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Mol. Cells, Vol. 16, No. 1, pp. 1-12 / Minireview M olecules and Cells KSMCB 2003 SM-20, EGL-9, and the EGLN Family of Hypoxia-inducible Factor Prolyl Hydroxylases Robert S. Freeman*, Daphne M. Hasbani†, Elizabeth A. Lipscomb‡, Jennifer A. Straub, and Liang Xie Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. (Received June 23, 2003; Accepted June 24, 2003) Key to the transduction of signals from the environment to the cell nucleus are enzymes that posttranslationally modify proteins. Modifications such as protein phosphorylation have long been known to regulate protein interactions, stability, and localization, as well as enzyme activity. Recent investigations into how cells respond to varying oxygen levels have identified a new mechanism for regulating signal transduction involving the post-translational hydroxylation of proline. The enzymes that catalyze this reaction comprise a novel family of prolyl hydroxylases, which include a growth-factor-responsive and cell-death-related protein (SM-20) in mammals, and a protein (EGL-9) in C. elegans important for normal egg laying. Introduction Cells respond to changes in their environment by activating networks of signal transduction pathways that culminate in adaptive changes in gene expression. Key to most (if not all) signal transduction pathways are enzymes that catalyze post-translational modifications. Over the last 25 years a nearly universal role for phosphorylation and dephosphorylation as mechanisms for altering the function, stability, and subcellular location of signaling proteins has been firmly established (Graves and Krebs, 1999). In more recent times, the transfer of a signal from the plasma membrane to the nucleus has been shown to involve other types of protein modifications including regulated proteolysis (Ebinu and Yankner, 2002), fatty acid acylation (Resh, 1996), ubiquitination (Yang and Yu, 2003), and acetylation (Magnaghi-Jaulin et al., 1999). Recent studies on the regulation of hypoxia-inducible factor (HIF), an important oxygen-sensing transcription factor in animals from nematodes to man, have now identified an additional mechanism for signal transduction involving a not-so-new post-translational modification, hydroxylation of proline residues. Enzyme catalyzed proline hydroxylation has long been known as a post-translational mechanism in collagen biosynthesis (Peterkofsky and Udenfriend, 1965), where it is important for stabilizing the triplehelical conformation in collagen fibers (reviewed by Kivirikko and Pihlajaniemi, 1998). Now with the recent discovery of a novel family of prolyl hydroxylases, proline hydroxylation has emerged as an important component in the signaling pathway that regulates how cells adapt to changes in oxygen (O2) levels. When exposed to abnormally low O2 concentrations (i.e., hypoxia), multicellular organisms respond by increasing their O2 transport capacity, glycolytic energy production, and cell survival mechanisms. These responses are a direct consequence of HIF-dependent transcriptional transactivation of genes such as erythropoietin, vascular endothelial growth factor (VEGF), glucose transporter-1, and insulin-like growth factor (see Semenza, 1999, for a review of HIF biology and biochemistry). † Present address: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA. ‡ Present address: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. Demo * To whom correspondence should be addressed. Tel: 1-585-273-4893; Fax: 1-585-273-2652 E-mail: [email protected] Abbreviations: Ang II, angiotensin II; ARNT, aryl hydrocarbon nuclear translocator; CODD, C-terminal oxygen-dependent degradation domain; CsA, cyclosporin A; EGLN, Egl-nine; EST, expressed sequence tag; HIF, hypoxia-inducible factor; HRE, hypoxia-inducible factor response element; MTS, mitochondria targeting sequence; NGF, nerve growth factor; NODD, Nterminal oxygen-dependent degradation domain; pVHL, von Hippel-Lindau tumor suppressor protein; VEGF, vascular endothelial growth factor. 2 Prolyl Hydroxylases and Signal Transduction / There are three distinct forms of HIF in mammalian cells, two of which (HIF-1 and HIF-2) are known to be induced by hypoxia. For HIF to be activated as a transcription factor, one of two unique HIF α-subunits (HIF-1α or HIF2α) must enter the nucleus and bind a common HIF βsubunit (identical to ARNT, the aryl hydrocarbon nuclear translocator). Both α- and β-subunits are members of the basic helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) superfamily, but unlike the α-subunit, the β-subunit can form complexes with various other bHLH/PAS domain proteins. Hypoxia-induced activation of HIF results from stabilization of the α-subunits, which otherwise have a half-life of just a few minutes under normoxic conditions. Maxwell et al. (1999) were the first to show that ubiquitination and proteasomal degradation of HIF-1α requires the von Hippel-Lindau tumor suppressor protein (pVHL), the substrate recognition subunit for an E3 ubiquitin ligase (see Conaway and Conaway, 2002, for a recent review). Subsequent studies indicated that stabilization of HIF was due to disruption of the HIF-pVHL interaction under hypoxic conditions (Cockman et al., 2000), but the oxygen-sensing mechanism underlying the change in affinity remained unknown. Then, two laboratories simultaneously reported that oxygen-dependent hydroxylation of a conserved proline residue in HIF-1α was critical for mediating its interaction with pVHL (Ivan et al., 2001; Jaakkola et al., 2001). (Note that Yu et al., 2001, reported similar findings a short time later.) Because the well-known collagen prolyl hydroxylases are located in the endoplasmic reticulum, whereas HIF is found in the cytoplasm and nucleus, the enzyme responsible for hydroxylating HIF was believed to be novel. Within months of the initial reports of post-translational modification of HIF by proline hydroxylation, Epstein et al. (2001) identified the HIF prolyl hydroxylase in C. elegans and provided evidence for a related family of enzymes in humans. In this review we describe the proteins that comprise the Egl-nine (EGLN) family of HIF prolyl hydroxylases. We begin with rat SM-20, the first family member identified, by reviewing what was known about its function prior to the discovery of the other EGLN proteins. The identification of egl-9 and the three mammalian EGLN genes is described next followed by a critical review of their function as prolyl hydroxylases and oxygensensitive regulators of HIF. We conclude with the hypothesis that proline hydroxylation may prove to be a general mechanism for regulating protein-protein interactions in a variety of signal transduction pathways. SM-20 is a growth factor regulated gene in muscle cells that can promote cell death in neurons Treatment of cells with growth factors and a variety of other stimuli induces the rapid and typically transient expression of so-called ‘immediate early genes’ such as cjun and c-fos. While comparing gene expression in rat vascular smooth muscle cells treated with or without platelet-derived growth factor, Taubman and colleagues (Wax et al., 1994) identified a novel gene called SM-20 that displayed characteristics of an immediate early gene. SM-20 mRNA levels peaked 1−2 h after treatment with serum, PDGF, angiotensin II (Ang II), isoproterenol, or forskolin and, like other immediate early genes, its expression was superinduced when stimulated in the presence of the protein synthesis inhibitor cycloheximide. SM-20 mRNA levels are also regulated by growth factors in C2C12 and C25 skeletal myoblast lines, although somewhat differently than in smooth muscle cells (Moschella et al., 1999). In skeletal myoblasts, SM-20 expression is enhanced during differentiation initiated in vitro by serum reduction. Upregulation of SM-20 expression in myoblasts was prevented by the addition of differentiation-blocking growth factors such as basic fibroblast growth factor. Based on these and other results described below, a role for SM-20 in myogenesis was postulated (Moschella et al., 1999). The rat SM-20 gene is predicted to encode a 355 amino acid protein (calculated molecular mass of 39.8 kDa). At the time it was first described, the predicted SM-20 protein lacked any resemblance to known proteins, although a region within its C-terminus shared a high degree of sequence identity with an uncharacterized open reading frame in the C. elegans genome (Wax et al., 1994). Antibodies generated against SM-20 detected a 40 kDa protein in immunoblots of rat aorta smooth muscle cells, consistent with its calculated molecular mass (Wax et al., 1996). In immunocytochemical studies, anti-SM-20 antibodies revealed a punctate or sometimes striated pattern in the cytoplasm of smooth muscle cells. Similar SM-20 immunoreactivity was detected in cardiac and skeletal muscle, in the smooth muscle cells of atherosclerotic plaques from humans, and in the neointima in a rat aorta balloon injury model. In addition to the relatively high levels of SM-20 antigen in muscle-rich tissues, lower levels were found in the epithelial cells of various organs (e.g., intestine, lung, pancreas, kidney, and skin) (Wax et al., 1996). SM-20 mRNA is also expressed in peripheral neurons and in brain (Lipscomb et al., 1999; Wax et al., 1994). In developing mouse embryos, low levels of SM-20 antigen were detected in proliferating myoblasts as early as embryonic day 8.5 (Moschella et al., 1999). However, SM-20 immunoreactivity increased substantially in postmitotic myocytes present by day 10.5 and it remained elevated in adult muscle. Using differential display, we found that expression of the SM-20 gene is upregulated in rat sympathetic neurons after withdrawal of nerve growth factor (NGF) (Lipscomb et al., 1999). Sympathetic and certain sensory neurons Robert S. Freeman et al. / require NGF for survival. Apoptosis initiated by NGF withdrawal is inhibited by agents that block RNA and protein synthesis, giving rise to the widely accepted idea that trophic factor deprivation-induced death requires the expression of genes that activate the apoptotic machinery. SM-20 is one of a small number of genes (others include c-jun, c-fos, cyclin D1, Bim, and DP5/HRK) whose mRNA levels increase in NGF-deprived neurons (reviewed by Freeman et al., 2003). SM-20 expression increases within 5 hours after NGF removal, peaking between 10 and 15 h. Other apoptosis-inducing stimuli also upregulate SM-20 expression in neurons, including the phosphatidylinositol 3-kinase inhibitor LY294002 and the anti-tumor drug cytosine arabinoside (Lipscomb et al., 1999). Over-expression of SM-20 is sufficient to promote cell death in NGF-maintained sympathetic neurons (Lipscomb et al., 1999; 2001). Induction of SM-20 expression in proliferating or NGF-differentiated neuronal PC12 cells stably expressing a tetracycline-inducible SM-20 transgene also leads to significant cell death (Straub et al., 2003). In both neurons and PC12 cells, SM-20-induced death is blocked by broad spectrum caspase inhibitors. Induction of SM-20 expression in PC12 cells is followed by an increase in cytosolic cytochrome c and an unexpected increase in the total amount of cytochrome c. Precisely how SM-20 expression leads to increased cytochrome c is still under investigation, but it is not accompanied by elevated cytochrome c mRNA expression nor an increase in several other mitochondrial proteins (Straub et al., 2003). We recently demonstrated that the N-terminus of SM20 contains a functional mitochondria targeting sequence (MTS) as defined by deletion analysis and its ability to target normally cytoplasmic proteins to mitochondria (Lipscomb et al., 2001). Unfortunately in performing these experiments we had to rely on over-expression of SM-20, as the anti-SM-20 antibodies available to us at the time were not sufficient for localizing endogenous SM-20 in cells. Nonetheless, a mitochondrial localization for at least a portion of the endogenous protein seems likely when images of smooth muscle cells stained with antiSM-20 antibodies (Wax et al., 1996) are compared with those of cells over-expressing SM-20 (Lipscomb et al., 2001). In addition to mitochondrial-localized SM-20, we always observe some cells that contain a pool of SM-20 in their nuclei. Nuclear-localized SM-20 has been detected in immunofluorescence experiments, in experiments with an SM-20-EGFP fusion protein, and in immunoblots of subcellular fractions enriched for nuclei (Figs. 1A and 1B). The SM-20 protein in nuclear fractions migrates at 40 kDa by SDS-PAGE while the majority of the SM-20 detected in mitochondria-enriched fractions migrates at 33–34 kDa - the molecular mass difference probably due to a cleavage event within mitochondria that removes the 3 A B a a a Fig. 1. Localization of SM20 to mitochondria and nuclei. A. CV-1 cells were transfected with an expression vector encoding an SM-20/GFP fusion protein. After 24 h, the cells were stained with Mitotracker Orange and analyzed by fluorescence microscopy (magnification is 400×). The cell marked with an arrow contains SM-20/GFP fluorescence in mitochondria and the nucleus. B. Subcellular fractions enriched for either mitochondria or nuclei were prepared from metabolically 35S-labeled NIH3T3 cells (see Lipscomb et al., 2001, for details) 24 h after transfection with SM-20 expression vector or empty vector. SM-20 protein was immunoprecipitated with a C-terminal anti-SM-20 antibody and then analyzed by SDS-PAGE and fluorography. Bands labeled ‘a’ and ‘b’ correspond to full-length SM-20 (40 kDa) enriched in the nuclear fraction and a truncated SM-20 protein (33–34 kDa) found only in the mitochondrial fraction. Aliquots of each fraction were immunoblotted for cytochrome c and proliferating cell nuclear antigen (PCNA) to indicate their purity. MTS (Lipscomb et al., 2001). It is feasible that nuclear localization of some of the over-expressed SM-20 occurs only when the mitochondrial import machinery or other processes involved in targeting SM-20 to mitochondria become saturated. However, much of the endogenous protein detected in muscle cells, sympathetic neurons, and PC12 cells migrates at 40 kDa, suggesting that at least some of the endogenous SM-20 may reside outside of mitochondria (Lipscomb et al., 1999; Wax et al., 1996; 4 Prolyl Hydroxylases and Signal Transduction / Wolf et al., 2002). Determining where SM-20 is localized in cells is important for at least two reasons. First, the SM-20 orthologs in humans and mice are predicted to encode smaller proteins that lack a classical N-terminal MTS (discussed below). If the localization of SM-20 in rat cells does not at least partially overlap with that of its ortholog in humans, then the relevance of studies on rat SM-20 to humans may be questionable. Second, a truncated form of SM-20 lacking its MTS retains the ability to promote cell death in sympathetic neurons, despite being diffusely localized throughout the cytoplasm and nucleus (Lipscomb et al., 2001). Ultimately, resolving the issue of SM-20 localization will require specific antibodies that are sufficiently sensitive to detect endogenous SM-20 under various conditions. Moreover, the possibility that SM-20 may move between mitochondria, nucleus, or other intracellular sites also warrants investigation. Evidence that much of the endogenous SM-20 protein in cells is unprocessed and that the over-expressed protein can be found in nuclei and mitochondria suggests that endogenous SM-20 will also be found in both organelles in rat cells. The EGLN gene family Recently, a family of three SM-20-like genes in the human and mouse genomes was described (Bruick and McKnight, 2001; Epstein et al., 2001; Taylor, 2001). Unfortunately, an equal number of acronyms were coined to describe the genes (Table 1). To avoid confusion in future reports, we recommend the adoption of the EGLN nomenclature (as done in this review) for its evolutionary significance, its acceptance by the Human Genome Organization (Taylor, 2001), and because it is less restrictive with respect to biochemical function. The human and mouse EGLN genes use conserved splice donor/acceptor sites to generate major transcripts consisting of 5 exons. Overall, the predicted EGLN proteins share 50% identity over the 239 amino acids that comprise the smallest family member, EGLN3. More striking, however, is a stretch of 109 amino acids encoded by exons 2–4 that is 75% identical between family members. As their name indicates, EGLN genes are homologous to the egl-9 gene in C. elegans (Darby et al., 1999), a gene required for normal egg laying. Over the 239 amino acid region of shared homology, the protein encoded by egl-9 is 40% identical to the mammalian EGLNs. The egl-9 gene is most similar to mammalian EGLN1, with the proteins encoded by each gene having two well-conserved MYND-type zinc finger domains of unknown function near their N-termini (Taylor, 2001). Because there is just a single EGLN family member in C. elegans (and apparently in Drosophila), the appearance of EGLN2 and EGLN3 is likely the result of gene duplication events in higher metazoans. Table 1. Alternative nomenclatures for EGLN family genesa. Egl-nine (EGLN)b Prolyl hydroxylase HIF prolyl hydroxydomainc (PHD) lased (HIF) EGLN1 PHD2 HPH2 EGLN2 PHD1 HPH3 EGLN3 PHD3 HPH1 a Names across each row stand for the same gene. Taylor (2001). c Epstein et al. (2001). d Bruick and McKnight (2001). b The egl-9 locus was originally characterized over 20 years ago together with a large number of other loci in which mutations resulted in defective egg laying (Trent et al., 1983). Sixteen years later, Darby et al. (1999) cloned the egl-9 gene while screening for C. elegans strains that were resistant to a rapid and lethal paralysis induced by Pseudomonas. The mutations in egl-9 conferring resistance to Pseudomonas toxicity are almost certainly lossof-function mutations since they prematurely terminate or otherwise disrupt the egl-9 open reading frame. An analysis of green fluorescence protein expression in transgenic animals containing a gfp reporter gene fused in frame with exons 1 and 2 of egl-9 suggests that EGL-9 is expressed in various muscle cells and sensory neurons in C. elegans. These results indicate that activation of EGL-9 or a pathway involving it underlies the toxin-induced paralysis and death of the worm. A more recent report from this laboratory describes the surprising finding that the toxic substance produced by Pseudomonas is cyanide (Gallagher and Manoil, 2001). Within the EGLN family, the human and mouse EGLN3 cDNAs (compiled from a large number of overlapping expressed sequence tags, or ESTs) contain open reading frames of 239 codons that are 98 and 100% identical, respectively, to amino acids 117–355 of rat SM-20 (Taylor, 2001)1. The extremely high degree of sequence identity between SM-20 and EGLN3 indicates that they represent orthologous proteins in rats, mouse and humans. Assuming the predicted EGLN3 open reading frames are correct, then translation of the mouse and human genes would initiate at a site downstream of the sequences en1 The SM-20 amino acid sequence used in the alignment reported by Taylor (2001) is based on the sequence originally described by Wax et al. (1994) and has a serine at amino acid 304. In ot er SM-20 EST and cDNA sequences and in the SM20 gene itself (GenBank accession No. NW_043947) there is a codon for phenylalanine (‘TTC’ instead of the ‘TCC’ reported in the original cDNA) at this position. Since all other EGLN genes including egl-9 encode phenylalanine at this position, amino acid 304 in SM-20 is almost certainly a phenylalanine. Robert S. Freeman et al. / coding the MTS in SM-20 (hereafter, the term SM-20 will be used exclusively for the rat gene). This would occur despite significant conservation at the nucleotide level between the 5′ ends of the EGLN3 cDNAs and the 5′ coding sequences in SM-20. In the mouse cDNA, a stop codon is present in place of the codon for Glu83 in SM-20. The corresponding region in human EGLN3 is not interrupted by a stop codon; instead, it lacks an upstream AUG analogous to the start site in SM-20. Therefore, barring alternative splicing in the 5′ exon or initiation of translation at a non-AUG codon, the first available AUG in mouse and human EGLN3 would correspond to Met117 in SM-20. Thus, the mouse and human EGLN3 proteins apparently lack the first 116 residues found in SM-20, although it should be noted that the endogenous proteins have not yet been studied. Expression of the 239-codon EGLN3 cDNA in COS-1 and U2OS osteosarcoma cells gives rise to a protein that is distributed throughout the cytoplasm and nucleus (Huang et al., 2002; Metzen et al., 2002), virtually identical to the distribution of an SM-20 deletion mutant lacking its MTS (Lipscomb et al., 2001). As with EGLN3, the predicted EGLN1 and EGLN2 proteins are highly conserved across species showing 80 and 86% identity, respectively, between their human and mouse orthologs. Cells transfected with EGLN1 or EGLN2 cDNAs (encoding proteins of 427 and 407 amino acids, respectively) reveal EGLN1 to be mostly in the cytoplasm while EGLN2 appears exclusively in the nucleus (Huang et al., 2002; Metzen et al., 2002). A handful of reports in addition to those discussed above are consistent with a role for EGLN proteins in controlling cell growth. For example, SM-20 has been identified as a gene that is upregulated in response to p53 activation in temperature-sensitive-mutant rat embryo fibroblasts (Madden et al., 1996). Colony formation assays in p53-deficient cell lines selected for stable expression of ectopic SM-20 resulted in reduced numbers of colonies, suggesting that SM-20 might negatively impact cell growth downstream of p53 function. However in PC12 cells, SM20 mRNA and protein levels are partially suppressed following treatment with Ang II, which was found to inhibit proliferation in these cells by acting through the angiotensin AT2-receptor (Wolf et al., 2002). This was opposite to the effect of Ang II in smooth muscle cells, where it induces immediate-early expression of SM-20 (Wax et al., 1994) by acting through AT1 receptors. Antisense inhibition of SM-20 by itself decreased proliferation of PC12 cells while over-expression of SM-20, which resulted in similarly decreased cell numbers in the presence or absence of Ang II, reversed the growth inhibitory effect of Ang II (Wolf et al., 2002). Moreover, a partial suppression of basal apoptosis was noted in the angiotensin-treated cells, which could also be related to inhibition of SM-20 expression given the finding that over-expression of SM-20 can pro- 5 mote cell death in PC12 cells (Straub et al., 2003). Expression of an incomplete mouse EGLN2 cDNA (called Falkor) was found to confer resistance to cisplatin-induced growth arrest in mouse embryo fibroblasts (Erez et al., 2002). Because over-expression of full-length EGLN2 reversed the effect, the authors suggested that the truncated protein encoded by the partial cDNA might counteract a growth inhibitory function of the endogenous EGLN2 protein induced by cisplatin. Treatment with antisense EGLN2 oligonucleotides modestly enhanced the growth of cells in the absence of cisplatin lending support to a possible growth inhibitory function for EGLN2. An opposite conclusion was reached concerning EGLN2 function in experiments with estrogen-responsive breast cancer cells. Using SAGE, EGLN2 was identified as an estrogen-induced gene in select estrogen-responsive breast cancer cell lines (Seth et al., 2002). In colony formation assays with these cells, stable expression of EGLN2 led to an increase in colony number irrespective of estrogen, suggesting that EGLN2 might stimulate growth in certain tumor cells. Together these diverse studies suggest growth regulatory functions for EGLN2 and SM-20, but whether they act to enhance or inhibit cell growth depends on the particular cell type and stimulus studied. EGLN genes are expressed in a quasi tissue-specific manner in adult humans (Fig. 2A). Two prominent EGLN1 transcripts are expressed at highest levels in skeletal muscle and heart and are also readily detected in kidney, liver, brain, and placenta. Roughly similar levels of a single EGLN2 mRNA are detected in heart, skeletal muscle, brain, kidney, and liver with somewhat lower levels in other tissues. The major EGLN3 transcript is most abundant in heart but is present at lower levels in other muscle containing tissues and in kidney, liver, and brain. In adult brain, EGLN3 appears to be expressed ubiquitously in all regions (Fig. 2B). Some differences with these expression patterns have been noted in mouse tissues; in addition, high levels of EGLN2 were seen in mouse testis where little if any EGLN1 or EGLN3 was detected (Lieb et al., 2002). EGLN proteins are novel iron- and 2-oxoglutarate-dependent dioxygenases A major breakthrough towards understanding SM-20 and EGLN function came when secondary structure prediction algorithms identified the gene products of egl-9 and an EST derived from the human EGLN3 gene as potential new members of the iron- and 2-oxoglutarate-dependent dioxygenase superfamily (Aravind and Koonin, 2001; Epstein et al., 2001). The defining structural features of this large and diverse family are an α-helix followed by 7–8 β-strands arranged in a jelly roll β-barrel topology. Situated within the large cavity that forms the active site is a conserved 2- 6 Prolyl Hydroxylases and Signal Transduction / A B Fig. 2. Expression of EGLN genes in human tissues. A. A human multi-tissue northern blot (Clontech) was hybridized (sequentially) with 32P-labeled cDNA probes for EGLN3, EGLN2, and EGLN1. Autoradiography was for 10 d (EGLN3), 3 d (EGLN2), or 23 h (EGLN1). Lane 1, brain; lane 2, heart; lane 3, skeletal muscle; lane 4, colon; lane 5, thymus; lane 6, spleen; lane 7, kidney; lane 8, liver; lane 9, small intestine; lane 10, placenta; lane 11, lung; lane 12, peripheral blood lymphocytes. B. An adult human brain northern blot (Clontech) was hybridized with a 32P-labeled EGLN3 cDNA probe. Lane 1, cerebellum; lane 2, cerebral cortex; lane 3, medulla; lane 4, spinal cord; lane 5, occipital pole; lane 6, frontal lobe; lane 7, temporal lobe; lane 8, putamen; lane 9, amygdala; lane 10, caudate nucleus; lane 11, corpus callosum; lane 12, hippocampus; lane 13, whole brain; lane 14, substantia nigra; lane 15, thalamus. The two blots (lanes 1−8 and 9−15) were hybridized and washed simultaneously and exposed to film for 15 h with an intensifying screen. Subsequent blotting with an actin cDNA probe indicated approximately equal RNA loading across the lanes in the blots in (A) and (B). histidine-1-carboxylate motif (H-X-D/E…H) involved in chelating nonheme iron. Iron(II) is critical for activating O2 and as a template for the orderly binding of reactants. In dioxygenase-catalyzed reactions both oxygen atoms of an O2 molecule are incorporated into substrate. The conserved C-terminal region of EGL-9 and EGLN3 showed greatest similarity to the catalytic domain of mammalian prolyl-4hydroxylase (Aravind and Koonin, 2001) (Fig. 3). In the reaction catalyzed by prolyl-4-hydroxylase, one oxygen atom is added to a peptidyl proline to form hydroxyproline while the other is used in a coupled decarboxylation reaction that converts 2-oxoglutarate to succinate. In addition to requirements for O2, iron(II), and 2-oxoglutarate, ascorbate (although not stoichiometrically consumed) is necessary for maximal prolyl hydroxylase activity (reviewed by Kivirikko and Pihlajaniemi, 1998). At the same time that EGL-9 and related proteins were suggested to be novel prolyl hydroxylases, researchers investigating the oxygen-dependent regulation of HIF were searching for an enzyme that could catalyze hydroxylation of HIF α-subunits at a critical proline residue (Mole et al., 2001). This proline (Pro564 in HIF-1α) lies within the C-terminal half of a previously characterized oxygen-dependent degradation domain, abbreviated ‘CODD’. Hydroxylation of Pro564 in HIF-1α had just been shown to mediate its interaction with the VHL protein present in an E3 ubiquitin ligase complex, resulting in its rapid ubiquitination and proteasomal degradation (Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001) (Fig. 4). In these studies, the hydroxyproline-dependent association of HIF-1α with pVHL was inhibited by hypoxia and by various other agents including iron chelators, cobalt(II), and structural analogs of 2-oxoglutarate. These compounds had been known for some time to mimic hypoxia and more recently to stabilize HIF-1α under normoxic conditions. Armed with HIF and pVHL homologs in C. elegans and the realization that egl-9 might encode a novel prolyl hydroxylase, Ratcliffe and colleagues (Epstein et al., 2001) carried out a series of genetic and biochemical experiments that elegantly established EGL-9 as a HIF prolyl hydroxylase in C. elegans. Analysis of endogenous HIF protein levels clearly demonstrated a defect in the ability of C. elegans strains with inactivating mutations in egl-9 to downregulate HIF expression under ambient O2 conditions. Inactivating mutations in the C. elegans collagen-prolyl-hydroxylase genes or in other potential HIFregulating genes did not affect the oxygen-mediated destabilization of HIF. Thus, loss of EGL-9 function specifically correlated with a failure to destabilize HIF protein in normoxic animals. Additional experiments showed that a recombinant EGL-9 protein (residues 359−723) could hydroxylate the conserved proline in C. elegans HIF in an O2, iron, and 2-oxoglutarate dependent manner. Interestingly, the four egl-9 inactivating alleles that resulted in constitutive HIF expression in normoxia were the same loss-of-function alleles found by Darby et al. (1999) to confer resistance to paralysis induced by Pseudomonas. Mammalian EGLN proteins function as HIF prolyl hydroxylases Along with the discovery that EGL-9 functions as a HIF prolyl hydroxylase, Epstein et al. (2001) and Buick and Robert S. Freeman et al. 7 / Fig. 3. The iron- and 2-oxoglutarate-dependent dioxygenase catalytic domain in EGLN family members. The amino acid sequence in the catalytic domains of rat SM20, three human EGLN proteins, and C. elegans EGL-9 are aligned with the corresponding domain in human prolyl-4-hydroxylase (P4H). The predicted secondary structure is indicated at the top of the alignment: H = α helix; E = β strand. Asterisks identify the conserved 2-histidine-1-carboxylate motif involved in coordinating iron(II) in the active site. Vertical bars indicate residues conserved in all six proteins. Fig. 4. Regulation of HIF-1α stability by oxygen-sensitive proline hydroxylation. Under normal oxygen concentrations, HIF-1α is hydroxylated at Pro402 and Pro564 by one or more HIF prolyl hydroxylases. The reaction requires O2 and iron(II) and utilizes peptidyl proline and 2-oxoglutarate as co-substrates; ascorbate is a cofactor needed for maximal activity. Hydroxyproline, succinate, and CO2 are produced. Proline hydroxylation of HIF-1α increases its affinity for pVHL, which targets HIF-1α for ubiquitination and degradation. Under hypoxic conditions, prolyl hydroxylase activity decreases and non-hydroxylated HIF-1α is free to accumulate in the nucleus where it associates with the HIF β-subunit (ARNT) to activate transcription of target genes such as VEGF. McKnight (2001) used in vitro pull-down assays to show that all three EGLN proteins could enhance the pVHLbinding activity of HIF-1α or synthetic HIF-1α peptides centered around Pro564. As expected, peptides mutated at Pro564 and incubated with EGLN proteins failed to bind pVHL, although this was not always the case when fulllength HIF-1α was used as the substrate (see below for a discussion of a second proline hydroxylation site in HIF1α). Importantly, the ability of a recombinant EGLN protein (in this case EGLN2) to enhance the HIF-1α-pVHL association showed a similar graded response to changes in O2 tension (0–21%) as the endogenous activity present in RCC4 renal carcinoma cell extracts (Epstein et al., 2001). In these and other studies (Ivan et al., 2002) the EGLN-dependent effects were shown to be inhibited by iron chelators, CoCl2, the 2-oxoglutarate analog N-oxalylglycine, and several newly described prolyl hydroxylase inhibitors. These compounds inhibit prolyl hydroxylases through distinct mechanisms; however, with the possible exception of the most recent compounds, their use is lim- ited due to lack of specificity (Günzler and Weidmann, 1998). Mutations in the critical iron-binding residues in the 2-histidine-1-carboxylate motif block the ability of EGLN proteins to increase the HIF-pVHL interaction. Purified recombinant EGLN proteins have been shown to directly hydroxylate Pro564 in synthetic peptide substrates confirming that the activity is intrinsic to the EGLNs (Bruick and McKnight, 2001; Epstein et al., 2001; Ivan et al., 2002). HIF-dependent transcriptional transactivation assays done in HEK293 and COS-1 cells under normoxic cell culture conditions (i.e., 21% O2) have shown that coexpression of either EGLN1, EGLN2, or EGLN3 together with wild-type HIF-1α significantly reduces transactivation of the HIF response elements (HREs) driving expression of the reporter genes (Bruick and McKnight, 2001; Huang et al., 2002). The ability of over-expressed EGLN proteins to repress HRE activation by over-expressed HIF-1α depended on an intact iron-chelating center in the enzyme and was inhibited when cells were cultured at O2 8 Prolyl Hydroxylases and Signal Transduction / < 1%2. Decreased HRE activity in normoxic cells overexpressing each of the EGLN proteins is accompanied by a reduction in the level of ectopic HIF-1α protein (Huang et al., 2002). This result, along with the simple observation that ectopically expressed HIF-1α can be detected in normoxic cells (in contrast to endogenous HIF-1α), is consistent with the notion that the endogenous prolyl hydroxylases are rate-limiting for destabilizing HIF-1α when O2 is not limiting. Together these results are consistent with a model whereby HIF-1α levels are kept low under normoxic conditions through the action of HIF prolyl hydroxylases but then increase dramatically during hypoxia when these same enzymes are inhibited by inadequate O2. Oddly enough, endogenous HIF-1α protein levels are not substantially increased by CoCl2 treatment or even hypoxia (O2 < 1%) in cells over-expressing EGLN1 or EGLN3 (Cioffi et al., 2003; Metzen et al., 2002), raising the possibility that such conditions do not completely inhibit these enzymes when over-expressed. Consequently, experiments involving over-expression should be interpreted with caution due to the possibility that a portion of the over-expressed enzyme or its substrate might escape its normal regulatory controls. Reports published in recent months raise the possibility that HIF prolyl hydroxylases are regulated in ways other than just changes in O2 tension. An endogenous HIF prolyl hydroxylase activity in Hep3B cells has been partially purified using the CODD of HIF-1α as substrate and a pVHL pull-down assay as an indirect measure of Pro564 hydroxylation (Wang et al., 2002). The prolyl hydroxylase activity was suppressed by CoCl2, iron chelators, or low levels of O2 as expected, but the suppression was reversed by nitric oxide (NO) donors such as sodium nitroprusside. NO had previously been implicated in the inactivation of HIF but the mechanism was unknown (see Semenza, 2001). Because treatment with NO donors led to reduced levels of endogenous HIF-1α protein and activity under conditions that would otherwise inhibit the prolyl hydroxylase and stabilize HIF (i.e., iron chelation or very low O2), NO was postulated to directly activate the prolyl hydroxylase (Wang et al., 2002). These results suggest that NO may function in a novel mechanism for activating prolyl hydroxylases at suboptimal O2 levels. Like NO, the immunosuppressant cyclosporin A (CsA) was recently shown to inhibit hypoxia-induced stabilization of endogenous HIF-1α (D’Angelo et al., 2003). Using an assay for 2-oxoglutarate-dependent dioxygenases, 2 Note that the O2 levels achieved in various studies may not be directly comparable as it is difficult to obtain reproducibly low O2 levels, especially < 1%, in cell culture experiments. In addition, the indicated O2 levels are not always maintained during the preparation of extracts and in reactions. This is particularly important since HIF-1α can undergo extensive degradation within minutes of re-oxygenation. a Pro564-dependent activity was identified in a ‘light mitochondria’ fraction from rat kidney that was enhanced by treatment with CsA. Incubating recombinant CODD with the light mitochondria fraction resulted in binding to [35S]-labeled pVHL that too was enhanced by CsA. Mutation of Pro564 in the CODD to alanine prevented the interaction with pVHL and its augmentation by CsA, providing evidence that CsA was acting via the prolyl hydroxylase. Although the molecular nature of the prolyl hydroxylase activity in the rat kidney extracts is unknown, the localization of SM-20 to mitochondria makes it an attractive candidate. The studies described above suggest that both CsA and NO may act to suppress hypoxiainduced responses by maintaining or enhancing the activity of HIF prolyl hydroxylases under conditions that would otherwise be inhibitory. Future experiments will obviously be aimed at identifying the prolyl hydroxylases implicated in these studies. In addition to Pro564, a second conserved proline (Pro402) located in the N-terminal half of the oxygendependent degradation domain (NODD) of HIF-1α has been found to be important for the interaction with pVHL (Masson et al., 2001). By mutating these residues either individually or in tandem, Epstein et al. (2001) showed that the two prolines are differentially utilized, at least in vitro, by the EGLN proteins. All three human EGLN proteins as well as rat SM-20 could hydroxylate HIF-1α mutated at Pro402 (as assessed by its ability to bind pVHL). In contrast, only EGLN1 and EGLN2 could hydroxylate HIF-1α mutated at Pro564. None of the EGLN proteins could enhance the affinity of HIF-1α for pVHL when both prolines were mutated, suggesting that these are the only sites hydroxylated by the EGLNs - or more precisely, the only hydroxylated residues permissive for pVHL binding. The presence of two distinct sites for proline hydroxylation may help explain, at least partly, the existence of multiple EGLN prolyl hydroxylases. More than one EGLN enzyme might also be necessary in order to modify the different HIF α-subunits. HIF-2α, which is also stabilized by hypoxia, contains prolines equivalent to the two hydroxylated residues in HIF-1α. In each case, the hydroxylated proline is contained within a Leu-X-XLeu-Ala-Pro motif (Ivan et al., 2001; Jaakkola et al., 2001). Recently, Chan et al. (2002) described an antibody that preferentially binds HIF-1α when it is hydroxylated at Pro564. This exciting reagent, and future antibodies designed to recognize other hydroxyproline-containing sequences, may prove as useful for studying pathways involved in HIF regulation as phosphorylation site-specific antibodies have in other aspects of signal transduction. The development of this antibody is remarkable for another reason; it may indicate that similar hydroxyprolinetargeting antibodies designed with less sequence specificity towards HIF could be used in the search for novel substrates of EGLN prolyl hydroxylases. Robert S. Freeman et al. / Three-dimensional structure determinations of Pro564containing peptides bound to the pVHL/E3 ligase complex reveal a single pocket in pVHL for binding hydroxyproline (Hon et al., 2002; Min et al., 2002). The pocket is lined by five amino acids highly conserved from C. elegans to humans, all of which are affected by missense mutations in VHL disease, an autosomal dominant cancer syndrome that predisposes afflicted individuals to a variety of highly vascularized tumors. Comparisons between Pro402 and Pro564 peptides suggest that both hydroxylation sites in HIF-1α bind the same hydroxyprolinebinding pocket in pVHL (Hon et al., 2002), providing a possible explanation for why hydroxylation at just one site may under certain conditions be sufficient for destabilizing HIF-1α (Pereira et al., 2003). Results from pVHL pull-down assays using HIF fragments and recombinant prolyl hydroxylases made in insect cells suggest a tentative order of reactivity with respect to hydroxylation of Pro564 - EGLN1 > EGLN3 > EGLN2 (Huang et al., 2002). The activities of these enzymes with respect to Pro402 and, even more importantly, their activities in vivo remain to be determined. Nonetheless, the relatively low activity of EGLN2 is interesting in light of other experiments showing that the EGLN1 and EGLN3 genes, but not the EGLN2 gene, are transcriptionally upregulated in various cells exposed to hypoxia (Cioffi et al., 2003; Epstein et al., 2001; Metzen et al., 2002). Thus, EGLN1 and EGLN3 may themselves be HIF target genes that are upregulated in response to hypoxia. Increased EGLN expression in cells while they are still hypoxic could be part of a negative feedback mechanism for the rapid hydroxylation and degradation of HIF-1α when O2 supplies are restored. The ability of different EGLN proteins to hydroxylate HIF-1α in vivo is likely to be a function of their intracellular location as well as the location of HIF-1α. Since HIF1α can be found in both the cytoplasm and the nucleus, and may even be regulated by mitochondrial function (Agani et al., 2000), all of the EGLN proteins could be involved in regulating HIF at distinct intracellular sites. For example, treatment of HeLa cells with the prolyl hydroxylase inhibitor dimethyloxalylglycine blocks not only HIF-1α degradation upon reoxygenation, but also its export from the nucleus (Groulx and Lee, 2002). Thus degradation of the nuclear pool of HIF-1α in reoxygenated cells appears to require a nuclear prolyl hydroxylase, possibly EGLN2 or EGLN3. Evidence that EGLNs are the endogenous HIF hydroxylases also comes from experiments using novel small molecule prolyl hydroxylase inhibitors and polypeptides derived from the hydroxylation sites in HIF-1α (Huang et al., 2002; Ivan et al., 2002; Willam et al., 2002). Treatment of a variety of normal and tumor cells with a novel prolyl hydroxylase inhibitor (FG0041) at concentrations that inhibit EGLN1 in vitro led to stabili- 9 zation of endogenous HIF-1α protein levels and increased synthesis of VEGF, a well-characterized HIF target (Ivan et al., 2002). In other studies, over-expression of the HIF1α NODD (containing Pro402) or CODD (containing Pro564) was found to enhance HIF-1α protein levels, transactivation activity, and expression of HIF target genes (Huang et al., 2002; Willam et al., 2002). Promising results from in vivo angiogenesis assays suggest that introduction of these HIF-1α fragments into mice can increase blood vessel growth and VEGF and glucose transporter-1 immunostaining, apparently as the result of HIF activation (Willam et al., 2002). Over-expression of these domains could block HIF-1α degradation by competitive inhibition with respect to either the prolyl hydroxylase or, maybe more likely, the HIF-binding pocket in pVHL. Ultimately, direct proof that an EGLN protein functions as an endogenous HIF prolyl hydroxylase will come from experiments in which the EGLN genes are individually, or perhaps collectively, inactivated. Nonetheless, recent demonstrations that inactivation of egl-9 in C. elegans and the EGLN homolog in Drosophila (Bruick and McKnight, 2001; Lavista-Llanos et al., 2002) results in normoxic stabilization of HIF strongly suggest that at least one member of the mammalian EGLN family serves a similar function. Although abundant data now implicate the EGLN proteins as HIF prolyl hydroxylases, the relative importance of these enzymes as critical regulators of HIF stability in vivo remains to be verified. Sensitive assays that directly measure EGLN prolyl hydroxylase activity will be needed in order to fully address this issue. The ability to directly measure EGLN activity is even more critical given recent reports that HIF’s ability to transactivate gene expression and its stabilization by hypoxia are regulated by factors in addition to proline hydroxylation. HIF-1α was recently shown to undergo an additional hydroxylation event, this time at an asparagine residue (Asn803) (Lando et al., 2002). Like proline hydroxylation, hydroxylation of asparagine is catalyzed by an iron- and 2-oxoglutarate-dependent dioxygenase, but in this case an asparaginyl hydroxylase that was originally identified as ‘Factor Inhibiting HIF-1’, or FIH-1 (Lando et al., 2002; Mahon et al., 2001). This reaction, which is also sensitive to O2, regulates the interaction between HIF-1α and the nuclear transcriptional coactivator CBP/p300, thus influencing HIF transcription factor activity. A third post-translational mechanism for regulating HIF-1α, also discovered within the last year, involves acetylation at a lysine residue in the oxygendependent degradation domain (Jeong et al., 2002). Acetylation of HIF-1α by a cytosolic enzyme called ARD1 enhanced its interaction with pVHL resulting in increased ubiquitination and ultimately decreased HIF stability. Mutation of the target lysine (Lys532) increased the stability of the over-expressed HIF-1α under normoxic conditions. Determining the relative contributions of proline hy- 10 Prolyl Hydroxylases and Signal Transduction / droxylation, asparagine hydroxylation, and lysine acetylation for the overall activity of HIF will undoubtedly take some time to resolve and may well depend on cell type, O2 levels, and other factors that influence HIF function. Conclusion HIF function is clearly important for a proper physiologic response to changes in O2 tension through its ability to regulate genes involved in glycolysis, angiogenesis, erythropoiesis, and cell survival (Maxwell and Ratcliffe, 2002). Moreover, it is highly likely that one or more EGLN proteins will have a critical role in regulating HIF function in oxygen-sensitive tissues, and perhaps in response to other stimuli under non-hypoxic conditions. But do all three EGLN proteins function exclusively as HIF prolyl hydroxylases? Clearly O2 plays an important role in regulating EGLN function but other factors (growth factors, neurotrophic factors, CsA, NO) also appear to modulate the expression or activity of EGLN proteins. Unpublished data from our laboratory showing that prolyl hydroxylase inhibitors can protect neurons from cell death under normoxic conditions support a role for EGLN proteins in neuronal apoptosis that may or may not involve HIF. A recent report that the large subunit (Rpb1) of RNA polymerase II can bind pVHL in a hydroxyproline-dependent manner hints that additional substrates for EGLN prolyl hydroxylation exist and will soon be identified (Kuznetsova et al., 2003). Even in C. elegans which has only one EGLN gene, the observation that one of four egl-9 mutants defective in HIF regulation and resistant to Pseudomonas toxicity does not appear to have an egg-laying defect (Darby et al., 1999), suggests that at least one egl-9-associated phenotype need not involve the HIF pathway. Data implicating EGLN proteins in such disparate processes as O2 sensing, neuronal cell death, cell proliferation, and muscle cell differentiation in mammals, as well as egg laying and toxin-induced paralysis in C. elegans, point to the realization that prolyl hydroxylation as a signal transduction mechanism will soon extend well beyond the interaction between HIF and pVHL. Acknowledgments We thank current and past members of the Freeman laboratory for their help in formulating the ideas expressed in this review. R. S. Freeman acknowledges support from the National Institutes of Health grants NS34400 and NS42224. References Agani, F. H., Pichiule, P., Chavez, J. C., and LaManna, J. C. (2000) The role of mitochondria in the regulation of hypoxiainducible factor 1 expression during hypoxia. J. Biol. Chem. 275, 35863−35867. Aravind, L. and Koonin, E. V. (2001) The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2oxoglutarate- and iron-dependent dioxygenases. 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