-luc reporter gene activity was stimulated around 10-fold under normoxic conditions, possibly due to a small but detectable pool of HIF-1 localized in the nucleus under these conditions (26). This reporter gene activity showed a modest (~1.5-fold) increase by hypoxia (Fig. 1A). However, as expected (5, 26), ectopic expression of CBP further enhanced hypoxia-dependent activation by HIF-1 in a dose-dependent manner compared with hypoxia-stimulated activity in the absence of exogenous coactivators (two- to threefold). Although expression of CBP slightly stimulated HIF-1 activity already at normoxia, its overall activity was almost entirely dependent on the exposure to hypoxia as has been described previously (5, 26). The effect was most likely limited to threefold due to the large quantities of endogenous CBP already present in the cell prior to transfection. Coexpression of HIF-1 together with TIF2 resulted in potent (around 12-fold) activation of the reporter gene over the levels observed at normoxia, thereby establishing that both TIF2 and CBP support transcription by the HIF-1-Arnt complex (Fig. 1A). In conclusion, TIF2 which was originally identified as a coactivator supporting hormone-dependent transcriptional activation by members of the steroid hormone receptor family, also functions as a conditionally regulated coactivator in hypoxia-dependent transcriptional activation by the HIF-1-Arnt complex.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   TIF2 enhances the HIF-1-mediated transactivation function and interacts with HIF-1 in vivo. (A) COS7 cells were transiently cotransfected as described in Materials and Methods with 0.5 µg of pT81/HRE-luc reporter construct, hHIF1- expression vector (pCMV4/HIF-1; 0.2 µg), Arnt expression vector (pCMV4/Arnt; 0.2 µg), and 0.75 to 1.5 µg of either TIF2 (pSG5/TIF2) or CBP (Rc/RSV-CBP-HA), as indicated. The total amount of DNA was kept constant by the addition of parental pCMV4 when appropriate. Following transfection cells were incubated either under normoxic (21% O2) or hypoxic (1% O2) conditions. Luciferase activities were normalized for transfection efficiency by cotransfection of alkaline phosphatase-expressing pRSV-AF. Data are presented as luciferase activity relative to cells transfected with pCMV4 and reporter gene only and incubated at normoxia. Values represent the mean ± SE of two independent experiments. (B) Intranuclear redistribution of the GFP-HIF-1 fusion protein in the presence of TIF2. GFP-HIF-1 was transfected into COS7 cells in the absence or presence of exogenous TIF2 (pSG5/TIF2). After 24 h of expression, either 100 µM 2,2'-dipyridyl or vehicle (H2O) was added to the culture medium and incubated for 2 h before observation. Subsequently, 2,2'-dipyridyl was withdrawn by washing the cells and changing the medium, and the cells were thereafter incubated for an additional 24 h. Cells were observed microscopically at different time points as indicated by arrows. Photographs were taken with a Zeiss fluorescence microscope.

Regulation of the hypoxia-inducible transactivation function of HIF-1 by SRC-1. As outlined above, TIF2 belongs to a growing family of the p160 family of coactivators including SRC-1. TIF2 and SRC-1 appear to have similar activities as coactivators to enhance the transcriptional potential of many members of the ligand-activated nuclear hormone receptors (18, 28, 42, 52). SRC-1 has been demonstrated to directly and constitutively interact with CBP (28, 60). Moreover, in analogy to CBP (6, 41), SRC-1 has been shown to possess intrinsic HAT activity (10, 47). Against this background we also tested the ability of SRC-1 to modulate HIF-1-dependent transcriptional activation of the minimal HRE-driven reporter gene. In transient transfection experiments using COS7 cells, HRE-dependent reporter gene activity was stimulated from two- to eightfold in a dose-dependent manner by SRC-1, compared with hypoxia-stimulated activity in the absence of exogenous coactivators (Fig. 2A). Transient expression of SRC-1 also enhanced the transcriptional activity of human HIF-1 (hHIF-1) under normoxic conditions, albeit to a lesser extent (around threefold [Fig. 2A]). Thus, SRC-1 and TIF2 have similar properties in enhancing the transcriptional potential of hHIF-1. To determine whether regulation of HIF-1 function by members of the p160 family of coactivators was cell type specific, we next carried out similar transfection experiments with the human 293 embryonic kidney cell line. In these experiments we observed that both SRC-1 (Fig. 2B) and TIF2 (data not shown) can significantly enhance hHIF-1-dependent transcriptional activation in 293 cells.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   SRC-1 stimulates HIF-1 activity in a hypoxia-dependent manner and interacts in vitro with HIF-1. COS7 (A) and human embryonic kidney 293 (B) cells were cotransfected with pT81/HRE-luc (0.5 µg), 0.2 µg of pCMV4/HIF-1 (hHIF1-), 0.2 µg of pCMV4/Arnt (Arnt), and 0.75 to 1.5 µg of SRC-1 (pSG5/SRC-1), as indicated. Six hours after transfection, cells were exposed to either 21 or 1% O2 for 36 h before harvest. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of three independent experiments performed in duplicate ± SE are shown. (C) The SRC-1 PAS domain is not required for functional interaction with HIF-1. (Top) Schematic representation of full-length SRC-1 and SRC-1PAS. (Bottom) pGAL4/HIF 71-826 was cotransfected into COS7 cells together with a reporter plasmid expressing the luciferase gene driven by the thymidine kinase minimal promoter under the control of five copies of GAL4 binding sites and 1.5 µg of an expression vector encoding either full-length SRC-1 or a deletion mutant of SRC-1, SRC-1PAS, lacking the PAS domain. Cells were exposed to 21 or 1% O2 for 36 h before harvest and reporter gene assays. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of two independent experiments performed in duplicate ± SE are shown. (D) In vitro interaction between SRC-1 and HIF-1. COS7 cells were transfected with 10 µg of the expression plasmid pGST-HIF-1 (GST-HIF) or empty GST expression vector (GST). Cells were exposed to either 100 µM CoCl2 or vehicle (H2O) for 24 h. Cell extracts were prepared and incubated with [35S]methionine-labeled in vitro-translated SRC-1 protein. The complexes were immobilized on glutathione-agarose beads for 2 h and eluted with the sample buffer by boiling. The eluted material was analyzed by SDS-PAGE (5% gel) and visualized by fluorography. Lane 1 represents one-fifth of the amount of [35S]methionine-labeled SRC-1 used in the binding reactions. Positions of molecular mass markers are shown on the left in kilodaltons.

Role of LXXLL motifs in HIF-1-SRC-1 interaction. SRC-1 contains four copies of the short sequence motif LXXLL. Three of these motifs were identified in the central domain of the protein, and the fourth motif was found in the eight most C-terminal amino acids of human SRC-1 (16). The LXXLL motifs have been shown to be necessary to mediate the binding of SRC-1 to ligand-occupied nuclear receptors (16, 38, 56). To investigate the role of the LXXLL motifs of SRC-1 in hypoxia-dependent transactivation by HIF-1, we compared the activities of wild-type SRC-1 and SRC-1 M1234, a mutant protein in which the conserved leucine couplets were changed to alanine at residues 636/7, 693/4, 752/3, and 1438/9 (16) in transient transfection experiments. As shown in Fig. 3A, the mutant protein in which all four functional binding motifs are disabled enhanced the activity of GAL4/HIF 71-826 to the same extent as the wild-type SRC-1 under hypoxic conditions. Moreover, transient expression of either wild-type SRC-1 or SRC-1 M1234 together with GFP-HIF-1 protein in COS7 cells had no effect on the subcellular distribution of fluorescence activity at normoxia. Under hypoxic conditions, SRC-1 induced relocalization of GFP-HIF-1 from a uniform nuclear distribution to discrete dot-like structures within the nucleus (Fig. 3B). This effect was also produced by the mutant SRC-1 M1234 protein (Fig. 3B), indicating that both proteins interact with HIF-1 in a hypoxia-dependent manner in vivo. Taken together, these results suggest that in contrast to nuclear receptor signaling, the LXXLL motif is not required for HIF-1-SRC-1 interaction.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   LXXLL motifs of SRC-1 are not required for HIF-1-mediated transactivation function and interaction with HIF-1 in vivo. (A) (Top) Schematic representation of full-length SRC-1 and mutant SRC-1 M1234. Black bars represent the approximate locations of the LXXLL motifs in the linear SRC-1 sequence; circles with numbers indicate sites of mutation of LXXLL motifs in which conserved leucine residues are replaced by alanines. (Bottom) pGAL4/HIF 71-826 was cotransfected into COS7 cells together with a GAL4-responsive reporter plasmid and 1.5 µg of either full-length SRC-1 or mutated SRC-1, SRC-1 M1234 expression vectors. Cells were exposed to 21 or 1% O2 for 36 h before harvest and reporter gene assays. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of two independent experiments performed in duplicate ± SE are shown. (B) Intranuclear redistribution of the GFP-HIF-1 fusion protein in the presence of SRC-1 and SRC-1 M1234. pGFP-HIF-1 was transfected into COS7 cells with or without SRC-1 or SRC-1 M1234 as indicated. After 24 h of expression, either 100 µM 2,2'-dipyridyl or vehicle (H2O) was added to the culture medium and incubated for 2 h. Cells were observed using a fluorescence microscope. Photographs of several cells for each condition were taken and representative cells are shown. Bar = 10 µm.

Definition of the functional domains of HIF-1 which interact with the coactivators CBP and SRC-1. To identify HIF-1 structures which could serve as interaction surfaces with the SRC-1 and CBP coactivator proteins, we constructed a series of GAL4 DBD fusion proteins containing HIF-1 fragments of various lengths (as schematically represented in Fig. 4A). These constructs were transiently transfected into COS7 cells together with a GAL4-responsive reporter gene. Fusion of full-length HIF-1 to the GAL4 DBD produced significant hypoxia-dependent induction of reporter gene activity in the absence of ectopically expressed coactivator proteins (26) (Fig. 4A). Coexpression of either CBP or SRC-1 strongly (around 8- to 10-fold) enhanced the transcriptional potential of HIF-1 in hypoxic cells. These coactivators also increased to a lesser extent the transcriptional activity of GAL4/HIF 1-826 under normoxic conditions. Neither CBP nor SRC-1 enhanced the transcriptional activity of the GAL4 DBD alone (data not shown). GAL4/HIF 71-826, which lacks the bHLH domain of HIF-1, induced relative luciferase activity about 3.5-fold over background levels (Fig. 4A). In the presence of coexpressed CBP or SRC-1, luciferase activity was induced under hypoxic conditions by GAL4/HIF 71-826 to a 30- or 34-fold-higher level, respectively, in comparison with the activity observed at hypoxia in the absence of exogenous coactivators.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Definition of HIF-1 structures which are regulated by the CBP and SRC-1 coactivators. (A) Different subregions of HIF-1 were fused to the GAL4 DBD and transfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of 1.5 µg of either CBP or SRC-1 expression plasmid. Cells were exposed to 21 or 1% O2 for 36 h before harvest. N-TAD and C-TAD, N- and C-terminal transactivation domains. (B) The C-terminal transactivation domain of Arnt mediates transcriptional activation by CBP and SRC-1. COS7 cells were cotransfected with the indicated GAL4-Arnt fusion proteins and 1.5 µg of CBP or SRC-1 expression vectors together with a GAL4-responsive reporter plasmid. Six hours after transfection, cells were exposed to either 21 or 1% O2 for 36 h before harvest and reporter gene assays. (C) HIF-1 prevents Arnt from functionally interacting with CBP and SRC-1. GAL4/Arnt 128-774 and a deletion mutant (GAL4/Arnt 128-603) were transfected into COS7 cells together with 0.2 µg of hHIF-1 expression vector and 1.5 µg of CBP and SRC-1 expression vectors, as indicated. Luciferase activity was measured following 36 h of exposure to either 21 or 1% O2. Normalized reporter gene activities are expressed relative to that of nonfusion GAL4 in normoxia. The results of three independent experiments performed in duplicate ± SE are shown.

SRC-1 and CBP act synergistically to enhance HIF-1-dependent transcription. The ability of either CBP or SRC-1 to enhance the transcriptional activity of HIF-1 prompted us to investigate the effect of coexpression of both coactivators on HIF-1 function. We initially investigated the effects of transient expression of SRC-1 and CBP individually and in combination with one another on hypoxia-dependent activation by HIF-1 of a minimal HRE-driven reporter gene. As observed in Fig. 2A and B, the effect of SRC-1 on HIF-1-dependent activation of the reporter gene was strictly dose dependent in COS7 and 293 cells, respectively. For instance, at a low concentration of SRC-1 expression vector (0.75 µg), no significant effect on HIF-1-dependent activation of the reporter gene was detected (Fig. 2A, 2B, and 5A). In a similar fashion, an identically low concentration of CBP expression vector produced no enhancement of the transactivation function of HIF-1 (Fig. 5A). Under all of these conditions, poor hypoxia-dependent inducibility of the reporter gene was observed. However, a potent, hypoxia-dependent activation response could be reconstituted upon transient coexpression of SRC-1 and CBP at doses which yielded no significant effect individually (Fig. 5A), indicating strong synergy between these two coactivators in enhancing HIF-1 function.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   CBP and SRC-1 cooperatively enhance HIF-1-mediated transcriptional activation. (A) COS7 cells were transiently cotransfected with pCMV4/HIF-1 and 0.75 to 1.5 µg of SRC-1 and/or 0.75 to 1.5 µg of CBP expression plasmids together with a hypoxia-responsive reporter gene (pT81/HRE-luc) and subsequently exposed to 21 or 1% O2. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of three independent experiments performed in duplicate ± SE are shown. (B) Cooperative activation by CBP and SRC-1 of GAL4-HIF-1 fusion proteins. COS7 cells were cotransfected with GAL4-HIF-1 fusion constructs together with a GAL4-responsive reporter plasmid in the absence or presence of CBP and/or SRC-1, as indicated (in micrograms). Cells were exposed to 21 or 1% O2 before harvest. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of two independent experiments performed in duplicate ± SE are shown. (C) p300 protein stimulates the activity of the two minimal transactivation domains of HIF-1 in a hypoxia-dependent manner. (Right) Schematic representation of full-length p300 (pCMV-p300-HA) and p300 (pCMV-p3001254-1376). (Left) The two minimal transactivation domains of HIF-1 were fused to GAL4 DBD and transfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of p300, p300, and/or SRC-1 expression vectors, as indicated. Cells were exposed to 21 or 1% O2 before harvest. Normalized reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of a representative experiment performed in duplicate are shown.

Ref-1 potentiates HIF-1 function in the presence of CBP and SRC-1. The nuclear redox regulator Ref-1 is known to stabilize the DNA binding activity of AP-1 by reduction of a conserved cysteine residue of Fos and Jun. Ref-1 is a bifunctional enzyme: it harbors both redox and endonuclease DNA repair activities (57) and has been implicated in up-regulation of HIF-1-dependent induction of gene expression under hypoxic conditions (13, 20). Against this background, we wanted to further investigate the effect of Ref-1 on HIF-1-dependent transcriptional activation and to examine its effect on the functional interaction of HIF-1 with the coactivators CBP and SRC-1. To investigate whether HIF-1 is a target of regulation by Ref-1, we initially used the different GAL4 DBD fusion proteins containing distinct subfragments of HIF-1. As shown in Fig. 6A, transient overexpression of Ref-1 markedly potentiated hypoxic induction of reporter gene expression by the fusion proteins GAL4/HIF 71-826 and GAL4/HIF 526-826, spanning both transactivation domains of HIF-1. The transcriptional activation function of GAL4/HIF 531-584, containing the N-terminal transactivation domain of HIF-1, was only very moderately but significantly stimulated by Ref-1 in hypoxic cells, whereas Ref-1 produced more potent regulation of hypoxia-inducible transactivation by GAL4/HIF 776-826, containing the C-terminal transactivation domain (Fig. 6A).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Ref-1 enhances HIF-1 function. (A) COS7 cells were cotransfected with different GAL4-HIF-1 fusion constructs together with a GAL4-responsive reporter plasmid in the absence or presence of 1.5 µg of Ref-1 (pCMV5/Ref-1), as indicated. Cells were exposed to 21 or 1% O2 before harvest. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities were expressed as relative to that of GAL4 in normoxia. The results of two independent experiments performed in duplicate ± SE are shown. (B) Ref-1 potentiates CBP and SRC-1 activation of HIF-1. The same GAL4- HIF-1 fusion proteins as shown in panel A were cotransfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of different combinations of Ref-1 (0.75 µg), CBP (0.75 µg), and/or SRC-1 (0.75 µg) expression vector, as indicated. The bottom panel shows an enlargement of the area marked with dots. (C) Effect of hypoxia treatment on subcellular localization of Ref-1. COS7 cells grown on coverslips were transiently transfected with 3 µg of pGFP/Ref-1. After 6 h of incubation, the medium was changed to fresh DMEM supplemented with 10% FCS and incubated for 24 h. Cells were then exposed to 21 or 1% O2 for 4 h. After being washed three times with PBS, cells were fixed with 4% paraformaldehyde in PBS for 2 h at room temperature, subsequently washed three times with PBS, and mounted. Cells were observed with a fluorescence microscope. Representative cells are shown. Bar = 10 µm.

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Ref-1 interacts with HIF-1 in vitro. [35S]methionine-labeled in vitro-translated GAL4/HIF 1-826 (A) and GAL4/HIF 531-584 (N-TAD) or GAL4/HIF 776-826 (C-TAD) (B) were incubated for 30 min at room temperature with recombinant GST/Ref-1 or GST in the presence or absence of diamide as indicated. Bound proteins were eluted in SDS sample buffer, run on SDS-7.5% (A) and 12.5% (B) polyacrylamide gels, and visualized by fluorography. Lane 6 in panel A represents 1/10 of the amount of the [35S]methionine-labeled HIF-1 protein used in the binding reactions; lane 4 represents [35S]methionine-labeled HIF-1 incubated with anti-GST affinity gel alone in the absence of GST/Ref-1. (C) Anti-GST Western blot. One-tenth input of proteins used in GST pull-down assays followed by elution and SDS-PAGE was analyzed by Western blotting using anti-GST antibodies. Positions of molecular mass markers are shown on the left in kilodaltons.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this report we demonstrate that two members of the p160 family, SRC-1 and TIF2, are able to interact with HIF-1 in a hypoxia-dependent manner and enhance its hypoxia-inducible transactivation potential. Moreover, low concentrations of SRC-1 can produce this effect in synergy with CBP, and importantly, this effect is greatly potentiated by the redox regulatory protein Ref-1, indicating that these three proteins are important components of the hypoxia signaling pathway.

Conditional recruitment of the SRC-1 and CBP classes of coactivators to HIF-1 is an important step in the hypoxia signaling pathway. TIF2 and SRC-1 are related proteins that have originally been identified as coactivators of nuclear hormone receptors and are expressed in most tissues (17-19, 28, 42, 52, 53). The two proteins could have redundant functions, or they could serve as coactivators for different or overlapping subsets of transcription factors. Our results indicate that both SRC-1 and TIF2 are able to functionally interact in very similar manners with HIF-1, a protein belonging to the bHLH/PAS family of transcription factors. In fact, SRC-1 and TIF2 are members of the same family of factors harboring a bHLH/PAS motif in their N termini. Whereas both the HLH (35) and PAS (34) motifs are potent dimerization interfaces mediating, for instance, HIF-1-Arnt heterodimerization (14, 54), the N-terminal bHLH/PAS motif of SRC-1 is not important for functional interaction with HIF-1. In agreement with this observation, the bHLH/PAS region of SRC-1 is irrelevant for enhancing steroid hormone receptor-dependent transcription or mediating physical interaction with the receptors (28, 42, 60). In the case of various members of these receptors, a conserved helix (helix 12) of the ligand binding domain is establishing a physical contact with the SRC-1 class of coactivators, and in turn, there are multiple motifs within the SRC-1 family of proteins mediating this interaction with receptors (19, 24, 38, 53, 56). These interaction interfaces appear to be characterized by the integrity of the short signature motif LXXLL, where L is leucine and X is any amino acid (16). Interestingly, our results indicate that an SRC-1 mutant protein with mutated LXXLL motifs that is unable to support estrogen receptor-dependent transcription (16) functionally interacts with HIF-1. Thus, HIF-1 may employ a mechanism of coactivator recruitment that is different from that of steroid receptors. In a similar fashion, the interaction between SRC-1 and the p50 subunit of NF-B occurs via a region that does not harbor an LXXLL motif (39). It will now be interesting to identify the structural motif(s) of SRC-1 which mediates interaction with the hypoxia-activated form of HIF-1.

Regulation of HIF-1 function by the redox regulatory protein Ref-1. What is the mechanism of hypoxia-inducible recruitment of the coactivators to HIF-1? We and others have previously observed that hypoxia-dependent activation of HIF-1 function is a multistep mechanism including massive up-regulation of HIF-1 protein levels (20, 21, 25, 44) by inhibition of ubiquitination of HIF-1 (27), nuclear translocation (26), dimerization with the constitutively nuclear factor Arnt (14, 54), and recruitment of CBP (5, 26). Moreover, we have recently demonstrated that GAL4-HIF-1 fusion proteins which show constitutive nuclear compartmentalization due to the nuclear localization signal contained within the GAL4 DBD require the hypoxic signal for functional interaction with CBP (26). In analogy to the steroid receptor system, it is an attractive scenario that the hypoxic signal determines a conformational change in HIF-1 which is critical for recruitment of the coactivators. This model of conformational regulation of HIF-1 function is supported by the present experiments which show that the integrity of the C terminus of HIF-1 containing both transactivation domains is important for cooperative regulation by SRC-1 and CBP. Moreover, the transactivation domain of Arnt is also able to functionally interact with both CBP and SRC-1 (this study and reference 30). Thus, conformational regulation may also provide a mechanism of conditional coactivator assembly on the individual transactivation domains within the heterodimeric HIF-1-Arnt complex. To understand the hypoxia signaling pathway, it is critical to identify what determines recruitment of the coactivators within the nucleus of hypoxic cells. It is possible that noncovalent modification of the C terminus of HIF-1 plays a role in determining this regulatory effect. Although this is a plausible mechanism, a protein kinase mediating such a modification has not yet been demonstrated. In the present study we observed that Ref-1 enhances the effects of SRC-1 and CBP on the transactivation potential of HIF-1. Ref-1 is known to stimulate the DNA binding activity of a number of transcription factors, including Fos, Jun, and p53, by a redox-dependent mechanism (22, 57). The stimulatory effect on Fos and Jun is elicited by reduction of a conserved cysteine residue located in the DNA binding domain of each protein (reference 57 and references therein). What is the target of regulation by Ref-1 in the hypoxia signaling pathway? Intriguingly, only the C-terminal transactivation domain of HIF-1 contains cysteine residues, whereas the N-terminal one located between amino acid residues 531 and 584 lacks cysteines. Transient overexpression of Ref-1 alone resulted in significant stimulation of the hypoxia-inducible a